The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway

Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin’s potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated.

Viable retinal pigment epithelial (RPE) cells are critical for normal photoreceptor cell metabolism and visual cycle. Hence, RPE degeneration is accompanied by concomitant photoreceptor degeneration 1 . Drusen, which are located between the RPE cells and Bruch's membranes, contain a number of proteins that relate to inflammation; and there is increasing body of evidence that oxidative stress 2 , local inflammation 3 , and activation of the complement system 4 play a part in the development of AMD. The choriocapillaris has also been suggested to play a role in the etiology of AMD 5 . The disorder is subdivided into a dry and a wet type. There are no established curative treatment options for the dry type of AMD, which constitutes more than 85% of the cases. Cell based restoration of RPE cells has been explored in several studies 6,7 . There are two main approaches for delivering cultured RPE to the submacular space: 1) injection of a RPE cell suspension; and 2) implantation of RPE as an intact cell sheet 8 . Benefits of the former include ease of procedure, while some of the disadvantages are increased apoptosis due to loss of cell-cell interaction and a disorganized RPE upon injection 8 . Implantation of RPE as an intact cell sheet should in theory prevent the mentioned disadvantages of injecting RPE.
Several culture protocols intended for driving the differentiation of human embryonic stem cells (hESC) and human induced pluripotent stem cells (iPSC) into RPE have been described [9][10][11][12] . Although protocols using hESC or iPSC have successfully produced differentiated and pigmented RPE cells, they are usually time-consuming,

Results
Microarray Analysis of hRPE Cultured With or Without Sericin. Microarray experiments were performed to assess the effect on hRPE of adding sericin to the culture medium in absence of FBS. Similarly as described in materials and methods, hRPE were seeded in complete EpiCM on Nunclon Δ surface plates and cultured for two days before replacing EpiCM with either 1) DMEM with 1% sericin or 2) DMEM. Both DMEM media were supplied with 10.000 U penicillin and 10 mg streptomycin at a final concentration of 1%.
Global Perspective. Gene expression differed considerably between the two culture groups, with a total of 438 significantly differentially regulated genes (fold change > 1.5; P < 0.05). In the sericin-supplemented group, 229 genes were down-regulated and 209 genes were up-regulated compared to the control, while 14419 transcripts were unchanged.
Substantially Regulated Genes. The C-X-C motif chemokine 10 (CXCL10, also known as IP-10; interferon gamma-induced protein 10) was the most up-regulated gene in the dataset, with a 55.5-fold up-regulation in the sericin-supplemented group compared to the control ( Table 2). Complement component 3 (C3) was the second most up-regulated gene in the sericin group, with a 34.7-fold up-regulation. The chemokine (C-C motif) ligand 2 (CCL2) was up-regulated 21.6-fold in the sericin-supplemented group ( Table 2). TNFAIP3 (tumor necrosis factor, alpha-induced protein 3; A20), which is involved in NF-κ B regulation, was up-regulated 10.4-fold in the sericin group.
Downstream Effects. The results obtained from the Ingenuity Pathway Analysis (IPA) predicted a downstream effect compatible with increased cell viability and survival in the sericin-supplemented group (Fig. 2). In the cell viability category, 37 of 57 genes had an expression direction consistent with increased cell viability, yielding a Z-score of 2.8. In the cell survival category, 38 of 61 genes had an expression direction consistent with increased cell survival, yielding a Z-score of 2.7.
RPE Related Gene Transcripts. Pigmentation. The (IPA) detected 11 differentially expressed genes related to cell pigmentation ( Table 3). Ten of the genes were up-regulated and one was down-regulated in hRPE cells cultured in DMEM with sericin compared to cells cultured in DMEM without sericin.
Visual Cycle. Several visual cycle genes were significantly up-regulated in hRPE cells cultured in DMEM with sericin compared to that of cells cultured in DMEM without sericin, while no visual cycle genes were significantly down-regulated (Table 4). Compared to cells cultured in DMEM without sericin, the cells that were cultured in DMEM with sericin displayed a 2.8-fold up-regulation of RPE65, a key isomerase of the visual cycle and an essential marker of RPE cell maturation 22,23 , and a 1.4-fold up-regulation of cellular retinaldehyde binding protein 1 (RLBP1; also known as CRALBP), which positions retinol for enzymatic turnover and thereby accelerates the process 24 . Both retinol dehydrogenase 10 (RDH10) and retinol dehydrogenase 11 (RDH11) play complementary roles as 11-cis-retinol dehydrogenases in the visual cycle [25][26][27] , and were up-regulated 1.9 and 1.6-fold, respectively, in cells cultured in DMEM with sericin compared to cells cultured in DMEM without sericin.

RT-PCR Validation.
To validate the microarray results, NFKBIA, RPE65, CSF1, NFKBIZ and RGR transcripts were quantified by RT-PCR in hRPE cells cultured in DMEM with or without 1% sericin for a total of 14 days. In Table 5, ΔΔCt values are transformed to fold change. All six transcripts were up-regulated in the Affymetrix and RT-PCR experiments (   Fig. 3f). Typical cobblestone morphology with essentially hexagonal cells was seen in the samples cultured in DMEM with sericin ( Fig. 3b), MEM-α with sericin ( Fig. 3e), MEM-α with FBS (Fig. 3d), and MEM-α alone (Fig. 3f). Cell confluence was superior in DMEM with 1% sericin (Fig. 3b) compared to DMEM alone (Fig. 3c) and DMEM with 1% FBS (Fig. 3a). Hence, these results show that after 12 days in culture, only cells cultured in media supplied with sericin become pigmented and sericin appears to support hRPE cell confluence.
Ultrastructure Following hRPE Culture With or Without Sericin. Scanning electron microscopy of hRPE cultured for 12 days in DMEM with 1% sericin appeared tightly adjoined, hexagonal and had apical microvilli (Fig. 3g), hence demonstrating a differentiated ultrastructure. Transmission electron microscopy of hRPE cultured for 12 days in DMEM with 1% sericin contained melanosomes of all four stages following seven days of culture (Fig. 3h), thereby supporting the validity of the light microscopy experiments.
Melanin Quantification Following hRPE Culture With or Without Sericin. As the pigment melanin absorbs light at a specific wave length, measurement of pigment quantity is commonly performed by spectrophotometry 28 . Following 12-day culture, spectrophotometry showed increased absorption at 562 nm in cells cultured in DMEM with 1% sericin (39 ± 6 melanin/protein; P < 0.001) compared to cells cultured in DMEM without sericin (Fig. 4). Thus, the results further supports increased pigmentation in sericin-cultured cells.

Protein Levels of CRALBP and RPE65 Following hRPE Culture With or Without Sericin. To ver-
ify the presence and to assess the quantity of RPE-related proteins quantitative immunofluorescence and western blot experiments were used to analyze the mean levels of CRALBP and RPE65. With quantitative immunofluorescence, CRALBP was most abundant in cells cultured for 14 days in DMEM with sericin, and present to a significantly higher degree than in cells cultured in DMEM alone, DMEM with FBS or DMEM with FBS and sericin (P < 0.01) (Fig. 5). Retinal pigment epithelial cells cultured in DMEM with sericin and FBS also demonstrated a significantly higher expression of CRALBP compared to the DMEM alone and DMEM with FBS groups (P < 0.01). Thus, quantitative immunofluorescence confirmed increased levels of CRALBP, as indicated by the microarray results (Table 4). On western blot experiments, however, the level of CRALBP protein in cells cultured in DMEM with sericin (87% ± 21%) relative to that in cells cultured in DMEM alone (100%; P = 0.38) was similar. The level of RPE65 protein, on the other hand, was significantly higher in the DMEM with sericin group (168% ± 11%) compared to in DMEM alone (100%; P = 0.008), as measured by western blot. Therefore, sericin increases RPE65 protein in cultured primary hRPE.
hRPE Cell Density and Cell Death Following Serum Starvation. As the microarray results predicted increased viability of culturing cells in DMEM with sericin compared to DMEM without sericin, cell density and  cell death after 12 days of cultivation with or without sericin was quantified by counting DAPI-stained cell nuclei and by microplate fluorometer measurements of EH-1, the latter which is indicative of dead cells. DMEM with sericin yielded the highest cell density and significantly higher density than DMEM alone (P = 0.006), DMEM with FBS (P < 0.001) and DMEM with sericin and FBS (P = 0.007) (Fig. 6a). Cultured hRPE were assayed with EH-1 to quantify the number of dead cells following 14 days of culture. The highest number of dead cells was obtained when using DMEM alone (Fig. 6b). Cells cultured in this medium   demonstrated a significantly higher amount of dead cells than when using DMEM with sericin or DMEM with FBS (P < 0.001). Thus, the cell density and cell death results supported the microarray prediction of increased viability when culturing hRPE in DMEM with sericin.

NF-κB Pathway and Melanization of Cultured hRPE Cells.
Since IPA identified both the NF-κ B complex, and TNF, a well known activator of NF-κ B 29 , as top upstream regulators, we focused on the role of NF-κ B signaling in melanization. The NF-κ B-inhibitor JSH-23 specifically inhibits nuclear translocation and activation of NF-κ B 30 . The addition of JSH-23 to a culture medium consisting of DMEM with 1% sericin prevented development of pigmented cells as demonstrated by light microscopy and spectrophotometry following seven days of culture, as opposed to control cells cultured in DMEM with sericin, but without JSH-23 (Figs 4 and 7a,b) (N = 3). However, stimulation of the NF-κ B pathway by adding PMA, a NF-κ B agonist, to a culture medium consisting of DMEM without sericin did not induce melanization (Figs 4 and 7c). Thus, NF-κ B activation appears to be necessary, but not sufficient, for sericin-induced melanization of hRPE.

Effect of Sericin on the Main and Alternate NF-κB Pathways.
To verify the microarray data, which indicated that sericin induces activation of the NF-κ B pathway, we next investigated sericin's effect on activation of the main (phospho-NF-κ B p65) and alternate (NF-κ B p52) pathways. Following 12 days, primary hRPE cells cultured in DMEM with 1% sericin exhibited a higher level of phospho-NF-κ B p65 divided by total protein (7.6 ± 0.7) compared to cells cultured in DMEM alone (5.9 ± 0.1; P = 0.01). To investigate if FBS inhibits sericin's stimulatory effect on phospho-NF-κ B p65, we next compared cells cultured in DMEM with 1% sericin with that of cells cultured in DMEM with 1% sericin and FBS. The addition of FBS caused an even higher level of phospho-NF-κ B p65 (12.4 ± 1.7; P = 0.01) compared to the DMEM with 1% sericin group.
Regarding the alternate pathway, the mean level of NF-κ B p52 protein in cells grown in DMEM with 1% sericin (2.0 ± 0.6; P = 0.12) relative to that in cells grown in DMEM alone was approximately two-fold higher, but the potential difference between the groups did not reach significance (N = 3) (Fig. 8). Cells cultured in DMEM with 1% sericin and FBS (2.7 ± 0.6; P = 0.04) demonstrated a more than two-fold increase in the level of NF-κ B p52 protein relative to cells grown in DMEM alone (Fig. 8). Collectively, these data verify that sericin activates the main NF-κ B pathway and that FBS do not inhibit sericin's activation.

Discussion
In the current study, sericin induced pigmentation of cultured hRPE by NF-κ B pathway activation while still preserving cell viability and improving RPE maturation, as indicated by pathway analysis of Affymetrix microarray, micro and ultra-structural studies, spectrophotometry, western blot, ELISA, and viability assays.
The NF-κ B pathway was, alongside TNF, IFN-γ and IL1ß, one of the top upstream sericin-induced regulators in this study. The NF-κ B pathway is involved in multiple cellular processes, including inflammation     and immunity. NF-κ B is also part of the TNF pathway and is regulated by IL1ß. A link between inflammatory cytokines, including IL1ß and TNF-α , and induction of pigmentation in chick RPE cells has been reported 31 . Interestingly, IFN-γ activation has been related to hypopigmentation in skin melanocytes 32 . In our study, sericin activated the main NF-κ B pathway. Furthermore, the addition of the NF-κ B activator inhibitor JSH-23 prevented pigmentation in sericin-cultured cells and thereby confirmed the role of NF-κ B in pigmentation of hRPE, whereas the relatively scarce pigmentation achieved with the NF-κ B agonist PMA alone suggested that sericin promotes pigmentation, albeit not exclusively, by activating the NF-κ B pathway. Ingenuity pathway analysis identified several genes that are potentially linked to sericin-induced pigmentation, including PROM1, C10orf11, SLC24A5, TGM2 and IRF1, which were all up-regulated by sericin. PROM1 is also related to maculopathy, as mutations in this gene may cause macular degeneration, including dominant bull's eye maculopathy 33,34 . Mutations in C10orf11 have been shown to decrease pigmentation of melanocytes and lead to human albinism 35 . Interestingly, down-regulation of SLC24A5 causes reduced melanin content in chick RPE 36 . Furthermore, SLC24A5 has also been related to melanin content in skin melanocytes and the gene product of SLC24A5 localizes to intracellular membranes, including melanosomes 37 . Inhibition of TGM2 has been reported to suppress melanogenesis in human melanoma cells 38 . Surprisingly, up-regulation of IRF1 has been associated with hypo-pigmentation in skin melanocytes 39 .
Previous reports have demonstrated that sericin inhibits tyrosinase, which is the main rate-limiting melanogenesis enzyme 17 , thus the induction of pigmentation by sericin in our study is unexpected. Tyrosinase catalyses the formation of dihydroxyphenylalanine (L-DOPA) from L-tyrosine 40 . L-DOPA is subsequently converted to melanin, aided by tyrosinase-related proteins 1 and 2 (TRP-1 and TRP-2) 41 . Tyrosinase is inhibited by acidic conditions, including those resulting from high metabolic activity 42 . The microarray data did not reveal any significant effect of sericin on the tyrosinase transcript TYR, or on TRP-1 and TRP-2 encoding genes, (TYRP1 and TYRP2, respectively), thereby suggesting that sericin's effect on pigmentation is unrelated to regulation of these genes. The production of melanin is a complex process, however, involving several steps where sericin could have a stimulating role that fully compensates for its acclaimed tyrosinase-inhibiting effect.
Pigmentation was almost exclusively seen in cells that had been cultured in sericin-supplemented basal media (either MEM-α or DMEM). Hexagonal cobblestone morphology was also achieved when using either MEM-α or DMEM supplemented with sericin. After culturing the cell line ARPE-19 for 98 days in DMEM with FBS, Ahmado and co-workers demonstrated pigmented and hexagonal cells 43 . To our knowledge, this medium has not been used for normal RPE. The MEM-α -based medium, however, has been shown to induce pigmentation in normal hRPE cells in study by Sonoda et al. 44 . In contrast to our study, the hRPE in their study was pigmented upon start of culture and, after initial depigmentation, became repigmented after 14 days. Both MEM-α and DMEM supplemented with FBS are known to induce maturation of RPE cells in prolonged culture (> 3 weeks 44 / months 43 ). Our culture time with sericin of seven to 14 days may, therefore, have been too short time for widespread melanogenesis to occur in these media.
In RPE, pigment can be seen within vesicles called melanosomes, which can be divided into four stages of development based on ultrastructure 45 . While stage I and II melanosomes are amelanotic, stage III and IV melanosomes are partially, and fully, pigmented, respectively. hRPE cultured in DMEM containing sericin displayed melanosomes of all four stages following 14 days of culture, which suggests that the process of melanogenesis was ongoing.
The tight junction barrier is involved in creating a polarized epithelium and is necessary for maintaining an apical-basal concentration gradient across the RPE 44 . Thus, as shown by TEM in the current study, sericin enabled the development of a polarized RPE, as indicated by presence of apical tight junctions and basal cell nuclei.
TNFAIP3, CXCL10, C3 and CCL2 were among the top five sericin-induced genes in this study. TNFAIP3 was also the top up-regulated gene in the NF-κ B pathway. It is anti-inflammatory and prevents NF-κ B and TNF-mediated apoptosis 46 . TNFAIP3 is induced by TNF and inhibits the NF-κ B pathway by de-ubiquitination and ubiquitination of the TNF receptor-interacting protein 47 . Zinc supplementation in human AMD patients, which up-regulates TNFAIP3 48 , has been shown to be associated with decreased risk of developing advanced AMD or neo-vascular AMD during a 10-year follow-up 49 . In rats, TNFAIP3 (A20) was identified as a candidate gene for development of retinopathy 50 . The CXCL10 protein is a potent inhibitor of angiogenesis 51 and an antitumor agent causing tumor necrosis 52 . C3 is commonly found in drusen of AMD patients 53 , and the presence of C3 is critical for protection of the retina 54,55 . Absence of C3 expression has deleterious effects on the retinal structure and leads to progressive retinal degeneration 54,55 . C3 can also initiate angiogenesis, thereby opposing the anti-angiogenic effect of CXCL10. CCL2 contributes in maintaining normal RPE morphology, and lack of the gene leads to RPE cell loss and stress 56 .
Inclusion of sericin in the culture medium leads to up-regulation of several genes related to the visual cycle, including RPE65, RDH10 and CRALBP. Retinal diseases can result from mutations or malfunction of key proteins in the visual cycle, in which the RPE serves as a critical component 24 . The RDH10 is essential for synthesis of embryonic retinoic acid and therefore for limb, craniofacial and organ development 57 . RPE65 is one of the key markers of RPE cells and is responsible for light-independent conversion of all-trans-retinyl esters into 11-cis-retinol 58 . The data for CRALBP, a marker of hRPE maturation that is involved in retinol recycling 43,59 , were inconclusive with respect to whether this protein was significantly changed by addition of sericin. RPE65, however, was upregulated by sericin, as indicated by microarray profiling, RT-PCR and western blot. Thus, sericin affects at least some of the key markers of the visual cycle in cultured primary hRPE. Viability analyses were performed to investigate whether the sericin-induced up-regulation of several inflammatory cytokines was accompanied with increased cell death. To reduce the effect of cell proliferation on cell density, the measurements of cell density were performed after 12 days of post-confluent culture with or without sericin or FBS. Sericin appeared to preserve cell density under serum-free conditions, and resulted in higher cell density than when adding either FBS or a combination of FBS and sericin to DMEM. Corroborating experiments with ETH-1 demonstrated that sericin promotes cell survival, which is in line with the down-stream prediction made by IPA. Our results are further supported by a study demonstrating that sericin protects against cell death following acute serum-deprivation 60 and studies showing that FBS can be replaced by sericin in cryopreservation media without compromising viability 19,61 . The increased survival of hRPE upon stimulation with sericin may be related to up-regulation of TNFAIP3 (A20), which inhibits TNF-induced apoptosis, or up-regulation of anti-oxidant genes, including the pigmentation-related gene SOD2. Downstream analysis by IPA also predicted a relationship between up-regulation of TNFAIP3 and SOD2 and increased cell viability and cell survival. In addition, a direct reactive oxygen species-scavenging effect of sericin has been reported elsewhere 62 . In RPE, inflammatory cytokines, such as IFN-γ and TNF-α , have been shown to induce SOD2, which promotes cell survival in the presence of oxidative stress 63 . Thus, even though sericin promoted augmented expression of the inflammatory NF-κ B pathway, cell survival was increased, possibly by the concomitant up-regulation of anti-apoptotic and anti-oxidant genes.
In conclusion, sericin promotes pigmentation of cultured hRPE by activating the main NF-κ B pathway. Sericin's potential role in culture protocols for rapid differentiation of RPE cells derived from embryonic or induced pluripotent stem cells should be investigated.
Cell Culture. Third passage hRPE were seeded (7000 cells/cm 2 ) in complete EpiCM on Nunclon Δ surface plates and cultured under routine conditions of 95% air and 5% CO 2 at 37 °C. A confluent layer was obtained after two days, at which time EpiCM was replaced with either: 1) DMEM with 1% FBS; 2) DMEM with 1% sericin; 3) DMEM without FBS or sericin; 4) MEM-α with 1% FBS; 5) MEM-α with 1% sericin; or 6) MEM-α without FBS or sericin. All culture media based on DMEM were supplied with 10.000 U penicillin and 10 mg streptymocin at a final concentration of 1% 43 . The MEM-α -based media were added taurine, triiodo-thyronine, non-essential amino acids, glutamine-penicillin-streptomycin, hydrocortisone, and N1 medium supplement, as described elsewhere 44 . The culture medium was changed every two to three days, and the hRPE were maintained in culture for  Light Microscopy. Cell morphology and presence of pigment was assessed after 14 days of culture by light microscopy at 200× magnification (N = 8). Photomicrographs were captured using a Leica DM IL LED microscope and Canon EOS 5D mark II camera or with a Nikon Eclipse microscope with a DS-Qi1 black-and-white camera.
Transmission Electron Microscopy. hRPE cells cultured for 12 days in DMEM with 1% sericin were processed for transmission electronmicroscopy (TEM) analysis as previously described 64 . In brief, ultrathin sections (60e70 nm thick) were cut on a Leica Ultracut Ultramicrotome (Leica, Wetzlar, Germany) and examined using a CM120 transmission electron microscope (Philips, Amsterdam, the Netherlands) (N = 4).
Scanning Electron Microscopy. hRPE cells cultured on glass coverslips in DMEM with 1% sericin for 12 days were used for scanning electron microscopy (SEM) as described previously 65 . Glutaraldehyde-fixed samples (N = 3) were dehydrated in increasing ethanol concentrations and then dried according to the critical point method (Polaron E3100 Critical Point Drier, Polaron Equipment Ltd., Watford, UK) with CO 2 as the transitional fluid. The specimens were attached to carbon stubs and coated with a 30-nm thick layer of platinum in a Polaron  Sunnyvale, CA). Soluene ® -350 was thereafter added to the remaining cell lysate (9:1). The cell lysate was subsequently incubated for 60 minutes at 80 °C, before being centrifuged for 10 minutes at 8600 g. Solubilized melanin was measured at 490 nm on the microplate reader, and the concentration was adjusted by multiplication with protein levels (N = 5).
Quantitative Immunofluorescence. Following 14 days of culture the cells were fixed in 100% methanol for 15 minutes and then washed three times with fresh PBS (N = 4-8). Fixed cells were incubated for 45 minutes at room temperature in a blocking buffer consisting of 10% goat serum, 1% BSA, 0.1% Triton X-100, 0.05% Tween-20, 0.05% sodium azide in PBS. Cells were then incubated overnight at 4 °C with the following antibody diluted in blocking buffer: anti-CRALBP (1:100), which targets a functional protein in the visual cycle and a marker for differentiated hRPE. The Cy3-conjugated secondary antibody, diluted in 0.2% PBST with 1% BSA, were incubated for one hour at room temperature. Negative control consisted of replacing the primary antibody with PBS. The cultures were thereafter rinsed three times in PBS and incubated with 1μg/mL DAPI in PBS to stain cell nuclei before a final wash with PBS. Photomicrographs of the cultures were captured at 200× magnification using a Nikon Eclipse Ti fluorescence microscope with a DS-Qi1 black-and-white camera. The exposure length and gain was maintained at a constant level for all samples, and the fluorescence intensities of the Cy3 fluorochromes, which were conjugated to the secondary antibodies, were within the dynamic range of the camera.
Phenotype was quantified using ImageJ (National Institutes of Health, Bethesda, MD) as described elsewhere 66,67 , with some modifications. In brief, mean fluorescence per cell was measured by enlarging regions of interest (ROI) created around the DAPI-stained nuclei to enclose the CRALBP-expressing cytosol. By using this method, we were able to normalize for differences in cell density in each photomicrograph.
Quantification of Cell Density. After 14 days in culture, the hRPE (N = 4-8) were fixed with methanol, as described above. The cultures were then rinsed three times in PBS and incubated with 1 μg/mL DAPI in PBS to stain cell nuclei before a final wash with PBS. Photomicrographs of the cultures were captured at 200× magnification with identical exposure length and gain. ImageJ was used to convert 16-bit images to binary images, as reported elsewhere 66 . The "Analyze particles" function in ImageJ was then used to automatically count cell nuclei per image.
Quantification of Cell Death. The amount of dead cells in the cultures after 14 days was quantified by incubating the samples with EH-1 for 30 minutes at 37 °C (N = 8). Ethidium homodimer-1 stains nuclei of dead cells red and its fluorescence was quantified by the microplate fluorometer with the excitation/emission filter pair 530/620. Background fluorescence, measured in wells incubated with EH-1-reagent, but without cells, was subtracted from all values before calculating mean fluorescence for the groups.
Western Blot. Protein levels were measured by Western blot analysis of primary hRPE cultured for 12 days.
In brief, cells were lysed in RIPA-buffer and proteins analyzed by SDS-PAGE followed by electroblotting onto PVDF membranes.
ELISA. Level of phospho-NF-κ B p65 was measured by enzyme-linked immunosorbent assay (ELISA) in primary hRPE cultured for 12 days according to the manufacturers instructions (RayBio ® Phospho-NF-κ B p65 (S536) and Total NF-κ B p65 ELISA kit). Briefly, samples were incubated with anti-phospho-NF-κ B p65 for 2.5 hours at ambient temperature, washed, incubated with primary antibody for 1 hour, washed and then incubated with HRP-conjugated anti-rabbit IgG. Following washing, the addition of TMB substrate solution and Stop Solution, the color changes were measured at 450 nm.
Statistical Analysis. One-way ANOVA with Tukey's (equal variances assumed) or Dunnett's T3 (equal variances not assumed) post hoc pair-wise comparisons were used to compare three or more groups, while Student's t-test was used to compare two groups (SPSS ver. 21.0). Data were expressed as mean ± standard deviation, and values were considered significant if P < 0.05.