This study was supported by the Student Research Grant (13-14) of University of Ulsan College of Medicine, Seoul, Korea and a grant (2014-464) from the Asan Institute for Life Sciences, Seoul, Korea.

1. Cell Line
<ol>
	<li>Culture 106 telomerase-immortalized human corneal epithelial cells (HCECs) in a 6-well plate (density: 1039.9 cell/mm2) in a 37 &#176;C incubator with a 5% CO2 atmosphere using bronchial epithelium growth medium (BEGM) until they reach 95% confluence.</li>
</ol>
2. Western Blot Analysis after Creating Epithelial Scratch Wounds
<ol>
	<li>Streak a sterile 200 &#181;l pipette tip across the surface of a well of confluent cultured HCECs four times per dish in a biological safety cabinet (Class II, Type A2) and incubate HCECs separately in a 37 &#176;C incubator with a 5% CO2 atmosphere for 1, 5, 10, 30, 60, and 120 min.</li>
	<li>Use one 6-well plate for six different samples according to the incubation time after corneal epithelial wounding.</li>
	<li>Wash wounded HCEC monolayers three times with 1x PBS and then add 2.0 ml BEGM to each well.</li>
	<li>Detach HCECs using 2 ml 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) per well for 5 min, centrifuge at 900 x g for 5 min in a 15 ml tube, and aspirate trypsin-EDTA using a 1 ml pipette.</li>
	<li>Suspend HCECs with 1 ml 1x PBS and transfer them to a 1.5 ml tube.</li>
	<li>Centrifuge HCECs at 10,000 x g for 15 sec and aspirate 1x PBS using a 1 ml pipette.</li>
	<li>Resuspend HCECs in 100 &#181;l ice-cold lysis buffer (10 mM Tris, 10 mM NaCl, 2 mM EDTA, 25 mM NaF, 2 mM Na3VO4, 1 mM phenylmethanesulfonyl fluoride [PMSF], protease inhibitors [1 &#181;M pepstatin A, 1 &#181;M leupeptin, and 0.1 &#181;M aprotinin] and 0.5% Triton X-100 [pH 7]) and mix them well.</li>
	<li>Incubate cells for 30 min on ice to induce cell lysis.</li>
	<li>Centrifuge lysates at 4 &#176;C at 10,000 x g for 15 min, and then transfer supernatants to fresh 1.5 ml tubes (90 &#181;l aliquots) and store them at -80 &#176;C.</li>
	<li>Determine total protein concentrations of cell lysates using the Bradford protein assay.9</li>
	<li>Load 30 &#181;g total cell proteins into a 10% or 12% acrylamide gel for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then electrophoretically transfer separated protein bands to nitrocellulose filters with a 200 mA current for 1 hr at 4 &#176;C to use in western blot assays.</li>
	<li>Block nitrocellulose filter membranes with 5% skimmed milk in Tris-buffered saline with Tween 20 (TBST) for 1 hr, add primary rabbit polyclonal antibodies against non-phosphorylated HSP27 (1:1,000 dilution) or primary rabbit polyclonal antibody against phosphorylated HSP27 (1:1,000 dilution) in 5% bovine serum albumin (BSA), and incubate the membranes overnight at 4 &#176;C on a shaker.</li>
	<li>Detect immunoreactive bands using horseradish peroxidase-conjugated goat anti-rabbit antibodies (1:10,000 dilution) in 5% BSA after washing for 10 min 3 times with TBST.</li>
	<li>Incubate the membrane in the western blotting luminol reagents (6-7 ml per 10 cm &#215; 5 cm membrane) for 1 min at room temperature.</li>
	<li>Remove the membrane from reagent solution, remove excess liquid with an absorbent towel, and place in a plastic sheet protector.</li>
	<li>Working in a dark room with a safe light, place covered membrane in a film cassette with protein side facing up.</li>
	<li>Place X-ray film on top of membrane and expose for 1 min.</li>
</ol>
3. siRNA Transfection Assay10
<ol>
	<li>Culture HCECs at 5&#215;105 cells/well in a 6-well plate in a 37 &#176;C incubator with a 5% CO2 atmosphere using BEGM until they reach 95% confluence.</li>
	<li>Dilute the transfection reagent (2.5 or 7.5 &#181;l) with 100 &#181;l reduced serum medium for transfection (the dilution factor was 41 or 14.3) and dissolve the HSP27-specific and scrambled control siRNA in 100 &#181;l reduced serum medium to create 10 or 50 nM of HSP27-specific and scrambled control siRNA.</li>
	<li>Mix 100 &#181;l siRNA solution with 100 &#181;l diluted transfection reagent (1:1 ratio) and incubate the mixture for 15 min at room temperature.</li>
	<li>Add the siRNA-lipid complexes to cells. Then after 4 hr change media to complete BEGM and incubate the cells for 2 days at 37 &#176;C.</li>
	<li>Analyze transfected cells by western blotting, as described from sections 4.1 to 4.7.</li>
</ol>
4. Western Blot Assay for siRNA-transfected Cells11
<ol>
	<li>Extract HSP27-specific and scrambled control siRNA-transfected HCECs in a biological safety cabinet using 100 &#181;l ice-cold lysis buffer (10 mM Tris, 10 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 25 mM NaF, 2 mM Na3VO4, 1 mM phenylmethanesulfonyl fluoride (PMSF), protease inhibitors, and 0.5% Triton X-100, pH 7).</li>
	<li>Incubate cells for 30 min on ice to induce cell lysis.</li>
	<li>Pellet lysates at 10,000 x g for 15 min and then transfer supernatants to fresh 1.5 ml tubes (90 &#181;l aliquots) and store them at -80 &#176;C.</li>
	<li>Determine protein concentrations of the cell lysates using the Bradford protein assay.9</li>
	<li>Load samples with equal amounts of total cell proteins on a 10% or 12% acrylamide gel, subject the gel to SDS-PAGE, and electrophoretically transfer the separated protein bands to nitrocellulose filters with a 200 mA current for 1 hr at 4 &#176;C to use in western blot assays.</li>
	<li>Block nitrocellulose filter membranes with 5% skimmed milk in Tris-buffered saline with Tween 20 (TBST) for 1 hr, add primary antibodies against phosphorylated and non-phosphorylated HSP27 (1:1,000 dilution), phosphorylated Akt (1:1,000 dilution), non-phosphorylated Akt (1:1,000 dilution, used as a cell survival marker), Bcl-2-associated X protein (1:1,000 dilution, used as a pro-apoptotic protein), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:200 dilution, used as a loading control) in 5% bovine serum albumin (BSA), and incubate the membranes overnight at 4 &#176;C on a shaker.</li>
	<li>Detect immunoreactive bands using horseradish peroxidase-conjugated goat anti-rabbit antibodies (1:10,000 dilution) in 5% BSA after washing 3 times with TBST, 10 min each wash.</li>
	<li>Incubate the membrane in the western blotting luminol reagents (6-7 ml per 10 cm &#215; 5 cm membrane) for 1 min at room temperature.</li>
	<li>Remove the membrane from reagent solution, remove excess liquid with an absorbent towel, and place in a plastic sheet protector.</li>
	<li>Working in a dark room with a safe light, place the covered membrane in a film cassette with the protein side facing up.</li>
	<li>Place X-ray film on top of the membrane and expose for 1 min.</li>
</ol>
5. Scratch-induced Directional Wounding Assay Evaluation of Cell Migration12
<ol>
	<li>In a biological safety cabinet, make a wound by dragging a sterile pipette tip across the surface of a well of confluent cultures of HSP27-specific siRNA-transfected or scrambled control siRNA-transfected HCECs.</li>
	<li>Immediately after wounding, wash the cells twice with 1x phosphate buffered saline (PBS) and maintain them in BEGM cultures in a 37 &#176;C incubator with a 5% CO2 atmosphere for 24 hr after wounding.</li>
	<li>Photograph HCEC images using an upright microscope at 100X magnification 24 hr after wounding and perform background flattening using the Filter command in image analysis software.</li>
	<li>Using the Select Measurements command, define the Area of Interest (AOI) with the same sized polygonal shape which can cover perpendicularly from end to end of initial wound, and determine three different AOI in the wounded area of each sample.</li>
	<li>Automatically count the cell number in each field using the Count/Size Measure menu options.</li>
</ol>
6. Flow Cytometry Analysis of Apoptosis
<ol>
	<li>Culture HSP27-specific siRNA-transfected and control siRNA-transfected HCECs containing each 10 nM siRNA at a concentration of 5&#215;105 cells/well in 6-well plates until the cells reach 95% confluence in a 37 &#176;C incubator with a 5% CO2 atmosphere in BEGM.</li>
	<li>Detach HCECs using 2 ml 0.25% trypsin-EDTA per well for 5 min, centrifuge at 900 x g for 5 min in a 15 ml tube, and aspirate trypsin-EDTA using a 1 ml pipette.</li>
	<li>Wash cells twice with cold PBS and then resuspend the cells in 1x binding buffer (0.1 M HEPES/NaOH [pH 7.4], 1.4 M NaCl, and 25 mM CaCl2) at a concentration of 106 cells/ml.</li>
	<li>Transfer 100 &#181;l cell suspension (1 x 105 cells) into a 5 ml culture tube.</li>
	<li>Add 5 &#181;l fluorescein isothiocyanate-conjugated Annexin V and 5 &#181;l propidium iodide.</li>
	<li>Gently vortex the cells and incubate them for 15 min at room temperature in the dark.</li>
	<li>Add 200 &#181;l of 1x binding buffer to each tube and analyze the cells by a flow cytometer within 1 hr.</li>
</ol>

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	<li>Dua, H. S., Gomes, J. A., Singh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+epithelial+wound+healing.">Corneal epithelial wound healing.</a> Br. J. Ophthalmol. 78 (5), 401-408 (1994).</li><li>Estil, S., Primo, E. J., Wilson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+in+shed+human+corneal+cells.">Apoptosis in shed human corneal cells.</a> Invest. Ophthalmol. Vis. Sci. 41 (11), 3360-3364 (2000).</li><li>Guay, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+actin+filament+dynamics+by+p38+map+kinase-mediated+phosphorylation+of+heat+shock+protein+27.">Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27.</a> J. cell. Sci. 110, 357-368 (1997).</li><li>Park, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+heat+shock+protein+mRNAs+under+in+vivo+glutathione+depletion+in+the+mouse+retina.">Differential expression of heat shock protein mRNAs under in vivo glutathione depletion in the mouse retina.</a> Neurosci. Lett. 413 (3), 260-264 (2007).</li><li>Rane, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+protein+27+controls+apoptosis+by+regulating+Akt+activation.">Heat shock protein 27 controls apoptosis by regulating Akt activation.</a> J. Biol. Chem. 278 (30), 27828-27835 (2003).</li><li>Shin, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+tumor+cell+migration+and+invasion+with+biphenyl+isoxazole+derivative+KRIBB3,+a+synthetic+molecule+that+inhibits+Hsp27+phosphorylation.">Blocking tumor cell migration and invasion with biphenyl isoxazole derivative KRIBB3, a synthetic molecule that inhibits Hsp27 phosphorylation.</a> J. Biol. Chem. 280 (50), 41439-41448 (2005).</li><li>Jain, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+phosphorylated+heat+shock+protein+27+during+corneal+epithelial+wound+healing.">Expression of phosphorylated heat shock protein 27 during corneal epithelial wound healing.</a> Cornea. 31 (7), 820-827 (2012).</li><li>Alekseev, O. M., Richardson, R. T., Alekseev, O., O'Rand, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+gene+expression+profiles+in+HeLa+cells+in+response+to+overexpression+or+siRNA-mediated+depletion+of+NASP.">Analysis of gene expression profiles in HeLa cells in response to overexpression or siRNA-mediated depletion of NASP.</a> Reprod. Biol. Endocrinol. 7, 45 (2009).</li><li>Park, H. Y., Kim, J. H., Lee, K. M., Park, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+prostaglandin+analogues+on+tear+proteomics+and+expression+of+cytokines+and+matrix+metalloproteinases+in+the+conjunctiva+and+corea.">Effect of prostaglandin analogues on tear proteomics and expression of cytokines and matrix metalloproteinases in the conjunctiva and corea.</a> Exp. Eye. Res. 94 (1), 13-21 (2012).</li><li>Voegeli, T. S., Currie, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA+knocks+down+Hsp27+and+increases+angiotensin+II-induced+phosphorylated+NF-kappaB+p65+levels+in+aortic+smooth+muscle+cells.">siRNA knocks down Hsp27 and increases angiotensin II-induced phosphorylated NF-kappaB p65 levels in aortic smooth muscle cells.</a> Inflamm. Res. 58 (6), 336-343 (2009).</li><li>Shi, B., Isseroff, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arsenite+pre-conditioning+reduces+UVB-induced+apoptosis+in+corneal+epithelial+cells+through+the+anti-apoptotic+activity+of+27+kDa+heat+shock+protein+(HSP27).">Arsenite pre-conditioning reduces UVB-induced apoptosis in corneal epithelial cells through the anti-apoptotic activity of 27 kDa heat shock protein (HSP27).</a> J. Cell. Physiol. 206 (2), 301-308 (2006).</li><li>Shen, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+corneal+epitheliotrophic+capacity+among+different+human+blood-derived+preparations.">Comparison of corneal epitheliotrophic capacity among different human blood-derived preparations.</a> Cornea. 30 (2), 208-214 (2011).</li><li>Song, I. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+protein+27+phosphorylation+is+involved+in+epithelial+cell+apoptosis+as+well+as+epithelial+migration+during+corneal+epithelial+wound+healing.">Heat shock protein 27 phosphorylation is involved in epithelial cell apoptosis as well as epithelial migration during corneal epithelial wound healing.</a> Exp Eye Res. 118 (1), 36-41 (2014).</li></ol>

The authors have no financial or proprietary interests in any materials or methods mentioned in this study.

In this present study, we evaluated the potential role of HSP27 in corneal epithelial wounding using in vitro approaches. The critical steps involved siRNA transfection for HSP27 knock-down to observe the function of HSP27 in cells subjected to stress. Notably, a role for HSP27 was revealed by these experiments in epithelial cell migration and apoptosis during corneal epithelial wound healing. Unlike previous studies10 that used rat HSP27-specific siRNA to transfect vascular smooth muscle cells, we used a siRNA transfection technique to modify gene expression in human CECs to effectively knock-down HSP27-specific gene expression and study HSP27 function. Although there were differences in the target sequence that we used as well as in the cell density, final siRNA concentration, and incubation time, the protocol recommended by the manufacturer was explicitly followed. In terms of alternative methods, HSP27 knock-out mouse may be used to show if HSP27 phosphorylation involves epithelial migration and cell apoptosis. However, it is difficult to monitor the change of HSP27 phosphorylation in mouse model, because its phosphorylation occurs in very short period during epithelial wound healing.
There were several limitations to the present study. First, the in vitro environment in which we cultured human CECs certainly differed from the in vivo environment for human CECs, especially regarding cell survival. Second, the siRNA used in this study was not specific to the phosphorylated form of HSP27 as it affected the overall expression levels of HSP27, including both phosphorylated and non-phosphorylated forms.
In the future, a clinical application of these procedures would be to apply HSP27 to live human wounded corneas. We hope that the current findings will help to advance treatments of corneal epithelial tissue damage.

Corneal epithelial cells (CECs) are continuously shed into tear film, while they are simultaneously replaced by cells from the limbus and corneal epithelial basal layers.1 Various extrinsic stressors can induce the apoptosis and desquamation of CECs.2 The heat shock proteins (HSPs) are highly conserved and can be divided into two families according to molecular size.3 The largest HSP family includes HSP90, HSP70, and HSP60, and the smaller family includes HSP27.4 The phosphorylation of HSP27 is known to play an important role in cell survival and is required for cell migration because of the role of this protein in actin remodeling.5-7 Therefore, we attempted to test the potential role of HSP27 phosphorylation in CEC migration and apoptosis in an in vitro model of epithelial wound healing.
RNA interference (RNAi) using either small or short interfering RNAs (siRNA) has generated interest in both basic and applied biology, as it potentially allows the expression of any gene of interest to be knocked-down.8 Herein, we used HSP27-specific siRNA to assess the contribution of HSP27 to CEC wound healing and apoptosis. Traditional RNAi methods for gene knock-down in cells use synthetic RNA duplexes, including two unmodified 21-mer oligonucleotides that can be assembled to create siRNAs. The RNAi siRNA that we used in this present study is a simple and highly efficient method to transfect cells, and this reagent works with various immortalized cell lines. In this present study, we demonstrate the methods used for this analysis, including a scratch-induced directional wound assay, western blotting, siRNA transfection assay, immunofluorescence assay, and flow cytometry.

<table><tbody><tr><td>Biological safety cabinet</td><td>CHC LAB Co.Ltd,&amp;nbsp; Daejeon, Republic of Korea&amp;nbsp;</td><td>CHC-777A2-06</td><td>Class II, Type A2&amp;nbsp;</td></tr><tr><td>Stealth RNAi&amp;trade; siRNA</td><td>Thermo Fisher Scientific, Inc., Waltham, MA</td><td></td><td>RNAi siRNA; scrambled control-siRNA and HSP27-specific siRNA</td></tr><tr><td>BEGM&lt;sup&gt;TM&lt;/sup&gt;</td><td>Lonza, Inc., Walkersville, MD</td><td>CC-3171, CC4175</td><td>Bronchial epithelium growth medium&amp;nbsp;</td></tr><tr><td>Protease inhibitor&amp;nbsp;</td><td>Sigma-Aldrich, Inc., St. Louis, MO</td><td>P8340 ,P7626</td><td>1 &amp;#956;M Pepstatin A, 1 &amp;#956;M Leupeptin, 0.1 &amp;#956;M Aprotinin</td></tr><tr><td>Bradford protein assay&amp;nbsp;</td><td>Bio-Rad Laboratories, Hercules, CA</td><td>#500-0001</td><td>Bradford protein assay&amp;nbsp;</td></tr><tr><td>Nitrocellulose filters&amp;nbsp;</td><td>Amersham, Little Chalfont, UK</td><td>RPN3032D</td><td>Western blotting membrane</td></tr><tr><td>Non-phosphorylated HSP27&amp;nbsp;</td><td>Abcam Inc., Cambridge, MA</td><td>ab12351</td><td>1:1,000 dilution (Total HSP27)</td></tr><tr><td>Phosphorylated HSP27 (Ser85)</td><td>Abcam Inc., Cambridge, MA</td><td>ab5594</td><td>1:1,000 dilution HSP27 was phosphorylated at Ser85</td></tr><tr><td>Lipofectamine&lt;sup&gt;&amp;reg; &lt;/sup&gt;RNAiMAX reagent&amp;nbsp;</td><td>Invitrogen, Carlsbad, CA</td><td>13-778-075</td><td>Transfection reagent</td></tr><tr><td>Phosphorylated Akt (Ser473)</td><td>Cell Signaling Technology, Danvers, MA</td><td>No. 4060</td><td>1:1,000 dilution Akt was phosphorylated at Ser473 (cell survival marker)</td></tr><tr><td>Non-phosphorylated Akt&amp;nbsp;</td><td>Cell Signaling Technology, Danvers, MA</td><td>No. 4061</td><td>1:1,000 dilution (Total Akt)</td></tr><tr><td>Bcl-2-associated X protein&amp;nbsp;</td><td>Cell Signaling Technology, Danvers, MA</td><td>No. 4062</td><td>1:1,000 (anti-apoptotic protein marker)</td></tr><tr><td>GAPDH</td><td>Santa Cruz Biotechnology, Santa Cruz, CA</td><td>No. 4063</td><td>1:1,000 loading control marker (house keeping gene)</td></tr><tr><td>Horseradish peroxidase-conjugated goat anti-rabbit antibodies</td><td>Thermo Fisher Scientific, Inc., Waltham, MA</td><td>NCI1460KR</td><td>1:10,000 dilution</td></tr><tr><td>OPTI-MEM</td><td>Invitrogen, Carlsbad, CA</td><td>31985</td><td>reduced serum medium for transfection</td></tr><tr><td>Image analysis software</td><td>Olympus, Inc., Tokyo, Japan</td><td>Image-Pro Plus 5.0</td><td></td></tr><tr><td>Skimed milk powder&amp;nbsp;</td><td>Carl Roth GmbH + Co. KG, Karlstruhe, Germany</td><td>T145.2</td><td></td></tr><tr><td>Tris&amp;nbsp;</td><td>Amresco LCC, Inc. Solon, OH</td><td>No-0497</td><td></td></tr><tr><td>Sodium Chloride&amp;nbsp;</td><td>Amresco LCC, Inc. Solon, OH</td><td>No-0241</td><td></td></tr><tr><td>Six well culture plate</td><td>Thermo Fisher Scientific, Inc., Waltham, MA</td><td>140675</td><td>35.00 mm diameter / well</td></tr><tr><td>24-well culuture dish</td><td>Thermo Fisher Scientific, Inc., Waltham, MA</td><td>142475</td><td></td></tr><tr><td>Orbital shaker</td><td>N-Bioteck, Inc., Seoul, South Korea</td><td>NB1015</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Santa Cruz Biotechnology, Santa Cruz, CA</td><td>sc-2323&amp;nbsp;</td><td></td></tr><tr><td>BDFACSCanto&lt;sup&gt;TM&lt;/sup&gt; II</td><td>BD Biosciences, Franklin Lakes, NJ</td><td></td><td>Flow cytometry</td></tr><tr><td>X-Ray Film</td><td>Kodak, Rochester, NY</td><td></td><td>Medical X-Ray Cassette with Green 400 Screen&amp;nbsp;</td></tr><tr><td>western blotting luminol reagent</td><td>Santa Cruz Biotechnology, Santa Cruz, CA</td><td>sc-2048&amp;nbsp;</td><td></td></tr><tr><td>FITC Annexin V Apoptosis Detection Kit I</td><td>BD Biosciences, Franklin Lakes, NJ</td><td>556547</td><td></td></tr></tbody></table>

rna interference-based investigation of the function of heat shock protein 27 during corneal epithelial wound healing

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.

The expression of phosphorylated HSP27 significantly increased at 5, 10, and 30 min after scratch wounding compared with unwounded HCECs13. Western blot analysis revealed that the expression of phosphorylated HSP27 and phosphorylated Akt were both significantly reduced, whereas the expression of Bax was significantly increased in HSP27-specific siRNA-transfected HCECs (Figure 1A-E). The phosphorylated HSP27 expression was reduced by 30% and 40% in 10 nM and 50 nM of HSP27-specific siRNA-transfected cells, respectively, compared with control siRNA-transfected cells, but the phosphorylated HSP27 expression was not reduced (Figure 1A-B). Moreover, the non-phosphorylated HSP27 expression was reduced by 20% and 30% in 10 nM and 50 nM of HSP27-specific siRNA transfected cells, respectively, but the non-phosphorylated HSP27 expression was not reduced (Figure 1A and C).
The scratch-induced directional wounding assay indicated that at 24 hr after wounding, HSP27-specific siRNA-transfected cells at 10 and 50 nM exhibited reduced migration (Figure 2). Moreover, HSP27-specific siRNA-transfected HCECs underwent more apoptotic and necrotic cell death compared with scrambled control siRNA-transfected cells by flow cytometry (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/54280/54280fig1.jpg" /> 
Figure 1. Western blot analysis using antibodies against phosphorylated HSP27 (p-HSP27), non-phosphorylated HSP27 (non-p-HSP27), phosphorylated Akt (p-Akt) as a cell-survival marker, non-phosphorylated Akt (non-p-Akt), Bcl-2eassociated X protein (Bax) as a pro-apoptotic protein, and GAPDH (A). The expression of phosphorylated and nonphosphorylated HSP27 and phosphorylated Akt significantly decreased (B-D), however, the expression of Bax significantly increased in the HSP27-specific siRNA-transfected HCECs (E), compared with that observed in the control siRNA-transfected cells (all p &#60; 0.05). The phosphorylated HSP27 expression was reduced by 30% and 40% in 10 nM and 50 nM of HSP27-specific siRNA-transfected cells compared with mock control, respectively, but the phosphorylated HSP27 expression was not reduced in 10 nM and 50 nM of control siRNA-transfected cells (B). **, *; &#8224;, &#8224;&#8224;; &#8225;, &#8225;&#8225;; &#167;, &#167;&#167;: a statistically significant difference among groups (p &#60; 0.05). The error bars represent standard deviation (SD). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54280/54280fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54280/54280fig2.jpg" /> 
Figure 2. Scratch-induced directional wounding assay to evaluate cell migration after wounding in siRNA-transfected HCECs. A scratch wound was created in control and HSP27-specific siRNA-transfected cells (A). Cells were removed from the &#39;dragged&#39; areas. At 24 hr after wounding, 10 and 50 nM of HSP27-specific siRNA-transfected cells exhibited lower numbers of migrating cells compared with the 10 and 50 nM control siRNA-transfected cells (B). ** and * indicate a statistically significant difference among groups (p&#60;0.05). The data are shown as means &#177; standard deviations. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54280/54280fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54280/54280fig3.jpg" /> 
Figure 3. Flow cytometry of 50 nM of scrambled control siRNA and HSP27-specific siRNA-transfected human corneal epithelial cells (HCECs) labeled with annexin V and PI (A and B). The percentage of the total cells in quadrants corresponded to early apoptotic cells (annexin V-positive and PI-negative cells, Q4, lower right), late apoptotic cells (annexin V-positive and PI-positive cells, Q2, upper right), and necrotic cells (annexin V-negative and PI-positive cells, Q1, upper left). HSP27-specific siRNA-transfect HCECs had more apoptotic and necrotic cell death than control siRNA-transfected cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54280/54280fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing at JoVE.com

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Herein, we present a protocol to use heat shock protein 27 (HSP27)-specific small interfering RNA to assess the function of HSP27 during corneal epithelial wound healing. RNA interference is the best method for effectively knocking-down gene expression to investigate protein function in various cell types.

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a ...

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Genetics

7.4K Views.  Saevit Eye Hospital. Herein, we present a protocol to use heat shock protein 27 (HSP27)-specific small interfering RNA to assess the function of HSP27 during corneal epithelial wound healing. RNA interference is the best method for effectively knocking-down gene expression to investigate protein function in various cell types.

Video: RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

Developmental Biology

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation results in calcium mobilization events that propagate from cell to cell, which are essential for initiating cellular motility into the wound bed, promoting efficient wound healing. The Trinkaus-Randall lab has developed a methodology for imaging the corneal wound healing response in ex vivo murine globes in real time. This approach involves enucleating an intact globe from a mouse that has been euthanized per established protocols and immediately incubating the globe with a calcium indicator dye. A counterstain that stains other features of the cell can be applied at this stage to assist with imaging and show cellular landmarks. The protocol worked well with several different live cell dyes used for counterstaining, including SiR actin to stain actin and deep red plasma membrane stain to stain the cell membrane. To examine the response to a wound, the corneal epithelium is injured using a 25 G needle, and the globes are placed in a 3D printed holder. The dimensions of the 3D printed holder are calibrated to ensure immobilization of the globe throughout the duration of the experiment and can be modified to accommodate eyes of different sizes. Live cell imaging of the wound response is performed continuously at various depths throughout the tissue over time using confocal microscopy. This protocol allows us to generate high-resolution, publication-quality images using a 20x air objective on a confocal microscope. Other objectives can also be used for this protocol. It represents a significant improvement in the quality of live cell imaging in ex vivo murine globes and permits the identification of nerves and epithelium.

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

The present work describes a novel experimental protocol that utilizes a 3D printed holder to enable high-resolution live cell imaging of enucleated globes. Through this protocol, the cellular calcium signaling activity in wounded corneal epithelium from ex vivo globes can be observed in real time.

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation ...

Biochemistry

Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of transparency due to fibrotic scarring. The tissue integrity of injured embryonic corneas is intrinsically restored with no detectable scar formation. Given its accessibility and ease of manipulation, the chick embryo is an ideal model for studying scarless corneal wound repair. This protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. First, eggs are windowed at early embryonic ages to access the eye. Second, a series of in ovo physical manipulations to the extraembryonic membranes are conducted to ensure access to the eye is maintained through later stages of development, corresponding to when the three cellular layers of the cornea are formed. Third, linear cornea wounds that penetrate the outer epithelial layer and the anterior stroma are made using a microsurgical knife. The regeneration process or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure. Studies to date using this model have revealed that wounded embryonic corneas display activation of keratocyte differentiation, undergo coordinated remodeling of ECM proteins to their native three-dimensional macrostructure, and become adequately re-innervated by corneal sensory nerves. In the future, the potential impact of endogenous or exogenous factors on the regenerative process could be analyzed in healing corneas by using developmental biology techniques, such as tissue grafting, electroporation, retroviral infection, or bead implantation. The current strategy identifies the embryonic chick as a crucial experimental paradigm for elucidating the molecular and cellular factors coordinating scarless corneal wound healing.

The present protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. The regenerating or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure.&#160;

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Medicine

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

An Epithelial Abrasion Model for Studying Corneal Wound Healing

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This corneal wound healing model is highly reproducible and is now being used to evaluate compromised corneal wound healing in the context of diseases.

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments for toxicant-induced corneal injury are generally supportive as no specific therapeutics exist to treat these injuries. In the efforts to develop treatments and therapeutics to care for exposure, it can be important to understand the molecular and cellular mechanisms of these injuries. We propose that utilization of high throughput small inhibitory RNA (siRNA) screening can be an important tool that could help to more rapidly elucidate the molecular mechanisms of chemical cornea epithelial injury. siRNA are double stranded RNA molecules that are 19-25 nucleotides long and utilize the post-transcriptional gene silencing pathway to degrade mRNA which have homology to the siRNA. The resulting reduction of expression of the specific gene can then be studied in toxicant exposed cells to ascertain the function of that gene in the cellular response to the toxicant. The development and validation of in vitro exposure models and methods for the high throughput screening (HTS) of hydrogen fluoride- (HF) and chloropicrin- (CP) induced ocular injury are presented in this article. Although we selected these two toxicants, our methods are applicable to the study of other toxicants with minor modifications to the toxicant exposure protocol. The SV40 large T antigen immortalized human corneal epithelial cell line SV40-HCEC was selected for study. Cell viability and IL-8 production were selected as endpoints in the screening protocol. Several challenges associated with the development of toxicant exposure and cell culture methods suitable for HTS studies are presented. The establishment of HTS models for these toxicants allows for further studies to better understand the mechanism of injury and to screen for potential therapeutics for chemical ocular injury.

High throughput small inhibitory RNA screening is an important tool that could help to more rapidly elucidate the molecular mechanisms of chemical cornea epithelial injury. Herein, we present the development and validation of exposure models and methods for the high throughput screening of hydrogen fluoride- and chloropicrin-induced cornea epithelial injury.

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments ...

Biology

Establishing a Severe Corneal Inflammation Model in Rats Based on Corneal Epithelium Curettage Combined with Corneal Sutures

Corneal inflammation, especially severe corneal inflammation, plays a significant role in the development of corneal limbal stem cell dysfunction. Constructing appropriate animal models can help us focus on the effects of severe inflammation on corneal limbal stem cells. A 2 mm rust remover&#160;was&#160;used to remove the central corneal epithelium of Sprague Dawley (SD) rats to create an injury. Then, the central stroma of the cornea was sutured with nylon sutures to induce persistent inflammation. In this way, a corneal inflammation model with central corneal epithelium abrasion and central stroma suturing was constructed, which induced severe corneal inflammation. The changes in corneal inflammation and the condition of the limbal stem cells at 1, 3, and 7 days post-modeling were observed. On the 3rd day after modeling, the rats&#39; corneal limbus was severely edematous, with obvious neovascularization and local hyperplasia, which are typical signs of limbal stem cell deficiency. By the 7th day, the corneal edema gradually worsened, and the neovascularization continued to increase. Through quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence staining, we found that the corneal epithelial inflammatory factors were significantly upregulated, the corneal epithelial differentiation was abnormal, the corneal epithelial stem cells were significantly reduced, and the cell proliferation and stemness had also decreased. Therefore, this model demonstrates that severe inflammation can induce limbal stem cell damage without directly damaging the limbal stem cells. The model is beneficial for observing the effects of severe inflammation on the biological mechanisms of stem cells and provides an ideal platform for studying the mechanisms of corneal epithelial stem cell dysfunction induced by inflammation.

We developed a rat model of severe corneal inflammation through corneal epithelium curettage combined with corneal sutures. The study evaluated corneal inflammation patterns, epithelial proliferation, and changes in limbal stem cells under inflammatory conditions.

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

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