We thank Dr. Swathi Arur (MD Anderson Cancer Center) for providing the let-60(n1046). We also thank Dr. David Reiner (Texas A&#38;M Health Science Center Institute of Biosciences &#38; Technology in Houston) for the lin-1 strain. Finally, we thank Dr. Danielle Garsin and her lab (The University of Texas, McGovern Medical School) for providing some of the reagents. Some worm strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). This Research was supported by the Cancer Prevention and Research Institute of Texas (CPRIT) grant RP200047 to JF Hancock.

1. Nematode growth medium (NGM) plate preparation
<ol>
	<li>Add 2.5 g of peptone and 3 g of NaCl to 970 mL of deionized water contained in a 2 L Erlenmeyer flask. Stir contents using a magnetic stir bar. Thereafter, add 20 g of agar to the flask. Autoclave the contents of the flask at 121 &#176;C and a pressure of 15 lb/in2 for 30 min. After sterilization, place the flask on a stir plate and allow the medium to cool until the temperature reaches 50 &#176;C.</li>
	<li>To prepare the NGM plates add the following reagents to the cooled medium: 25 mL of 1 M potassium phosphate buffer (pH = 6.0), 1 mL of 1 M MgSO4, 1 mL of 1 M CaCl2, 1 mL of (5 mg/mL in 95% ethanol) cholesterol, 1 mL of (10% v/w in ethanol) nystatin, and 1 mL of 25 mg/mL streptomycin.</li>
	<li>Under a laminar flow, pour the cooled medium into 60 mm x 15 mm sterile Petri dishes. Let the plates solidify for 2 h. These plates can be kept for 1 month at 4 &#176;C.</li>
</ol>
2. Propagation of C. elegans
<ol>
	<li>Spot 100 &#956;L of overnight grown E. coli OP50 onto the center of each NGM plate and allow the plates to dry for 24 h in a laminar hood. Subsequently, the plates can be stored in polystyrene container. 
	NOTE: The E. coli OP50 culture is grown in Luria-Bertani (LB) media at 37 &#176;C media prior to seeding of the plates. The culture can be stored at 4 &#176;C for 1&#8211;2 months and used for periodic seeding of plates.</li>
	<li>Using a sterile worm pick, gather 10&#8211;12 gravid adult worms from a previously grown plate under a dissecting microscope. Transfer these worms to a fresh NGM plate seeded with E. coli OP50 and incubate the plate for 24 h at 20 &#176;C.</li>
	<li>After 24 h, using a sterile worm-pick remove adult worms from the plates. Incubate the plates at 20 &#176;C for ~ 3 days. Embryos will develop into gravid adult worms.</li>
</ol>
3. Preparation of a synchronous C. elegans culture
<ol>
	<li>Collect gravid adult worms into a 15 mL conical tube by washing 2&#8211;4 plates with M9W.
	<ol>
		<li>Preparation of M9W: Dissolve 5 g of NaCl, 6 g of Na2HPO4, and 3 g of KH2PO4 in deionized water to a final volume of 1 L. Autoclave the solution at 121 &#176;C at a pressure of 15 lb/in2 for 30 min. Add 1 mL of 1 M MgSO4 to the cooled solution.</li>
	</ol>
	</li>
	<li>Pellet the worms by centrifuging the tube at 450 x g for 1 min. Decant the supernatant without disturbing the worm pellet.</li>
	<li>Prepare worm lysis solution by combining 400 &#956;L of 8.25% sodium hypochlorite (household bleach) and 100 &#956;L of 5 N NaOH. Add this solution to the worm pellet and flick the tube to mix contents of the tube. Prevent overbleaching of the embryos in the tube by observing the lysis of the worms under a dissecting microscope.</li>
	<li>Add 10 mL of M9W to the conical tube to dilute the lysis mix when 70% of the adult worms have lysed.</li>
	<li>Centrifuge the tube at 450 x g for 1 min. Replace the supernatant with 10 mL of M9W.</li>
	<li>Repeat step 3.5 two times.</li>
	<li>After completing the washing steps, add 3&#8211;5 mL of M9W to resuspend the egg pellet. Rotate the tube overnight at a speed of 18 rpm on a tube rotator at room temperature (RT).</li>
	<li>After overnight incubation, remove the tube from the rotator, pellet the L1 larvae by centrifuging the tube at 450 x g for 1 min. Aspirate the M9W until 250 &#956;L is left in the tube.</li>
	<li>Shake the tube to resuspend the larvae. Add three 5 &#956;L drops containing the larvae onto a Petri dish lid. Using a dissecting microscope count the number of worms in each drop and determine the number of worms in 1 &#956;L.</li>
</ol>
4. Preparation of drug assays
NOTE: The steps in this assay are shown in Figure 1.
<ol>
	<li>Grow 30 mL of E. coli OP50 in a 50 mL conical tube at 37 &#176;C overnight in an orbital shaker at 150 rpm.</li>
	<li>Spin overnight grown E. coli OP50 culture at 4,000 x g for 10 min to pellet the cells. Remove the supernatant and resuspend the pellet in 3 mL of M9W to concentrate the culture.</li>
	<li>Prior to preparing the working solutions for the drug assay, add 0.1 mL of (5 mg/mL in 95% ethanol) cholesterol into 100 mL of M9W.</li>
	<li>Prepare working solutions of each experimental drug by diluting each drug plus vehicle (dimethyl sulfoxide; DMSO) in 4.8 mL M9W supplemented with cholesterol to obtain the appropriate concentrations. Dissolve DMSO in 4.8 mL of M9W supplemented with cholesterol to prepare the vehicle control.</li>
	<li>Add 200 &#956;L of concentrated E. coli OP50 culture to each tube containing the vehicle control or drugs and vortex tubes to ensure they are mixed.</li>
	<li>To perform the experiment, add 2 mL of each working drug solution or vehicle control to each well in a 12-well tissue culture plate. Test each drug concentration and vehicle control in duplicate.</li>
	<li>Add ~100 L1 larvae per well (see synchronous C. elegans culture steps) using a sterile micropipette. Limit the volume of worms to 10 &#956;L.</li>
	<li>Incubate the plates at 20 &#176;C.</li>
	<li>Supplement wells with 50 &#956;L of 10x concentrated E. coli OP50 on day 3 of the assay if needed.</li>
</ol>
5. Agarose pad preparation for microscopy
<ol>
	<li>Prepare a 2% w/v solution of agarose by dissolving 0.1 g of agarose in 5 mL of deionized water. Heat the contents in a microwave to dissolve the agarose. 20 slides can be prepared from 5 mL of agarose solution.</li>
	<li>Place strips of lab tape along two glass slides. The tape will act as spacers limiting the thickness of the agarose pads. Thereafter, place a third clean glass slide between the taped slides.</li>
	<li>To make an agarose pad, spot 100 &#956;L of molten agarose onto the center of the clean slide. Place another clean glass slide across and on top of the agarose and gently press down to form a pad. Remove the top slide when the pad has solidified.</li>
</ol>
6. Observation of the Muv phenotype in the let-60, let-23 and lin-1 strains
NOTE: Only candidate drugs that suppress the Muv phenotypes in let-23 and let-60 strains will be assayed using the lin-1 strain to determine if the inhibition occurs at the level of RAS or EGFR.
<ol>
	<li>When the appropriate stage of the life cycle is reached (3 days for the let-60 and let-23 strains, 5 days for the lin-1 strain), remove the plates from the incubator. and collect the worms in 15 mL conical tubes using a pipette.</li>
	<li>Centrifuge the tubes at 450 x g for 1 min. Remove M9W without disturbing the worm pellet and add 5 mL of fresh M9W.</li>
	<li>Repeat step 6.2 two times.</li>
	<li>Without disturbing the worm pellet, remove all remaining M9W by aspiration. Thereafter, add 500 &#956;L of 2 mM sodium azide or 2 mM tetramisole hydrochloride. Allow tubes to incubate at RT for 15 min. 2 mM sodium azide or 2 mM tetramisole hydrochloride will anesthetize the worms for imaging. 
	CAUTION: Make sure to wear personal protective equipment (PPE) when handling sodium azide. The solution must be prepared under a chemical hood.</li>
	<li>Add 10 &#956;L of the anesthetized worm suspension onto the center of an agarose pad. Place a #1.5 coverslip gently over the worm suspension. If needed, fix the coverslip with some nail polish to prevent drying out.</li>
	<li>Use a DIC microscope to observe the let-60(n1046), let-23(sa62) and lin-1(sy254) strains. Image the worms at the 10x and 20x magnifications.</li>
	<li>For let-60(n1046) and let-23(sa62), score the adult worms based on the presence or absence of the Muv phenotype. While for the lin-1 strain, count the number VPCs that adopted 1&#176; or 2&#176; cell fates on the ventral side of L4 larvae using a high resolution DIC microscope.</li>
	<li>After scoring the micrographs for each strain and drug treatment, perform Student&#8217;s t-test to determine the statistical differences between the treatments.</li>
</ol>

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The authors declare no competing financial interests.

The assays we describe using the worm are simple and inexpensive to identify inhibitors of EGFR and RAS function. C. elegans is an attractive model for drug discovery because it is easy to grow in the lab due to the short life cycle (3 days at 20 &#176;C) and the ability to generate large numbers of larvae. More importantly, the EGFR-RAS-ERK MAPK pathway is evolutionarily and functionally conserved with mammals providing a genetically tractable system to analyze the effects of EGFR and RAS inhibitors. Further, the transparent nature of the worms enables an investigator to visualize distinct structures and the localization of Green fluorescent Protein (GFP) or other fluorophore fused to proteins of interest by DIC and fluorescent microscopy.
The protocols we used to propagation and maintain the various C. elegans used in this study were previously established20,21. However, in the preparation of NG plates we incorporated streptomycin and nystatin to prevent bacterial and fungal contamination. The addition of these antimicrobial agents did not impede the development of the worms and the induction of the Muv phenotype.
There are several advantages for obtaining L1 larvae by the worm synchronization protocol. Many larvae can be obtained from 2 or more plates containing gravid adults and the larvae collected are all age synchronized. This ensures development of the worms is consistent within the population. Some mutant strains displaying the Muv phenotype are poor egg layers resulting in low yields of larvae as seen in the null mutation harbored in the Ets family transcription factor lin-119. Bleach treatment of the lin-1 gravid adults will significantly increase the number of larvae required for the assay.
It is vital to observe the lysis of the worms during the preparation of a synchronous C. elegans culture. The thick eggshell partially protects the embryos from the action of the bleach-sodium hydroxide mix even as the cuticle of the larvae and adults dissolve. However, prolonged exposure to the lysis solution will penetrate this protective casing leading to the death of the embryos. Hence it is important to stop the action of the bleach-sodium hydroxide mix when 70% of the adult worms have lysed. This is achieved by diluting the lysis solution with M9W. The maintenance of the arrested L1 larvae is another step to consider in the synchronous C. elegans culture protocol. Prolonged incubation of the L1 arrested larvae in M9W can cause them to transform into the dauer stage due to accumulation of the dauer pheromone. To avoid the formation of the dauer stage, it is suggested to use the L1 arrested larvae within 1&#8211;2 days of collecting the embryos.
The basal Muv phenotype is 60%&#8211;90% and 90% for the let-60(n1046) and let-23(sa62) worms, respectively. This suggests the let-60(n1046) worms are subject to phenotypic drift and, therefore, it is important to report the basal levels of expression of the Muv phenotype. Treatment of let-60(n1046) and let-23(sa62) worms with R-fendiline and other K-RAS inhibitors reduce the percentage of worms expressing the Muv phenotype. However, it is also reported that some inhibitors of the EGFR-RAS-ERK MAPK pathway may reduce the number of pseudovulvae per worm alone or may affect both the expression and the number of pseudovulvae17. Hence it is important to count the number of pseudovulvae in the Muv worms in both drug and DMSO treated worms. The Muv phenotype expressed in let-60(n1046) and let-23(sa62) adult worms is clearly visible under a dissecting microscope. However, the lin-1 (null) strain is relatively unhealthy, developmentally impaired and the vulval protrusions are poorly distinguished in the adult worms. Therefore, instead of the ectopic vulval protrusions, in lin-1 (null) worms, VPCs that adopt a adopted 1&#176; or 2&#176; cell fates on the ventral side in the L4 stage, can be counted using a high resolution DIC microscope.
The assay is inexpensive, easy, and not time consuming to setup. To further improve the processing time, the worms can be anesthetized to a final concentration of 2 mM sodium azide within the wells of tissue culture plate and imaged using a dissecting microscope equipped with a camera. Another modification of the assay would be to use heat killed E. coli OP50 instead of live bacteria23. It has been shown that bacteria can metabolize certain small molecules leading to reduced bioactivities24.
Previous studies have shown that the induction of the vulva in the worm is dependent on certain environmental cues. The vulvaless phenotypes of lin-3(n378) and let-23(n1045) have shown to be partially suppressed by starvation and exiting the dauer stage14. Furthermore, a study by Moghal et al., showed that vulvaless lin-3(n378), let-23(sy1) and let-60(sy95dn) mutants grown in M9 buffer had a higher number of VPCs assuming vulval cell fates compared with animals grown on standard NG plates24. The data suggests worms grown in a liquid environment influences vulval induction. However, in our studies we did not observe&#160;the suppression of the Muv phenotype in a liquid environment.
In this protocol we demonstrate the use of C. elegans to evaluate the anti-RAS properties of fendiline. In a previous study, we have shown that multiple acid sphingomyelinase inhibitors, including tricyclic antidepressants such as desipramine, imipramine, and amitriptyline inhibit the Muv phenotype18. Furthermore, inhibitors of the sphingomyelin and ceramide biosynthetic pathways suppress the Muv phenotype expressed in the let-60(n1046) worms. These findings using the worm were validated in mammalian cell lines.
In conclusion, we demonstrate the use of C. elegans to identify inhibitors of EGFR and RAS activity in a liquid-based assay. Furthermore, the worm provides another system to identify and characterize the mechanisms of action of anti-RAS and EGFR therapeutics.

The cellular pathways that regulate developmental events within organisms are highly conserved among all metazoans. One such pathway is the EGFR-RAS-ERK mitogen activated protein kinase (MAPK) signaling cascade which is a critical pathway that governs cell proliferation, differentiation, migration and survival1,2. Defects in this signaling pathway can lead to pathological or disease states such as cancer. The epidermal growth factor receptor (EGFR) has shown to be highly expressed in human tumors, including 50% of oral squamous cell carcinomas, and contributes to the development of malignant tumors3,4,5. Whereas mutations in the three RAS isoforms H-, K- and N-RAS are major drivers for malignant transformation in multiple human cancers. Amongst these three RAS isoforms, oncogenic mutations in K-RAS are most prevalent6,7,8. For EGFR and RAS to function, they must localize to the plasma membrane (PM). Preventing the localization of these molecules to the PM can completely abrogate the biological activity of this signal pathway9,10. Hence the inhibition of the localization of these proteins to the PM is a therapeutic strategy to block the downstream signaling and the resulting adverse outcomes. Using a high-content screening assay, fendiline, an L-type calcium channel blocker, was identified as an inhibitor of K-RAS activity11. Nanoclustering of K-RAS to the inner leaflet of the PM is significantly reduced in the presence of fendiline. Furthermore, K-RAS is redistributed from the plasma membrane to the endoplasmic reticulum (ER), Golgi apparatus, endosomes, and cytosol. More importantly, the proliferation of pancreatic, colon, lung, and endometrial cancer cell lines expressing oncogenic mutant K-RAS is blocked by the inhibition of downstream signaling by fendiline11. These data suggest fendiline functions as a specific K-RAS anticancer therapeutic that causes the mis-localization of the RAS protein to the PM.
The nematode Caenorhabditis elegans has been extensively studied in the context of development. Many of the signal pathways that govern development in the worm are evolutionary and functionally conserved. For example, the EGFR mediated activation of RAS and the subsequent activation of the ERK MAPK signal cascade is conserved in the worm12. The cascade is represented by the following proteins: LET-23 &#62; LET-60 &#62; LIN-45 &#62; MEK-2 &#62; MPK-1. LET-60 is homologous to RAS, while LET-23 is homologous to EGFR. In the worm, this pathway regulates the development of the vulva13. The vulva is an epithelial aperture on the ventral body wall of the worm that allows fertilized eggs to be laid. The formation of the vulva in the worm is dependent on the exposure of the vulval precursor cells (VPC) to a gradient of activation of the EGFR-RAS-MAPK signal cascade. During the normal development, the proximal VPCs receive strong signals from the gonadal anchor cells to differentiate into 1&#176; and 2&#176; cell fates which give rise to a functional vulva12. Whereas distal VPCs differentiate into 3&#176; cell fates that fuse to the hypodermal syncytium and do not form vulva due to depleted signaling. In the absence of signaling, all VPCs differentiate into 3&#176; cell fates resulting in the formation of no vulva. However, constitutive signaling leads to the formation one or more non-functional vulva due to the induction of all VPCs to assume&#160;1&#176; and 2&#176; cell fates.
Mutations that cause defective or excessive vulval induction have been identified for many of the genes that encode for proteins representing this pathway. Defective vulval induction results in a vulvaless (Vul) phenotype, while excessive vulval induction results in a multivulva (Muv) phenotype that is represented by the development of numerous nonfunctional ectopic pseudovulvae throughout the ventral body wall. The Muv phenotype expressed by the let-60(n1046) strain is due to a gain of function mutation in RAS, while in the let-23(sa62) strain it is due an activating mutation in EGFR14,15. The strong Muv phenotype in these mutant strains has been shown to be perturbed by pharmacological interventions as demonstrated by the treatment of let-60(n1046) worms with the MEK-1 inhibitor U012616,17. Interestingly, we have shown that R-fendiline and inhibitors that affect sphingomyelin metabolism suppress the Muv phenotype in the worm18. To demonstrate these inhibitors block let-60 signaling at the level of RAS, the lin-1 null strain has been utilized17. Lin-1 is an Ets-like inhibitory transcription factor that functions as a repressor in the development of the vulva19. Strong reversion of the Muv phenotype in let-60(n1046) worms and no effect on lin-1 null worms suggest that these inhibitions occur at the level of RAS.
In this protocol, we demonstrate the use of C. elegans as a model to identify inhibitors of RAS and EGFR proteins. Using a liquid-based assay, we demonstrate the inhibitory effects of R-fendiline by suppressing the Muv phenotypes in the let-60(n1046) and let-23(sa62) mutant strains of C. elegans. This assay validates the use of C. elegans as a tool in the initial phase of drug discovery for anticancer therapeutics.

<table><tbody><tr><td>&lt;strong&gt;Media and chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agarose&amp;nbsp;</td><td>Millipore Sigma&amp;nbsp;</td><td>A9539-50G</td><td></td></tr><tr><td>Bacto Peptone&amp;nbsp;</td><td>Fisher Scientific</td><td>DF0118-17-0</td><td></td></tr><tr><td>BD Difco Agar&amp;nbsp;</td><td>Fisher Scientific</td><td>DF0145-17-0</td><td></td></tr><tr><td>BD Difco LB Broth</td><td>Fisher Scientific</td><td>DF0446-17-3</td><td></td></tr><tr><td>Calcium Chloride</td><td>Fisher Scientific</td><td>BP510-500</td><td></td></tr><tr><td>Cholesterol</td><td>Fisher Scientific</td><td>ICN10138201</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Fisher Scientific</td><td>BP213-1</td><td></td></tr><tr><td>Nystatin</td><td>Acros organics</td><td>AC455500050</td><td></td></tr><tr><td>Potassium Phosphate Dibasic</td><td>Fisher Scientific</td><td>BP363-500</td><td></td></tr><tr><td>Potassium pPhosphate Monobasic</td><td>Fisher Scientific</td><td>BP362-500</td><td></td></tr><tr><td>R-Fendiline</td><td></td><td></td><td>Commercially Synthesized (Pharmaceutical grade)</td></tr><tr><td>Sodium Azide</td><td>Millipore Sigma&amp;nbsp;</td><td>S2002-25G</td><td></td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>Fisher Scientific</td><td>BP358-1</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Fisher Scientific</td><td>SS266-1</td><td></td></tr><tr><td>8.25% Sodium Hypochlorite&amp;nbsp;</td><td></td><td></td><td>Bleach</td></tr><tr><td>Sodium Phosphate Dibasic&amp;nbsp;</td><td>Fisher Scientific</td><td>BP332-500</td><td></td></tr><tr><td>Streptomycin Sulfate&amp;nbsp;</td><td>Fisher Scientific</td><td>BP910-50</td><td></td></tr><tr><td>(&amp;minus;)-Tetramisole Hydrochloride</td><td>Millipore Sigma&amp;nbsp;</td><td>L9756</td><td></td></tr><tr><td>UO126 (MEK inhibitor)</td><td>Millipore Sigma&amp;nbsp;</td><td>19-147</td><td></td></tr><tr><td>&lt;strong&gt;Consumables&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>15mL Conical Sterile Polypropylene Centrifuge Tubes&amp;nbsp;</td><td>Fisher Scientific</td><td>12-565-269</td><td></td></tr><tr><td>50mL Conical Sterile Polypropylene Centrifuge Tubes</td><td>Fisher Scientific</td><td>12-565-271</td><td></td></tr><tr><td>Disposable Polystyrene Serological Pipettes 10mL</td><td>Fisher Scientific</td><td>07-200-574</td><td></td></tr><tr><td>Disposable Polystyrene Serological Pipettes 25mL</td><td>Fisher Scientific</td><td>07-200-575</td><td></td></tr><tr><td>No. 1.5&amp;nbsp; 18 mm X 18 mm Cover Slips</td><td>Fisher Scientific</td><td>12-541A</td><td></td></tr><tr><td>Petri Dish with Clear Lid (60 x 15 mm)</td><td>Fisher Scientific</td><td>FB0875713A</td><td></td></tr><tr><td>Petri Dishes with Clear Lid (100X15mm)</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>Plain Glass Microscope Slides (75 x 25 mm)</td><td>Fisher Scientific</td><td>12-544-4</td><td></td></tr><tr><td>12- Well Tissue Culture Plates</td><td>Fisher Scientific</td><td>50-197-4804</td><td></td></tr><tr><td>&lt;strong&gt;Software&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Prism</td><td>Graphpad</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Bacterial Strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; OP50</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Worm Strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Strain&lt;/strong&gt;</td><td>&lt;strong&gt;Genotype&lt;/strong&gt;</td><td>&lt;strong&gt;Transgene&lt;/strong&gt;</td><td>&lt;strong&gt;Source&lt;/strong&gt;</td></tr><tr><td>MT2124 &amp;nbsp;</td><td>&lt;em&gt;let-60(n1046) IV.&lt;/em&gt;</td><td></td><td>CGC</td></tr><tr><td>MT7567</td><td>&lt;em&gt;lin-1(sy254) IV.&lt;/em&gt;</td><td></td><td>CGC</td></tr><tr><td>PS1839</td><td>&lt;em&gt;let-23(sa62) II.&lt;/em&gt;</td><td></td><td>CGC</td></tr></tbody></table>

identification of egfr and ras inhibitors using caenorhabditis elegans

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

We first demonstrate that R-fendiline is able to suppress the Muv phenotype in the let-60(n1046) mutant strain compared to the DMSO treated worms. Our data shows that R-fendiline is able to block the Muv phenotype in the let-60(n1046) in a dose-dependent manner (Figure 2A,B). However, non-reversal of the Muv phenotype was observed in the lin-1 null mutant strain in response to increasing concentrations of R-fendiline (Figure 2B). The data suggests that R-fendiline blocks activated let-60 signaling at the level of RAS in C. elegans. Similarly, we observed the Muv phenotype was significantly reduced in the let-23(sa62) strain in response to 3, 10 and 30 &#181;M R-fendiline treatment relative to the DMSO treated worms (Figure 2C,D). In all experiments, Students t-test was used to determine the statistical significance.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61788/61788fig01.jpg" /> 
Figure 1: Flowchart representing the steps involved in preparing the drug assays using let-60(n1046), let-23(sa62) and lin-1(sy254) strains. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61788/61788fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61788/61788fig02.jpg" /> 
Figure 2: R-fendiline alters let-60 and let-23 function in C. elegans in a dose-dependent manner.&#160;(A) Representative images of let-60(n1046) worms in the presence of vehicle (DMSO) or 30 &#956;M R-fendiline. (B) Quantification of Muv phenotype in let-60(n1046) worms treated in the presence of DMSO, 3, 10 and 30 &#181;M R-fendiline, or 30 &#956;M U0126. (C) Representative images of let-23(sa62) worms in the presence of vehicle (DMSO) or 30 &#181;M R-fendiline. (D) Quantification of Muv phenotype in let-23(sa62) worms treated in the presence of DMSO, 3, 10 and 30 &#181;M R- fendiline, or 30 &#956;M U0126. In all images the pseudovulva are indicated by white arrows and normal vulva by white asterisks. A total of 60 worms were imaged for each treatment. The experiment was repeated 3 times. (*** P&#60;0.001 and ** P&#60;0.01 were considered significant) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61788/61788fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans at JoVE.com

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR proteins.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

identification-of-egfr-and-ras-inhibitors-using-caenorhabditis-elegans

Nanyang Technological University

Research

JoVE Journal

Cancer Research

2.9K Views.  University of Texas Health Science Center. The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR proteins.

Video: Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a&#160;model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism&#39;s genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

Developmental Biology

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress.

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

Genetics

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network function1. These proteins are found at the presynaptic and postsynaptic membranes 2. Studies in mice indicate that neurexins and neurologins have an essential role in synaptic transmission 1. Recent reports have shown that altered neuronal connections during the development of the human nervous system could constitute the basis of the etiology of numerous cases of autism spectrum disorders 3.
Caenorhabditis elegans could be used as an experimental tool to facilitate the study of the functioning of synaptic components, because of its simplicity for laboratory experimentation, and given that its nervous system and synaptic wiring has been fully characterized. In C. elegans nrx-1 and nlg-1 genes are orthologous to human NRXN1 and NLGN1 genes which encode alpha-neurexin-1 and neuroligin-1 proteins, respectively. In humans and nematodes, the organization of neurexins and neuroligins is similar in respect to functional domains.
The head of the nematode contains the amphid, a sensory organ of the nematode, which mediates responses to different stimuli, including osmotic strength. The amphid is made of 12 sensory bipolar neurons with ciliated dendrites and one presynaptic terminal axon 4. Two of these neurons, named ASHR and ASHL are particularly important in osmotic sensory function, detecting water-soluble repellents with high osmotic strength 5. The dendrites of these two neurons lengthen to the tip of the mouth and the axons extend to the nerve ring, where they make synaptic connections with other neurons determining the behavioral response 6.
To evaluate the implications of neurexin and neuroligin in high osmotic strength avoidance, we show the different response of C. elegans mutants defective in nrx-1 and nlg-1 genes, using a method based on a 4M fructose ring 7. The behavioral phenotypes were confirmed using specific RNAi clones 8. In C. elegans, the dsRNA required to trigger RNAi can be administered by feeding 9. The delivery of dsRNA through food induces the RNAi interference of the gene of interest thus allowing the identification of genetic components and network pathways.

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexins and neuroligins are membrane-neuron adhesion proteins which perform essential roles in synaptic differentiation and transmission. Neuroligin deficient mutants of C. elegans are defective in detecting osmotic strength, but when they also contain a mutation in the gene coding neurexin, they recover the wild type phenotype.

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network ...

Biology

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or identification of new ones can provide invaluable information about the function of a protein of interest. Some of the traditional methods for extract preparation frequently require labor-intensive and time-consuming techniques. Here, a modified extract preparation protocol using a bead mill homogenizer and metal beads is described as a rapid alternative to traditional protein preparation methods. This extract preparation method is compatible with downstream co-immunoprecipitation studies. As an example, the method was used to successfully co-immunoprecipitate&#160;C. elegans microRNA Argonaute ALG-1 and two known ALG-1 interactors: AIN-1, and HRPK-1. This protocol includes descriptions of animal sample collection, extract preparation, extract clarification, and protein immunoprecipitation. The described protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

This method describes a protocol for high-throughput protein extract preparation from Caenorhabditis elegans samples and subsequent co-immunoprecipitation.

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and &#947;-radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

This protocol demonstrates how to track a protein nuclear translocation under heat stress by using a green fluorescence protein (GFP) fusion protein as a marker and 4',6-diamidino-2-phenylindole (DAPI) staining. The DAPI staining protocol is fast and preserves the GFP and protein subcellular localization signals.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

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High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans