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Retinal Cell Biology  |   October 2013
Migration of Retinal Pigment Epithelium Cells Is Regulated by Protein Kinase Cα In Vitro
Author Affiliations & Notes
  • Suo Qiu
    The Second Affiliated Hospital, Wenzhou Medical University, Wenzhou, China
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Zhaoxin Jiang
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Zhen Huang
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Xiaoqing Chen
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Xiaobing Qian
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Qianying Gao
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Haihua Zheng
    The Second Affiliated Hospital, Wenzhou Medical University, Wenzhou, China
  • Correspondence: Qianying Gao, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, China; gaoqy@mail.sysu.edu.cn
  • Haihua Zheng, The Second Affiliated Hospital, Wenzhou Medical University, Wenzhou, 325027, China; eyezhh@126.com
Investigative Ophthalmology & Visual Science October 2013, Vol.54, 7082-7090. doi:10.1167/iovs.13-12099
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      Suo Qiu, Zhaoxin Jiang, Zhen Huang, Xiaoqing Chen, Xiaobing Qian, Qianying Gao, Haihua Zheng; Migration of Retinal Pigment Epithelium Cells Is Regulated by Protein Kinase Cα In Vitro. Invest. Ophthalmol. Vis. Sci. 2013;54(10):7082-7090. doi: 10.1167/iovs.13-12099.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Retinal pigment epithelium (RPE) cell migration and proliferation are considered key elements in proliferative vitreoretinopathy (PVR). Downregulation of protein kinase Cα (PKCα) can inhibit RPE cell proliferation. Here, we sought to analyze whether PKCα affects the migration of RPE cells.

Methods.: Human RPE (hRPE) cells were cultured, confirmed by immunofluorescence staining, and divided into four groups: control, thymeleatoxin, non–small interfering RNA (siRNA), and siRNA-PKCα. Thymeleatoxin was used to activate PKCα, and siRNA-PKCα was used to knock it down. Expression of PKCα was confirmed by quantitative RT-PCR (qRT-PCR). Cell migration ability was analyzed by wound healing assay and transwell chamber assay. Expression of zonula occludens (ZO)-1 and occludin was determined by immunofluorescence.

Results.: Pure populations of hRPE cell cultures were observed using light and fluorescence microscopy. The mRNA levels of PKCα were not significantly increased by thymeleatoxin, but were reduced by siRNA-PKCα as determined by qRT-PCR assay. The wound healed faster in the thymeleatoxin group than in the control group at time points 12, 15, and 20 hours. The wound healed more slowly in the siRNA-PKCα group than in the non-siRNA group at the three time points. A similar tendency among the four groups was consistently observed in regard to cell numbers counted in the transwell chamber assay. The expression of ZO-1 was highest in the siRNA-PKCα group, similar in the control and non-siRNA groups, and lowest in the thymeleatoxin group. After migration, the fluorescence intensity of ZO-1 was reduced to similarly weak levels among the four groups.

Conclusions.: Retinal pigment epithelium cell migration is enhanced by a PKCα agonist and suppressed by a PKCα antagonist. The results suggest that a PKCα-mediated signal transduction pathway plays a crucial role in hRPE cell migration and may be a potential therapeutic target against hRPE cell migration and PVR disease.

Introduction
Proliferative vitreoretinopathy (PVR), one of the primary causes of blindness, was found in developed countries to have occurred in 3.9% to 13.7% of patients with rhegmatogenous retinal detachment who had not yet undergone vitreoretinal surgery. 13 Proliferative vitreoretinopathy is characterized by the growth and contraction of cellular membranes on both the inner and outer surfaces of the retina; therefore, it is generally considered to be a modified wound healing process. 47  
Retinal pigment epithelium (RPE) cells are considered to be a key element in this process and are present in almost 100% of epiretinal membranes. 810 When a retinal break occurs, the serum components and inflammatory cells enter into the subretinal space, and RPE cells are then exposed to a variety of cytokines. 11 In response, RPE cells are activated and migrate through the provisional extracellular matrix and retinal holes to form pathologic membranes on both surfaces of the neural retina. 12 However, the cellular mechanisms involved in this migration process are not well understood. Understanding how this process is regulated by the cytokine environment would provide insight into potential therapeutic targets against the pathophysiology of PVR. 
Protein kinase C (PKC) is a multigene family of phospholipid-dependent serine-threonine kinases that mediate the phosphorylation of numerous protein substrates in signal transduction. 13 The family is classified into three groups on the basis of the arrangement of their W2 regulatory domains. Conventional isoforms (cPKC: α, β, γ) contain a diacylglycerol (DAG)/phorbol ester-binding C1 domain and a Ca2+-binding C2 domain. Novel isoforms (nPKC: δ, ε, η, θ) also contain C1 domains, but their C2 domains are unable to bind Ca2+. Atypical isoforms (aPKC: ζ and ι/λ) lack a C2 domain and have an atypical C1 domain; they are therefore regulated independently of Ca2+ and DAG. 1417 It has been well documented that the PKC family is involved in the processes of proliferation, migration, phagocytosis, and gel contraction in RPE cells. 1821  
At least 12 isoforms of PKC have been cloned to date, all displaying different enzymatic properties, tissue expression, and intracellular localization. 22,23 Inhibitors of PKC may restrain all isoforms simultaneously, 24,25 while only several isoforms are involved in PVR. 26,27 Our previous study characterized the expression pattern of all 12 PKC isoforms and showed that 10 isoforms (PKCα, PKCβI, PKCβII, PKCδ, PKCε, PKCθ, PKCμ, PKCζ, PKCλ, and PKCι) were present in cultured human RPE cells. 28 In addition, we have demonstrated that, among the 10 isoforms of PKC, PKCα was both necessary and sufficient to promote RPE cell cycle progression through downregulation of p27kip1. 29 In this study, we sought to further investigate whether PKCα might affect the migration of RPE cells. 
Materials and Methods
Cell Culture of Human RPE
Eyes were isolated from human donors within 24 hours after death and were obtained from the Zhongshan Ophthalmic Center, Sun Yat-sen University. None of the donors had a known history of eye disease. This project was approved by the Ethics Committee of the Zhongshan Ophthalmic Center and followed the tenets of the Declaration of Helsinki. Human RPE cells were harvested according to a previously described procedure. 29 The anterior segment was removed by incision of the eyeball 2 mm behind the ora serrata. The vitreous body and neurosensory retina were then carefully peeled away from the RPE-choroid-sclera with fine forceps, and an eye cup was made. The eye cup was rinsed three times with PBS and filled with trypsin (0.05%)–EDTA (0.02%) solution at 37°C for 30 minutes. A culture medium with 10% fetal bovine serum (FBS; Invitrogen-Gibco, Karlsruhe, Germany) was added to stop the enzyme reaction; then, we gently aspirated the eye cup with a Pasteur pipette and collected the cell suspension. The cell suspension was centrifuged at 1000g for 10 minutes. Cells were then resuspended in 4 mL Dulbecco's modified Eagle's medium (DMEM; Invitrogen-Gibco) containing 10% FBS, penicillin G (100 μg/mL), and streptomycin sulfate (100 mg/mL). The cells were then transferred to a 25-cm2 plate and grown in an incubator in saturating humidity and 5% CO2 at 37°C. Experimentation was performed using 80% to 90% confluent cells at a cell passage of 3 to 6. The same cell preparation was used for experimental replicates or cells from different donors. 
To confirm that the cell population was pure and of epithelial origin, we observed the morphological characteristics and presence of pigment in the cells under an inverted optical microscope (Olympus, Tokyo, Japan). In addition, the cells were confirmed by immunofluorescence staining for cytokeratin (CK) using a pan-cytokeratin (PCK) antibody and for nucleus using DAPI (4′6-diamidino-2-phenylindole dihydrochloride; Roche, Mannheim, Germany). 
Knockdown of PKCα by Small Interfering RNA (siRNA)
To determine whether PKCα contributes to the migration of hRPE cells, we used small interfering RNA (siRNA) to inhibit the expression of PKCα in these cells. SiRNA-PKCα and nonsilencing siRNA were purchased from Ribuo Biotech (Guangzhou, China). The sequence of siRNA-PKCα was positive-sense strand 5′GGCGUCCUGUUGUAUGAAAdAdT3′ and antisense strand 3′dTdACCGCAGGACAACAUACUUU5′. Chemically synthesized siRNA-PKCα was mixed with the suspension of two deoxyribose nucleotides to increase the stability at its 3′ end. Lyophilized siRNA was dissolved in dH2O into a stock concentration of 20 μM and stored at −20°C. 
Human RPE cells were transfected with 100 nM nonsilencing siRNA and siRNA-PKCα using lipofectamine 2000 (Invitrogen, Carlsbad, CA) as delivery medium, according to the manufacturer's protocol. For standard six-well plates, 10 μL stored siRNA was dissolved in 250 μL Opti-MEM (Invitrogen-Gibco), and 5 μL lipofectamine 2000 was dissolved in 250 μL Opti-MEM and incubated for 5 minutes. These two solutions were mixed together and incubated for 20 minutes. Cultures of hRPE cells were incubated in an antibiotic-free, serum-free medium for 24 hours to ensure a cell density of 50% to 70%. The mixed solution and another 1.5 mL growth DMEM were then added to every well. The solution containing siRNA was removed 4 to 6 hours later, and then culture medium with 10% FBS was added for cell culture. After transfection, the hRPE cells were incubated for another 24 hours to ensure that the hRPE cells were equilibrated in their original environment. Next, quantitative RT-PCR (qRT-PCR), wound healing assay, transwell chamber assay, and immunofluorescence were carried out. 
Quantitative Reverse Transcriptase–Polymerase Chain Reaction
To screen for PKCα expression, qRT-PCR was performed. Total RNA was extracted using Trizol reagent (Life Technologies, Gaithersburg, MD). The purity and the quantity of the RNA were measured by a spectrophotometer (Eppendorf, Hamburg, Germany). Two μg RNA was reverse transcribed following the protocol of the Super Script First-Strand Synthesis System (Takara, Tokyo, Japan). Primer sequences were designed using primer 3. 30 For PKCα, the forward primer was 5′-ATCCGCAGTGGAATGAGTCCTTTACAT-3′, and the reverse primer was 5′-TTGGAAGGTTGTTTCCTGTCTTCAGAG-3′. A housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used as the internal control. The forward primer was 5′-ACCCAGAAGACTGTGGATGG-3′, and the reverse primer was 5′-TGCTGTAGCCAAATTCGTTG-3′. Quantitative RT-PCR was performed in a 20-μL reaction mixture using the FastStart Universal SYBR Green Master reagent (Roche, Basel, Switzerland) on an ABI PRISM 7000 sequence detection system (Applied Biosystems, Carlsbad, CA). The PCR conditions were as follows: 1 cycle at 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. The ΔΔCt method was applied to estimate relative transcript levels. Levels of GAPDH amplification were used to normalize each sample Ct (threshold cycle) value. Units are expressed as relative quantification (RQ). 
RPE Cell Migration Assay
The cultured hRPE cells were divided into four groups: control, thymeleatoxin (Sigma-Aldrich, St. Louis, MO), non-siRNA (Ribuo Biotech), and siRNA-PKCα (Ribuo Biotech); they were incubated with DMEM (Invitrogen-Gibco) containing 10% FBS (Invitrogen-Gibco) or containing 10% FBS and 100 nM thymeleatoxin, 29,31,32 100 nM non-siRNA, or 100 nM siRNA-PKCα, respectively. Cell migration ability was analyzed by wound healing assay and transwell chamber assay. 
Wound Healing Assay
Human RPE cells at a density of 15,000 to 20,000/well were seeded into 25-mm wells in 12-well plates; the cells were then grown to confluence. To measure growth-inhibited migration, hRPE cells were pretreated with mitomycin C (10 μg/mL). Mitomycin C was applied to the cells for 2 hours and removed with three washes of PBS. A cross-stripe scratch wound was made on the cell surface with a yellow micropipette tip. The wound area was photographed with an inverted fluorescence microscope (Carl Zeiss, Oberkochen, Germany) at time points 0, 12, 15, and 20 hours with a ×10 objective. The microscope was able to record the same exact area at each time point by memorizing the xy directions through a computer-controlled, motorized head stage. AxioVision software (AxioVision release 4.7.2, Oberkochen, Germany) was used to draw the lines of the wound edges and to measure the distance between the two edge lines. The wound healing rate was defined as the change of wound distance per hour. 
Transwell Chamber Assay
The cell migration assay was performed using a 24-well transwell chamber. Ten thousand cells of each group were seeded on the inner chamber of a transwell plate containing an 8-μm pore size membrane. The cells were then incubated with the same medium in the inner chamber and lower chamber. After 24 hours of incubation, the cells on the upper membrane were scraped off; the migrated cells on the lower surface of the filter were fixed with 4% paraformaldehyde at room temperature for 10 minutes, stained with DAPI for 5 minutes, and then photographed with a Zeiss fluorescence microscope (Carl Zeiss) with ×5 objective. Imgview software (Imgview 2.0; MichaelGraphics Software Tools, Hochschulstrasse, Germany) was used to count the cell numbers in the immunofluorescence micrograph. Each experiment was repeated at least three times. 
Immunofluorescence
Human RPE cells were grown on glass coverslips, cultured with DMEM containing 10% FBS or containing 10% FBS and 100 nM thymeleatoxin, 100 nM non-siRNA, or 100 nM siRNA-PKCα. When the cells grew to confluence, a cross-stripe scratch wound was made on the cell surface; then, the cells were cultured for 24 hours in dishes containing 10% FBS DMEM medium. 
The subcellular distribution of zonula occludens (ZO)-1 and occludin was determined by indirect immunofluorescence. Cells were fixed at 4°C by washing twice with 100% ethanol (for 30 minutes total) and then rinsed three times in PBS. All reagent incubations were performed in a humidified chamber. After blocking nonspecific antibody binding with 10% bovine serum albumin, cells were incubated for 1 hour at 37°C with mouse anti-ZO-1 or mouse anti-occludin (Invitrogen). After washing three times for 5 minutes in PBS, the primary antibodies were revealed with Alexa Fluor 488 goat anti-mouse IgG (1:1000; Invitrogen) at 37°C for 30 minutes. Then, DAPI was incubated for 5 minutes at room temperature. After three rinses in PBS, the coverslips were mounted onto glass slides. The specimens were examined and analyzed using a Zeiss laser scanning confocal microscope (LSCM 510 META; Oberkochen, Germany). 
Statistical Analysis
All data were expressed as the mean ± standard deviation (SD). The level of significance was determined by using two-sample Student's t-tests. P < 0.05 was considered statistically significant. 
Results
Human RPE Cells Identified Under Inverted Optical Microscope and Fluorescence Microscope
Light microscopy showed that most of the isolated hRPE cells of passage 0 were hexagonal shaped and pigmented (Fig. 1). With increasing passage number, the pigment decreased and morphology flattened in the cells through passage 4. The cytokeratin and nuclei of the cell cultures were stained uniformly. 
Figure 1. 
 
Identification of human RPE cells. (A) Inverted optical microscope shows the pigmentation and the morphology of hRPE cells of passage 0 (4 hours). With increasing passage number, the pigment decreased and morphology flattened through cells of passage 4 (10 hours). (B) Fluorescence photomicrographs show the cytokeratin (green) and cell nuclei (blue) of hRPE cells (40 hours, scale bar: 50 μm).
Figure 1. 
 
Identification of human RPE cells. (A) Inverted optical microscope shows the pigmentation and the morphology of hRPE cells of passage 0 (4 hours). With increasing passage number, the pigment decreased and morphology flattened through cells of passage 4 (10 hours). (B) Fluorescence photomicrographs show the cytokeratin (green) and cell nuclei (blue) of hRPE cells (40 hours, scale bar: 50 μm).
PKCα mRNA Expression Not Upregulated Significantly by Thymeleatoxin but Downregulated by siRNA-PKCα as Assessed by qRT-PCR
Quantitative RT-PCR showed that thymeleatoxin increased the mRNA levels of PKCα compared to the control group at 24 hours, but there was not a significant difference between the two groups (P = 0.093) (Fig. 2). On the other hand, siRNA-PKCα significantly reduced the mRNA levels of PKCα compared with the non-siRNA group at 24 hours (P < 0.05). The vertical axis is the RQ level of PKCα. 
Figure 2. 
 
PKCα mRNA expression in hRPE cells. Quantitative RT-PCR shows that thymeleatoxin increases the mRNA levels of PKCα compared to the control group at 24 hours, but there is no significant difference between the two groups (P = 0.093). On the other hand, siRNA-PKCα significantly reduces the mRNA levels of PKCα compared with the non-siRNA group at 24 hours (P < 0.05). The vertical axis is the relative quantification (RQ) level of PKCα.
Figure 2. 
 
PKCα mRNA expression in hRPE cells. Quantitative RT-PCR shows that thymeleatoxin increases the mRNA levels of PKCα compared to the control group at 24 hours, but there is no significant difference between the two groups (P = 0.093). On the other hand, siRNA-PKCα significantly reduces the mRNA levels of PKCα compared with the non-siRNA group at 24 hours (P < 0.05). The vertical axis is the relative quantification (RQ) level of PKCα.
Faster Healing of Scratch Wounds in the Thymeleatoxin Group and Slower Healing in the siRNA-PKCα Group
During the 20-hour observation of wound healing, micrographs of the healing wounds and gap widths were recorded. The gap widths and wound healing rates of the four groups are listed in the Table
Table. 
 
Gap Widths and Wound Healing Rates of the Four Groups in the Wound Healing Assay
Table. 
 
Gap Widths and Wound Healing Rates of the Four Groups in the Wound Healing Assay
Groups Gap Widths, μm Wound Healing Rates, μm/h
0 h 12 h 15 h 20 h 0 h 12 h 15 h 20 h
Control 576.5 ± 29.0 393.3 ± 35.8 363.7 ± 43.3 295.5 ± 62.4 15.3 ± 3.2 17.7 ± 3.7 23.4 ± 5.5
Thymeleatoxin 518.6 ± 82.1 133.8 ± 149.3 74.4 ± 133.0 34.0 ± 87.8 24.6 ± 7.1 29.0 ± 7.8 41.3 ± 5.7
Non-siRNA 528.2 ± 60.4 297.5 ± 107.5 221.0 ± 117.9 72.8 ± 115.4 18.0 ± 6.2 23.3 ± 5.9 38.4 ± 9.6
SiRNA-PKCα 539.3 ± 52.0 394.8 ± 67.2 351.5 ± 66.6 258.6 ± 103.1 12.0 ± 3.9 15.6 ± 3.6 23.4 ± 6.9
In the wound healing assay, the thymeleatoxin group healed faster than the control group, with significant differences at time points 12, 15, and 20 hours (P < 0.05) (Fig. 3, Table). The siRNA-PKCα group healed more slowly than the non-siRNA group, with significant differences at each of the three time points (P < 0.05). 
Figure 3. 
 
Effects of PKCα on wound healing rate in hRPE cells. (A) Scratch wound heals faster in the thymeleatoxin group than the control group at time-points of 12, 15, and 20 hours under the microscope (10 hours, scale bar: 100 μm). (B) Scratch wound heals slower in the siRNA-PKCα group than the non-siRNA group at the three time-points under the microscope (10 hours, scale bar: 100 μm). (C) The graphical representations show that the wound healing rates are significantly different between the thymeleatoxin and the control groups (P < 0.05), and between the siRNA-PKCα and the non-siRNA groups (P < 0.05).
Figure 3. 
 
Effects of PKCα on wound healing rate in hRPE cells. (A) Scratch wound heals faster in the thymeleatoxin group than the control group at time-points of 12, 15, and 20 hours under the microscope (10 hours, scale bar: 100 μm). (B) Scratch wound heals slower in the siRNA-PKCα group than the non-siRNA group at the three time-points under the microscope (10 hours, scale bar: 100 μm). (C) The graphical representations show that the wound healing rates are significantly different between the thymeleatoxin and the control groups (P < 0.05), and between the siRNA-PKCα and the non-siRNA groups (P < 0.05).
Cell Migration Higher in the Thymeleatoxin Group and Lower in the siRNA-PKCα Group in the Transwell Assay
At 24 hours of incubation, fluorescence photomicrographs showed that more cells had migrated through the membrane in the thymeleatoxin group than in the control group, and the cell migration numbers were significantly different between the two groups (P < 0.05) (Fig. 4). Fewer migrated cells were observed in the siRNA-PKCα group than in the non-siRNA group at 24 hours, and the migrated cell numbers were significantly different between these groups (P < 0.05). 
Figure 4. 
 
Effects of PKCα on the number of migrated hRPE cells at 24 hours. (A) Fluorescence photomicrographs show the nucleus of hRPE cells that migrated through the membrane to the bottom chamber. More cells migrated through the membrane in the thymeleatoxin group than the control group (×5, scale bar: 100 μm). (B) Fewer migrated cells are observed in the siRNA-PKCα group than in the non-siRNA group (×5, scale bar: 100 μm). (C) Graphical representation shows that the amount of migrated cells is significantly different between the thymeleatoxin and the control groups (P < 0.05) and between the siRNA-PKCα and the non-siRNA groups (P < 0.05). The vertical axis represents the number of cells that migrated into the bottom chamber.
Figure 4. 
 
Effects of PKCα on the number of migrated hRPE cells at 24 hours. (A) Fluorescence photomicrographs show the nucleus of hRPE cells that migrated through the membrane to the bottom chamber. More cells migrated through the membrane in the thymeleatoxin group than the control group (×5, scale bar: 100 μm). (B) Fewer migrated cells are observed in the siRNA-PKCα group than in the non-siRNA group (×5, scale bar: 100 μm). (C) Graphical representation shows that the amount of migrated cells is significantly different between the thymeleatoxin and the control groups (P < 0.05) and between the siRNA-PKCα and the non-siRNA groups (P < 0.05). The vertical axis represents the number of cells that migrated into the bottom chamber.
Expression of ZO-1 and Occludin in the Four Groups Under the Fluorescence Microscope
Before migration, the expression of ZO-1 (green) was highest in the siRNA-PKCα group, similarly and moderately intense in the control and non-siRNA groups, and lowest in the thymeleatoxin group (Fig. 5). After migration, fluorescence intensity of ZO-1 was reduced to similarly weak levels among the four groups. 
Figure 5. 
 
Expression of ZO-1 and occludin in hRPE cells under the fluorescence microscope. (A) Before migration, the expression of ZO-1 (green) is highest in the siRNA-PKCα group, similarly and moderately intense in the control and non-siRNA groups, and lowest in the thymeleatoxin group. After migration, the fluorescence intensity of ZO-1 is reduced to similarly weak levels among the four groups. The cell nuclei stained with DAPI are blue (×40, scale bar: 50 μm). (B) Before migration, the expression of occludin (green) is more prevalent in the thymeleatoxin group than the control group. Occludin expression is seldom observed in the non-siRNA and the siRNA-PKCα groups. The expression of occludin changes little before and after migration (×40, scale bar: 50 μm).
Figure 5. 
 
Expression of ZO-1 and occludin in hRPE cells under the fluorescence microscope. (A) Before migration, the expression of ZO-1 (green) is highest in the siRNA-PKCα group, similarly and moderately intense in the control and non-siRNA groups, and lowest in the thymeleatoxin group. After migration, the fluorescence intensity of ZO-1 is reduced to similarly weak levels among the four groups. The cell nuclei stained with DAPI are blue (×40, scale bar: 50 μm). (B) Before migration, the expression of occludin (green) is more prevalent in the thymeleatoxin group than the control group. Occludin expression is seldom observed in the non-siRNA and the siRNA-PKCα groups. The expression of occludin changes little before and after migration (×40, scale bar: 50 μm).
Before migration, the expression of occludin (green) was more prevalent in the thymeleatoxin group than in the control group (Fig. 5). Occludin expression was seldom observed in the non-siRNA and siRNA-PKCα groups. The expression of occludin changed after migration compared to before. 
Discussion
Migration of RPE cells into the vitreous body plays an important role in the onset and development of PVR. Protein kinase C has been demonstrated to regulate both the migration and proliferation of RPE cells. 21 Ten isoforms are present in cultured human RPE cells, and PKCα has been confirmed to regulate RPE cell cycle progression and proliferation. Here, we further demonstrated that PKCα regulates the migration of RPE cells in vitro. 
Protein kinase C is a family of isoenzymes that play a central role in cellular processes such as proliferation, differentiation, mitosis, and inflammation. 13 The differences in the function of specific PKC isoforms are mainly due to their subcellular localization, activation, or inhibition by different stimuli and transcriptional regulation. 33 Murphy et al. 21 reported that migration of hRPE cells, stimulated by phorbol 12-myristate 13-acetate (PMA) and inhibited by calphostin C, is regulated by PKC in vitro. Our previous study characterized the expression pattern of all 12 PKC isoforms and showed that 10 of these are present in cultured human RPE cells. 28 Additionally, we found that PKCα was both necessary and sufficient to promote cell cycle progression after being stimulated with PMA; that is, downregulation of PKCα can inhibit RPE cell proliferation. 29 Moreover, siRNA-PKCα could be released sustainably from a novel foldable capsular vitreous body (FCVB) to inhibit PKCα expression in RPE cells in vitro. 34 Therefore, the role of PKCα in migration will be an interesting and essential research point after its proliferative role is ascertained. 
The mechanism through which PKCα stimulates migration relies on its structure. Protein kinase Cα is a conventional PKC that has a C2 domain containing a putative Ca2+ binding site. The binding of Ca2+ to the five aspartates orients the bulky aromatics to interact with the membrane. 35 Thymeleatoxin, a chemically related phorbol compound, selectively activates PKCα, β, and γ and increases the calcium sensitivity in intact cells. Moreover, 100 nM thymeleatoxin effectively activates PKCα. 36 Ryves et al. 37 demonstrated that thymeleatoxin enhances the kinase activity of PKCα through kinase activation assays. Nishizuka 38 and Miyawaki and Ashraf 39 showed that translocation is a key index of PKC activation as it enables the interaction of PKC with membrane phospholipids. Newton 33 and Zidovetzki and Lester 40 showed that PKCα was activated through interactions with the cell membrane, with allosteric effects of phosphatidylserine, DAG, and Ca2+ on the enzyme. Western blot analysis confirmed that PKCα was specifically translocated by thymeleatoxin from the soluble cytosolic fraction to the membranous fraction, 4143 while our previous and current studies both showed that the PKCα mRNA level was not significantly upregulated by thymeleatoxin treatment. 29 Based on this evidence, we speculate that thymeleatoxin did not increase the mRNA levels of PKCα but instead translocated it from the cytosolic fraction to the membranous fraction and enhanced its kinase activity. 
However, the roles of PKCα in proliferation and migration might overlap and interact with each other. Murphy et al. 21 reported that PMA and calphostin C affected the migration of RPE cells equally in growth-inhibited and growth-dependent migration. Similarly, mitomycin C treatment in this study enabled us to inhibit proliferation and thereby evaluate migration primarily based on cell motility. Protein kinase Cα is knocked down by siRNA, and the non-siRNA group was set as the negative control to redress the influences of siRNA. Small interfering RNAs, achieving target-specific gene silencing via double-stranded RNA-mediated RNA interference, have attracted much attention as a new therapeutic technique. 44,45 Previous research has demonstrated that 100 nM siRNA-PKCα has the most effective inhibitive influence among doses in the 10 to 200 nM range, with no significant toxicity (data not shown). However, the wound healed faster in the non-siRNA group than in the control group. Similar patterns were observed in the transwell chamber assay. The reasons are difficult to identify, and more in-depth research will be conducted in the future. 
Tight junction proteins play key roles in migration and can be divided into two groups. 46,47 Occludin belongs to the first group (integral membrane proteins), 48,49 and ZO-1 belongs to the second group (plaque proteins). 5052 The classical junctional scaffold protein ZO-1 is widely recognized for its vital role in the assembly of cell–cell adhesion complexes. 53 Zonula occludens-1 associates with the subcellular C-terminal tail of occludin, and interaction between these proteins is crucial for tight junctions. 54 In our study, the expression of ZO-1 was highest in the siRNA-PKCα group, moderately intense in the control and non-siRNA groups, and lowest in the thymeleatoxin group, indicating that a PKCα agonist might decrease the expression of ZO-1. After migration, fluorescence intensity of ZO-1 was reduced to similarly weak levels among the four groups, indicating that migration might decrease the expression of ZO-1. 
Some effective therapy involving PKCα has recently been studied. A new siRNA-based therapeutic strategy targeting the PKCα gene has been designed to overcome the chemoresistance of ovarian cancer. 55 Berberine, an isoquinoline alkaloid inhibiting PKCα in breast cancer cells, may be used as a candidate drug for the inhibition of metastasis of human breast cancer. 56 We have demonstrated that siRNA-PKCα can be released sustainably from a novel FCVB to inhibit PKCα expression in RPE cells in vitro. 34 As PKCα is the only isoform associated with the proliferation of RPE cells, combining its role in migration and inhibition of PKCα might be a rational approach for therapy against PVR disease. 
In conclusion, RPE cell migration is enhanced by a PKCα agonist and is suppressed by a PKCα antagonist. These results suggest that a PKCα-mediated signal transduction pathway plays a crucial role in RPE cell migration and might be useful as a potential therapeutic target against RPE cell migration and PVR disease. 
Acknowledgments
Supported by the project of Wenzhou Municipal Science and Technology Bureau in Zhejiang Province (Grant H20080029) and the National Key Technology R&D Program (Grant 2012BA108B02). 
Disclosure: S. Qiu, None; Z. Jiang, None; Z. Huang, None; X. Chen, None; X. Qian, None; Q. Gao, None; H. Zheng, None 
References
Ryan SJ. The pathophysiology of proliferative vitreoretinopathy in its management. Am J Ophthalmol . 1985; 100: 188–193. [CrossRef] [PubMed]
Ryan SJ. Traction retinal detachment. XLIX Edward Jackson Memorial Lecture. Am J Ophthalmol . 1993; 115: 1–20. [CrossRef] [PubMed]
Tseng W Cortez RT Ramirez G Stinnett S Jaffe GJ. Prevalence and risk factors for proliferative vitreoretinopathy in eyes with rhegmatogenous retinal detachment but no previous vitreoretinal surgery. Am J Ophthalmol . 2004; 137: 1105–1115. [CrossRef] [PubMed]
Weller M Wiedemann P Heimann K. Proliferative vitreoretinopathy--is it anything more than wound healing at the wrong place? Int Ophthalmol . 1990; 14: 105–117. [CrossRef] [PubMed]
Pastor JC. Proliferative vitreoretinopathy: an overview. Surv Ophthalmol . 1998; 43: 3–18. [CrossRef] [PubMed]
Hiscott P Hagan S Heathcote L Pathobiology of epiretinal and subretinal membranes: possible roles for the matricellular proteins thrombospondin 1 and osteonectin (SPARC). Eye (Lond) . 2002; 16: 393–403. [CrossRef] [PubMed]
Sadaka A Giuliari GP. Proliferative vitreoretinopathy: current and emerging treatments. Clin Ophthalmol . 2012; 6: 1325–1333. [PubMed]
Pastor JC de la Rua ER Martin F. Proliferative vitreoretinopathy: risk factors and pathobiology. Prog Retin Eye Res . 2002; 21: 127–144. [CrossRef] [PubMed]
Baudouin C Gastaud P. Vitreoretinal proliferation. I. Clinicopathological aspects [in French]. J Fr Ophtalmol . 1994; 17: 789–799. [PubMed]
Umazume K Liu L Scott PA Inhibition of PVR with a tyrosine kinase inhibitor, dasatinib, in the swine. Invest Ophthalmol Vis Sci . 2013; 54: 1150–1159. [CrossRef] [PubMed]
Campochiaro PA. Pathogenic mechanisms in proliferative vitreoretinopathy. Arch Ophthalmol . 1997; 115: 237–241. [CrossRef] [PubMed]
Hiscott P Sheridan C Magee RM Grierson I. Matrix and the retinal pigment epithelium in proliferative retinal disease. Prog Retin Eye Res . 1999; 18: 167–190. [CrossRef] [PubMed]
Parker PJ Murray-Rust J. PKC at a glance. J Cell Sci . 2004; 117 (pt 2): L131–L132. [CrossRef]
Konopatskaya O Poole AW. Protein kinase Cα: disease regulator and therapeutic target. Trends Pharmacol Sci . 2010; 31: 8–14. [CrossRef] [PubMed]
Dempsey EC Newton AC Mochly-Rosen D Protein kinase C isozymes and the regulation of diverse cell responses. Am J Physiol Lung Cell Mol Physiol . 2000; 279: L429–L438. [PubMed]
Gould CM Newton AC. The life and death of protein kinase C. Curr Drug Targets . 2008; 9: 614–625. [CrossRef] [PubMed]
Santana LF Navedo MF Amberg GC Nieves-Cintrón M Votaw VS Ufret-Vincenty CA. Calcium sparklets in arterial smooth muscle. Clin Exp Pharmacol Physiol . 2008; 35: 1121–1126. [CrossRef] [PubMed]
Kishi H Mishima HK Yamashita U. Growth regulation of retinal pigment epithelial (RPE) cells in vitro. Curr Eye Res . 1994; 13: 661–668. [CrossRef] [PubMed]
Qiao H Sakamoto T Hinton DR Interferon beta affects retinal pigment epithelial cell proliferation via protein kinase C pathways. Ophthalmologica . 2001; 215: 401–407. [CrossRef] [PubMed]
Kishi H Mishima HK Yamashita U. Involvement of the protein kinase pathway in melanin synthesis by chick retinal pigment epithelial cells. Cell Biol Int . 2000; 24: 79–83. [CrossRef] [PubMed]
Murphy TL Sakamoto T Hinton DR Migration of retinal pigment epithelium cells in vitro is regulated by protein kinase C. Exp Eye Res . 1995; 60: 683–695. [CrossRef] [PubMed]
Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature . 1988; 334: 661–665. [CrossRef] [PubMed]
Ono Y Fujii T Ogita K Kikkawa U Igarashi K Nishizuka Y. The structure, expression, and properties of additional members of the protein kinase C family. J Biol Chem . 1988; 263: 6927–6932. [PubMed]
Ding RQ Tsao J Chai H Mochly-Rosen D Zhou W. Therapeutic potential for protein kinase C inhibitor in vascular restenosis. J Cardiovasc Pharmacol Ther . 2011; 16: 160–167. [CrossRef] [PubMed]
Matz M Naik M Mashreghi MF Glander P Neumayer HH Budde K. Evaluation of the novel protein kinase C inhibitor sotrastaurin as immunosuppressive therapy after renal transplantation. Expert Opin Drug Metab Toxicol . 2011; 7: 103–113. [CrossRef] [PubMed]
Chen YJ Tsai RK Wu WC He MS Kao YH Wu WS. Enhanced PKCδ and ERK signaling mediate cell migration of retinal pigment epithelial cells synergistically induced by HGF and EGF. PLoS One . 2012; 7: e44937. [CrossRef] [PubMed]
Er H Turkoz Y Mizrak B Parlakpinar H. Inhibition of experimental proliferative vitreoretinopathy with protein kinase C inhibitor (chelerythrine chloride) and melatonin. Ophthalmologica . 2006; 220: 17–22. [CrossRef] [PubMed]
Yu K Ma P Ge J Expression of protein kinase C isoforms in cultured human retinal pigment epithelial cells. Graefes Arch Clin Exp Ophthalmol . 2007; 245: 993–999. [CrossRef] [PubMed]
Gao Q Tan J Ma P PKC alpha affects cell cycle progression and proliferation in human RPE cells through the downregulation of p27kip1 . Mol Vis . 2009; 15: 2683–2695. [PubMed]
Rozen S Skaletsky HJ. Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S Misener S eds. Bioinformatics Methods and Protocols: Methods in Molecular Biology . Totowa, NJ: Humana Press; 2000: 365–386.
Rahm A-K Gierten J Kisselbach J PKC-dependent activation of human K2P18.1 K+ channels. Br J Pharmacol . 2012; 166: 764–773. [CrossRef] [PubMed]
Stross C Helmer A Weissenberger K Protein kinase C induces endocytosis of the sodium taurocholate cotransporting polypeptide. Am J Physiol Gastrointest Liver Physiol . 2010; 299: 320–328. [CrossRef]
Newton AC. Protein kinase C: structure, function, and regulation. J Biol Chem . 1995; 270: 28495–28498. [CrossRef] [PubMed]
Chen X Liu Y Jiang Z Zhou L Ge J Gao Q. Protein kinase Cα downregulation via siRNA-PKCα released from foldable capsular vitreous body in cultured human retinal pigment epithelium cells. Int J Nanomedicine . 2011; 6: 1303–1311. [CrossRef] [PubMed]
Nakashima S. Protein kinase C alpha (PKC alpha): regulation and biological function. J Biochem . 2002; 132: 669–675. [CrossRef] [PubMed]
Roivainen R Messing RO. The phorbol derivatives thymeleatoxin and 12-deoxyphorbol-13-O-phenylacetate-10-acetate cause translocation and down-regulation of multiple protein kinase C isozymes. FEBS Lett . 1993; 319: 31–34. [CrossRef] [PubMed]
Ryves WJ Evans AT Olivier AR Parker PJ Evans FJ. Activation of the PKC-isotypes alpha, beta 1, gamma, delta and epsilon by phorbol esters of different biological activities. FEBS Lett . 1991; 288: 5–9. [CrossRef] [PubMed]
Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science . 1992; 258: 607–614. [CrossRef] [PubMed]
Miyawaki H Ashraf M. Ca2+ as a mediator of ischemic preconditioning. Circ Res . 1997; 80: 790–799. [CrossRef] [PubMed]
Zidovetzki R Lester DS. The mechanism of activation of protein kinase C: a biophysical perspective. Biochim Biophys Acta . 1992; 113: 261–272. [CrossRef]
Micol V Sánchez-Piñera P Villalaín J de Godos A Gómez-Fernández JC. Correlation between protein kinase C alpha activity and membrane phase behavior. Biophys J . 1999; 76: 916–927. [CrossRef] [PubMed]
Besson A Yong VW. Involvement of p21(Waf1/Cip1) in protein kinase C alpha-induced cell cycle progression. Mol Cell Biol . 2000; 20: 4580–4590. [CrossRef] [PubMed]
Nakano T Sekine S Ito K Horie T. Correlation between apical localization of Abcc2/Mrp2 and phosphorylation status of ezrin in rat intestine. Drug Metab Dispos . 2009; 37: 1521–1527. [CrossRef] [PubMed]
Kleinman ME Yamada K Takeda A Sequence- and target-independent angiogenesis suppression by siRNA via TLR3. Nature . 2008; 452: 591–597. [CrossRef] [PubMed]
Cho WG Albuquerque RJ Kleinman ME Small interfering RNA-induced TLR3 activation inhibits blood and lymphatic vessel growth. Proc Natl Acad Sci U S A . 2009; 106: 7137–7142. [CrossRef] [PubMed]
D'Atri F Citi S. Molecular complexity of vertebrate tight junctions. Mol Membr Biol . 2002; 19: 103–112. [CrossRef] [PubMed]
Gonzalez-Mariscal L Betanzos A Nava P Jaramillo BE. Tight junction proteins. Prog Biophys Mol Biol . 2003; 81: 1–44. [CrossRef] [PubMed]
Furuse M Hirase T Itoh M Nagafuchi A Yonemura S Tsukita S. Occludin: a novel integral membrane protein localizing at tight junctions. J Cell Biol . 1993; 123: 1777–1788. [CrossRef] [PubMed]
Furuse M Fujita K Hiiragi T Fujimoto K Tsukita S. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol . 1998; 141: 1539–1550. [CrossRef] [PubMed]
Gonzalez-Mariscal L Betanzos A Avila-Flores A. MAGUK proteins: structure and role in the tight junction. Semin Cell Dev Biol . 2000; 11: 315–324. [CrossRef] [PubMed]
Stevenson B Siliciano J Mooseker M Goodenough D. Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Biol . 1986; 103: 755–766. [CrossRef] [PubMed]
Itoh M Furuse M Morita K Kubota K Saitou M Tsukita S. Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins. J Cell Biol . 1999; 147: 1351–1363. [CrossRef] [PubMed]
Tsukita S Katsuno T Yamazaki Y Umeda K Tamura A. Roles of ZO-1 and ZO-2 in establishment of the belt-like adherens and tight junctions with paracellular permselective barrier function. Ann N Y Acad Sci . 2009; 1165: 44–52. [CrossRef] [PubMed]
Tsukita S Furuse M Itoh M. Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol . 2001; 2: 285–293. [CrossRef] [PubMed]
Zhao LJ Xu H Qu JW Zhao WZ Zhao YB Wang JH. Modulation of drug resistance in ovarian cancer cells by inhibition of protein kinase C-alpha (PKCα) with small interference RNA (siRNA) agents. Asian Pac J Cancer Prev . 2012; 13: 3631–3636. [CrossRef] [PubMed]
Kim S Han J Lee SK Berberine suppresses the TPA-induced MMP-1 and MMP-9 expressions through the inhibition of PKC-α in breast cancer cells. J Surg Res . 2012; 176: e21–e29. [CrossRef] [PubMed]
Figure 1. 
 
Identification of human RPE cells. (A) Inverted optical microscope shows the pigmentation and the morphology of hRPE cells of passage 0 (4 hours). With increasing passage number, the pigment decreased and morphology flattened through cells of passage 4 (10 hours). (B) Fluorescence photomicrographs show the cytokeratin (green) and cell nuclei (blue) of hRPE cells (40 hours, scale bar: 50 μm).
Figure 1. 
 
Identification of human RPE cells. (A) Inverted optical microscope shows the pigmentation and the morphology of hRPE cells of passage 0 (4 hours). With increasing passage number, the pigment decreased and morphology flattened through cells of passage 4 (10 hours). (B) Fluorescence photomicrographs show the cytokeratin (green) and cell nuclei (blue) of hRPE cells (40 hours, scale bar: 50 μm).
Figure 2. 
 
PKCα mRNA expression in hRPE cells. Quantitative RT-PCR shows that thymeleatoxin increases the mRNA levels of PKCα compared to the control group at 24 hours, but there is no significant difference between the two groups (P = 0.093). On the other hand, siRNA-PKCα significantly reduces the mRNA levels of PKCα compared with the non-siRNA group at 24 hours (P < 0.05). The vertical axis is the relative quantification (RQ) level of PKCα.
Figure 2. 
 
PKCα mRNA expression in hRPE cells. Quantitative RT-PCR shows that thymeleatoxin increases the mRNA levels of PKCα compared to the control group at 24 hours, but there is no significant difference between the two groups (P = 0.093). On the other hand, siRNA-PKCα significantly reduces the mRNA levels of PKCα compared with the non-siRNA group at 24 hours (P < 0.05). The vertical axis is the relative quantification (RQ) level of PKCα.
Figure 3. 
 
Effects of PKCα on wound healing rate in hRPE cells. (A) Scratch wound heals faster in the thymeleatoxin group than the control group at time-points of 12, 15, and 20 hours under the microscope (10 hours, scale bar: 100 μm). (B) Scratch wound heals slower in the siRNA-PKCα group than the non-siRNA group at the three time-points under the microscope (10 hours, scale bar: 100 μm). (C) The graphical representations show that the wound healing rates are significantly different between the thymeleatoxin and the control groups (P < 0.05), and between the siRNA-PKCα and the non-siRNA groups (P < 0.05).
Figure 3. 
 
Effects of PKCα on wound healing rate in hRPE cells. (A) Scratch wound heals faster in the thymeleatoxin group than the control group at time-points of 12, 15, and 20 hours under the microscope (10 hours, scale bar: 100 μm). (B) Scratch wound heals slower in the siRNA-PKCα group than the non-siRNA group at the three time-points under the microscope (10 hours, scale bar: 100 μm). (C) The graphical representations show that the wound healing rates are significantly different between the thymeleatoxin and the control groups (P < 0.05), and between the siRNA-PKCα and the non-siRNA groups (P < 0.05).
Figure 4. 
 
Effects of PKCα on the number of migrated hRPE cells at 24 hours. (A) Fluorescence photomicrographs show the nucleus of hRPE cells that migrated through the membrane to the bottom chamber. More cells migrated through the membrane in the thymeleatoxin group than the control group (×5, scale bar: 100 μm). (B) Fewer migrated cells are observed in the siRNA-PKCα group than in the non-siRNA group (×5, scale bar: 100 μm). (C) Graphical representation shows that the amount of migrated cells is significantly different between the thymeleatoxin and the control groups (P < 0.05) and between the siRNA-PKCα and the non-siRNA groups (P < 0.05). The vertical axis represents the number of cells that migrated into the bottom chamber.
Figure 4. 
 
Effects of PKCα on the number of migrated hRPE cells at 24 hours. (A) Fluorescence photomicrographs show the nucleus of hRPE cells that migrated through the membrane to the bottom chamber. More cells migrated through the membrane in the thymeleatoxin group than the control group (×5, scale bar: 100 μm). (B) Fewer migrated cells are observed in the siRNA-PKCα group than in the non-siRNA group (×5, scale bar: 100 μm). (C) Graphical representation shows that the amount of migrated cells is significantly different between the thymeleatoxin and the control groups (P < 0.05) and between the siRNA-PKCα and the non-siRNA groups (P < 0.05). The vertical axis represents the number of cells that migrated into the bottom chamber.
Figure 5. 
 
Expression of ZO-1 and occludin in hRPE cells under the fluorescence microscope. (A) Before migration, the expression of ZO-1 (green) is highest in the siRNA-PKCα group, similarly and moderately intense in the control and non-siRNA groups, and lowest in the thymeleatoxin group. After migration, the fluorescence intensity of ZO-1 is reduced to similarly weak levels among the four groups. The cell nuclei stained with DAPI are blue (×40, scale bar: 50 μm). (B) Before migration, the expression of occludin (green) is more prevalent in the thymeleatoxin group than the control group. Occludin expression is seldom observed in the non-siRNA and the siRNA-PKCα groups. The expression of occludin changes little before and after migration (×40, scale bar: 50 μm).
Figure 5. 
 
Expression of ZO-1 and occludin in hRPE cells under the fluorescence microscope. (A) Before migration, the expression of ZO-1 (green) is highest in the siRNA-PKCα group, similarly and moderately intense in the control and non-siRNA groups, and lowest in the thymeleatoxin group. After migration, the fluorescence intensity of ZO-1 is reduced to similarly weak levels among the four groups. The cell nuclei stained with DAPI are blue (×40, scale bar: 50 μm). (B) Before migration, the expression of occludin (green) is more prevalent in the thymeleatoxin group than the control group. Occludin expression is seldom observed in the non-siRNA and the siRNA-PKCα groups. The expression of occludin changes little before and after migration (×40, scale bar: 50 μm).
Table. 
 
Gap Widths and Wound Healing Rates of the Four Groups in the Wound Healing Assay
Table. 
 
Gap Widths and Wound Healing Rates of the Four Groups in the Wound Healing Assay
Groups Gap Widths, μm Wound Healing Rates, μm/h
0 h 12 h 15 h 20 h 0 h 12 h 15 h 20 h
Control 576.5 ± 29.0 393.3 ± 35.8 363.7 ± 43.3 295.5 ± 62.4 15.3 ± 3.2 17.7 ± 3.7 23.4 ± 5.5
Thymeleatoxin 518.6 ± 82.1 133.8 ± 149.3 74.4 ± 133.0 34.0 ± 87.8 24.6 ± 7.1 29.0 ± 7.8 41.3 ± 5.7
Non-siRNA 528.2 ± 60.4 297.5 ± 107.5 221.0 ± 117.9 72.8 ± 115.4 18.0 ± 6.2 23.3 ± 5.9 38.4 ± 9.6
SiRNA-PKCα 539.3 ± 52.0 394.8 ± 67.2 351.5 ± 66.6 258.6 ± 103.1 12.0 ± 3.9 15.6 ± 3.6 23.4 ± 6.9
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