We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.
Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.
The model described here, offers a useful approach to evaluate the efficiency ofbiopharmaceutical agents for reducing oxidative stress in cells. We have used the model to investigate the protective effects of PEDF and GM-CSF on the H2O2-mediated oxidative stress on retinal pigment epithelial cells, which are exposed to high levels of O2, and visible light, and the phagocytosis of photoreceptor outer segment membranes, generating significant levels of reactive oxygen species (ROS)1,2. They are considered a major contributor to the pathogenesis of avascular age-related macular degeneration (aAMD)3,4,5,6,7,8. Besides, there is a decrease in RPE-synthesized neuroprotective factors, specifically the pigment epithelium-derived factor (PEDF), insulin-like growth factors (IGFs), and granulocyte macrophage-colony-stimulating factor (GM-CSF) leading to the dysfunction and loss of RPE cells, followed by photoreceptor and retinal ganglion cell (RGC) death3,4,5. AMD is a complex disease that results from the interaction between metabolic, functional, genetic, and environmental factors4. The lack of treatments for aAMD is the major cause of blindness in patients older than 60 years of age in industrialized countries9,10. The reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of genetically modified RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation and supporting cell survival11,12,13,14,15,16. Even though there are several methodologies to deliver genes to cells, we have chosen the non-viral hyperactive Sleeping Beauty transposon system to deliver the PEDF and GM-CSF genes to RPE cells because of its safety profile, the integration of the genes into the host cells' genome, and its propensity to integrate the delivered genes in non-transcriptionally active sites as we have shown previously17,18,19.
Cellular oxidative stress can be induced in cells cultured in vitro by several oxidative agents, including hydrogen peroxide (H2O2), 4-hydroynonenal (HNE), tertbutylhydroperoxide (tBH), high oxygen tensions, and visible light (full spectrum or UV irradiation)20,21. High oxygen tensions and light require special equipment and conditions, which limits transferability to other systems. Agents such as H2O2, HNE, and tBH induce overlapping oxidative stress molecular and cellular changes. We chose H2O2 to test the antioxidant activity of PEDF and GM-CSF because it is convenient and biologically relevant since it is produced by RPE cells as a reactive oxygen intermediate during photoreceptor outer segment phagocytosis22 and it is found in ocular tissues in vivo23. Since the oxidation of glutathione may be partially responsible for the production of H2O2 in the eye, we have analyzed the levels of GSH/glutathione in our studies, which are linked to H2O2-induced oxidative stress and the regenerative capacity of cells21,22. The analysis of glutathione levels is especially relevant since it participates in the anti-oxidative protective mechanisms in the eye24. Exposure to H2O2 is used frequently as a model to examine the oxidative stress susceptibility and antioxidant activity of RPE cells1,25,26,27,28,29,30, and, additionally, it shows similarities to light-induced oxidative stress damage, a "physiological" source of oxidative stress21.
To evaluate the functionality and the effectiveness of neuroprotective factors, we have developed an in vitro model that allows for the analysis to quantify the anti-oxidative effect of growth factors expressed by cells genetically modified to overexpress PEDF and GM-CSF. Here, we show that RPE cells transfected with the genes for PEDF and GM-CSF are more resistant to the harmful effects of H2O2 than are non-transfected control cells, as evidenced by cell morphology, density, viability, intracellular level of glutathione, and expression of UCP2 gene, which codes for the mitochondrial uncoupling protein 2 that has been shown to reduce reactive oxygen species (ROS)31.
Procedures for the collection and use of human eyes were approved by the Cantonal Ethical Commission for Research (no. 2016-01726).
1. Cell isolation and culture conditions
Medium (mL) | ||||||
Area (cm²) | Seeding density for ARPE-19 cells (cells/well) | Application | For cell culture | To stop trypsin | Volume of trypsin (mL) | |
Flask T75 | 75 | 5,00,000 | ARPE-19 cell growth | 10 | 7 | 3 |
6 Well plate | 9.6 | 1,00,000 | Seeding of transfected ARPE-19 cells | 3 | 1 | 0.5 |
24 Well plate | 2 | 50,000 | Seeding of transfected hRPE cells | 1 | 0.8 | 0.2 |
96 Well plate | 0.32 | 5,000 for oxidative stress experiments with transfected cells (Fig. 1) | Oxidative stress experiments | 0.2 | ||
3,000 for oxidative stress experiments with non-transfected cells plus proteins (Fig. 1) |
Table 1: Cell culture volumes. Recommended media volumes for cell culture plates and flasks for the culture of ARPE-19 and primary human RPE cells.
No | age | gender | death to preservation (hours) | death to isolation | cultivation | cultivation | Symbol in graph | |
(days) | before transfection (days) | after transfection (days) | ||||||
2 | 80 | M | 20.7 | 8 | 140 | 36 | ![]() |
|
3 | 86 | F | 12.8 | 8 | 85 | 45 | ![]() |
|
4 | 86 | F | 8.5 | 5 | 26 | 133 | ![]() |
|
8 | 83 | F | 8.9 | 6 | 18 | 27 | ![]() |
|
mean | 83.8 | 12.7 | 6.8 | 67.3 | 60.3 | |||
SD | 2.9 | 5.7 | 1.5 | 57.0 | 49.1 |
Table 2: Demographics of human donors for retinal pigment epithelial cells.
2. Electroporation of ARPE-19 and primary human RPE cells
3. Oxidative stress induction (H2O2 treatment) and neuroprotection (PEDF and/or GM-CSF treatment)
Figure 1: Timelines of the H2O2 assay in the three different experimental approaches. 3,000 non-transfected cells treated with the conditioned medium/recombinant proteins or 5,000 transfected cells were seeded in 96-well plates for treatment with H2O2. To determine the effect of conditioned medium, cells were cultured in 100% cultured medium for 10 consecutive days, changing medium every day. To determine the effect of recombinant growth factors, cells were cultured by adding the appropriate amount of growth factors each day for 3 consecutive days. Note that non-transfected cells were seeded at 3,000 cells per well to avoid overgrowth during the longer culture duration compared to transfected cells. Please click here to view a larger version of this figure.
4. Analysis of oxidative stress level and antioxidant capacity
Induction of oxidative stress in human Retinal Pigment Epithelial cells
ARPE-19 and primary hRPE cells were treated with varying concentrations of H2O2 for 24 h and the intracellular level of the antioxidant glutathione was quantified (Figure 2A,B). H2O2 at 50 µM and 100 µM did not affect glutathione production, whereas at 350 µM there was a significant decrease of glutathione in ARPE-19 and primary hRPE cells. Analysis of cytotoxicity showed that 350 µM is the lowest concentration of H2O2 that causes a significant decrease in cell viability (Figure 2C). Morphologically, ARPE-19 cells treated with H2O2 appear less spread and more rounded, characteristics that become more obvious with increasing H2O2 concentration (Figure 3). The effect was less prominent for PEDF- and GM-CSF-transfected cells treated with H2O2 (Figure 3). To demonstrate the effect of cell number on H2O2-mediated oxidative stress, 5,000 and 10,000 ARPE-19 cells per well were seeded in a white 96-well plate; the day after, cells were treated with 350 µM H2O2 for 24 h and the levels of glutathione were determined. Figure 4 shows that the level of glutathione was decreased only in the wells (n = 3) seeded with 5,000 cells. For experiments to determine the effect of antioxidants of H2O2-generated ROS, it is essential to consider the number of cells; for the specific protocol presented in this report 3,000-5,000 cells/well (96-well plates) treated for 24 h with 350 µM H2O2 are appropriate to show significant cell damage while retaining the capacity to recover mimicking a sub-acute response to oxidative stress-induced cell damage.
Figure 2: Oxidative stress level evidenced as glutathione level and cell viability, in human RPE cells treated with H2O2. (A) ARPE-19 cells exposed to several concentrations of H2O2 showed significantly decreased glutathione levels (in brackets) at 350 µM (0.66 µM), 500 µM (0.022 µM), and 700 µM (0.002 µM) compared to H2O2-non-treated cells (2.9 µM) (p < 0.0001 for 350, 500, and 700 µM H2O2). (B) Primary human RPE cells showed decreased levels of glutathione; however, the effect was less prominent than for ARPE-19 but still statistically significant compared to the controls at 350, 500, and 700 µM H2O2. 350 µM was the lowest H2O2 concentration that produced significant oxidative damage as shown by decreased glutathione levels compared with non-treated control cells (p = 0.0022). Glutathione levels decreased with increasing H2O2 concentrations (500 µM: p = 0.022; 700 µM: p = 0.0005). (C) Cytotoxicity analysis showed that 350 µM H2O2 was the lowest concentration that produced a significant decrease in the percentage of viable cells (p < 0.0001 for 350, 500, and 700 µM). Data is presented as mean ± SD (n = 3 replicates) and significant differences are indicated with (*); post-hoc calculations of the ANOVA were performed using Tukey's multi-comparison test comparing C- with the H2O2-treatment groups. C-: H2O2 non-treated cells. This figure has been modified from Bascuas et al.37. Please click here to view a larger version of this figure.
Figure 3: Morphology of non-transfected and PEDF- or GM-CSF-transfected ARPE-19 cells treated with H2O2. Cells treated with increasing concentrations of H2O2 show fewer cells in the culture wells and display a more rounded, less spread morphology, a known sign of cellular stress. Note that for PEDF- or GM-CSF-transfected cells, cellular stress is less prominent and grow similar to non-treated control cells. C-: non-treated control cells. Please click here to view a larger version of this figure.
Figure 4: Influence of cell number on the effect of H2O2-induced oxidative stress. 5,000 and 10,000 ARPE-19 cells/well were seeded in 96-well plates. After 24 h, cells were treated with 350 µM H2O2 for 24 h. Significant differences in glutathione levels were observed in the wells seeded with 5,000 cells (p = 0.031, t-test) but not in the wells seeded with 10,000 cells. C-: non-treated cells. Please click here to view a larger version of this figure.
Analysis of the antioxidant effect of PEDF and GM-CSF delivered by SB100X-transfected human RPE cells in oxidative stress conditions
As positive controls, ARPE-19 and primary human RPE cells were treated with 5, 50, or 500 ng/mL commercially available PEDF or GM-CSF for 2 days before and during the 24 h H2O2 treatment. ARPE-19 cells treated with 500 ng/mL PEDF or 50 ng/mL GM-CSF produced significantly more glutathione compared to untreated controls under oxidative conditions (H2O2-treated) (Figure 5A); comparable PEDF and GM-CSF purified from culture media of transfected ARPE-19 cells showed a similar effect (Figure 5B). In primary hRPE cells, the addition of 500 ng/mL PEDF, 50 ng/mL GM-CSF, or 500 ng/mL PEDF plus 50 ng/mL GM-CSF whether commercial or purified from media conditioned by PEDF- or GM-CSF transfected ARPE-19 cells reduced cell damage as reflected by a significant increase in glutathione levels (Figure 5C). Primary hRPE cells treated for 10 days with conditioned medium from transfected ARPE-19 cells also showed higher glutathione levels compared to control cells (Figure 5D). Based on these results, further experiments have been done with 500 ng/mL for PEDF and 50 ng/mL for GM-CSF.
ARPE-19 and primary hRPE cells were transfected with the genes coding for PEDF and/or GM-CSF using the Sleeping Beauty transposon system combined with electroporation. Following transfection and analysis of gene expression by RT-qPCR, WB, ELISA, and immunohistochemistry (see Supplementary Material, Figure S1, and Figure S2), transfected ARPE-19 cells exposed to 350 µM H2O2 for 24 h showed significant higher glutathione levels than non-transfected H2O2-treated cells (Figure 6A). For primary hRPE cells, there is a significant increase in glutathione levels in PEDF-transfected cells compared with non-transfected cells treated with H2O2 when all donors were included in the analysis. Moreover, donors 2 and 3 show a significant increase in glutathione levels for all transfected groups (PEDF, GM-CSF, PEDF, and GM-CSF) (data not shown).
The study of the UCP2 gene expression completed the analysis by examination of mitochondrial oxidative stress. A proof-of-concept series was carried out in transfected ARPE-19 cells treated with 350 µM H2O2 for 24 h. As shown in Figure 7, in transfected ARPE-19 cells, the levels of UCP2 gene expression after H2O2 treatment are increased but the increase is not statistically significant. Figure 8 shows a WB of phosphorylated Akt (pAkt) from a lysate of GM-CSF-transfected cells exposed to H2O2; the normalized data shows only a small decrease compared with the untreated control, indicating that GM-CSF can protect the cells from oxidative stress damage.
Figure 5: Glutathione level as a marker of the antioxidant capacity of PEDF and GM-CSF. (A) Treatment of ARPE-19 cells with 500 ng/mL PEDF or 50 ng/mL GM-CSF for 3 days before and during 24 h H2O2 exposure increased the level of glutathione from 0.83 µM (C) to 1.83 µM (PEDF) and 1.3 µM (GM-CSF), p = 0.026 and p = 0.031, respectively. At a concentration of 5 ng/mL no increase in glutathione was observed; the difference in the level of glutathione between 50 and 500 ng/mL was not significant for either PEDF or GM-CSF. (B) PEDF (500 ng/mL) and GM-CSF (50 ng/mL) purified from conditioned media of transfected ARPE-19 cells showed an effect similar to commercially available PEDF or GM-CSF (p = 0.018, ANOVA). (C) The addition of 500 ng/mL PEDF, 50 ng/mL GM-CSF, or 500 ng/mL PEDF plus 50 ng/mL GM-CSF for 3 days before and during 24 h H2O2 treatment to the culture medium of primary hRPE cells significantly increased the levels of glutathione in cells treated with PEDF (2.6 µM [commercial], 2.5 µM [purified]), GM-CSF (2.9 µM [commercial], 3.3 µM [purified]), and PEDF plus GM-CSF (3.0 µM [commercial], 2.9 µM [purified]) compared to non-treated cells (1.9 µM) (p = 0.006, Kruskal-Wallis test). (D) A significant increase in glutathione levels was observed for hRPE cells cultured for 10 days in conditioned medium from PEDF-, GM-CSF-, or PEDF-GM-CSF-transfected ARPE-19 cells before the cells were treated with H2O2 (p = 0.003, Kruskal-Wallis test) (data showed for one donor). Data are expressed as mean ± SD (n = 3 replicates). Significant differences are indicated with (*); post-hoc calculations of the analyses of variance were performed by calculating Tukey's or Dunnett's multi-comparison tests comparing "C" with the PEDF-/GM-CSF-treated groups. C: cells treated only with H2O2, P: cells treated with PEDF, G: cells treated with GM-CSF, P+G: cells treated with PEDF plus GM-CSF. This figure has been modified from Bascuas et al.37. Please click here to view a larger version of this figure.
Figure 6: Glutathione level as a marker of the antioxidant capacity of PEDF- and GM-CSF-transfected human RPE cells. (A) The levels of glutathione of transfected ARPE-19 cells exposed to 350 µM H2O2 for 24 h (56 days post-transfection) were significantly higher compared to non-transfected cells (1.9 µM), i.e., 3.0 µM for PEDF- and GM-CSF-transfected cells, and 3.4 µM for double transfected cells (p = 0.0001, ANOVA). Data is expressed as mean ± SD (n = 3 replicates). (B) The dot plot shows the mean glutathione values for four different donors (C: 0.77 µM; P: 1.45 µM; G: 1.16 µM; P+G: 1.2 µM), which differs significantly between non-transfected and PEDF-transfected cells (p = 0.028, post-hoc calculations of the ANOVA were performed using Tukey's multi-comparison tests comparing "C" with the PEDF-/GM-CSF-treated groups). When the donors are analyzed separately, donor N°2 and N°3 (see Table 2 for symbol in the graph) show significant differences for all transfected groups compared to the non-transfected control (significances are not shown) treated with H2O2. C: non-transfected cells, P: PEDF-transfected cells, G: GM-CSF-transfected cells, P+G: PEDF- and GM-CSF-transfected cells. This figure has been modified from Bascuas et al.37. Please click here to view a larger version of this figure.
Figure 7: UCP2 gene expression in transfected ARPE-19 cells treated with H2O2. Since UCP2 gene expression can be used to examine mitochondrial oxidative damage, we examined the effect of the overexpression of PEDF and GM-CSF by transfected ARPE-19 cells. Transfected ARPE-19 cells treated with H2O2, even though not statistically significant, show increased UCP2 gene expression compared with the non-transfected control indicating oxidative stress reduction, the fold-increase was 1.57 for PEDF-, 1.51 for GM-CSF-, and 2.36 for PEDF- plus GM-CSF-transfected cells compared with the non-transfected control. Please click here to view a larger version of this figure.
Figure 8: Western Blot of phosphorylated Akt (Ser473) from a cell lysate of GM-CSF-transfected ARPE-19 cells. The WB demonstrated that GM-CSF enhances the phosphorylation of Akt in both, untreated and H2O2-treated cultures (UT: 3.32; H2O2: 2.69). The values are normalized to non-transfected non-H2O2-treated cells (C/UT). C: non-transfected, G: GM-CSF-transfected cells, UT: cells non-treated with H2O2, H2O2: cells treated with H2O2. Please click here to view a larger version of this figure.
Table S1: Primer pair sequences and annealing time/temperature used for RT-qPCR. Please click here to download this table.
Figure S1: PEDF and GM-CSF gene expression analysis in transfected hRPE cells. The RT-qPCR verified that transfected primary hRPE cells showed a significant increase in PEDF (p = 0.003, Kruskal-Wallis test) and GM-CSF (p = 0.013, Kruskal-Wallis test) gene expression compared with non-transfected cells. 2^(-ΔΔCT) method was used in this case36. Data is expressed as mean ± SD (n = 4 donors). Each dot represents the average of three replicates. This figure has been modified from Bascuas et al.37. Please click here to download this file.
Figure S2: Protein secretion in transfected primary hRPE and ARPE-19 cells. (A) The quantification of secreted proteins by ELISA showed that transfected hRPE cells secreted significantly more PEDF and GM-CSF than non-transfected cells (p = 0.014 for PEDF, and p = 0.006 for GM-CSF, Kruskal-Wallis test). Data is presented as mean ± SD (n = 4 donors). Each dot represents the average of three replicates. (B) The PEDF-GM-CSF double staining confirmed the co-secretion of PEDF and GM-CSF in double-transfected ARPE-19 cells (merged figure). This figure has been modified from Bascuas et al.37. Please click here to download this file.
Supplementary material. Please click here to download this file.
The protocol presented here offers an approach to analyze the anti-oxidative and protective function of PEDF and GM-CSF produced by transfected cells, which can be applied to cells transfected with any putative beneficial gene. In gene therapeutic strategies that have the objective to deliver proteins to tissue by transplanting genetically modified cells, it is critical to obtain information as to the level of protein expression, the longevity of expression, and the effectiveness of the expressed protein in a model of the disease. In our laboratory, the protocol presented here has been useful to define the effectiveness of PEDF and GM-CSF on oxidative stress, which has been hypothesized as an important element in the pathogenesis of aAMD6,7. Specifically, we have used the protocol to define the anti-oxidative effect of SB100X-mediated PEDF/GM-CSF-transfected primary hRPE cells. Several investigators have shown that H2O2 induces significant symptoms of oxidative stress but still allows cell regeneration28,29,38, similar to the results of our experiments that have shown that 350 µM for 24 h induces effective oxidative stress in human ARPE-19 and primary RPE cells that can be used to analyze the protective effect of the PEDF and GM-CSF. H2O2 as oxidative agent has been chosen for the study because of its physiological presence in the eye and corresponding defense mechanisms, e.g., glutathione metabolism20,21. Our laboratory has examined other models of oxidative stress such as treatment of cells with tBH, which initiates lipid peroxidation in the presence of redox-active metal ions1; however, oxidative stress was negligible. In the experiments presented here, cells were treated with H2O2 for 24 h because we found that shorter treatment times of 2-6 h is sufficient to induce changes in gene expression20, but subsequent consequences, e.g., cell proliferation, cell viability, and glutathione levels, might not be visible yet. Otherwise, the small size of the wells, necessary for the cytotoxicity and glutathione assays, rapidly leads to a confluent culture well; this might lead to contact inhibition and a masking of the effect of the oxidative agent. Therefore, a long incubation with H2O2 seems not useful, though the degeneration seen in aAMD is caused by chronic oxidative stress6,7.
A limitation of the experiments presented here is that the number of cells seeded influences the oxidative effect of H2O2, i.e., for the same H2O2 treatment, significant differences in the glutathione levels between H2O2-treated and non-treated cells were observed when 5,000 cells but not when 10,000 cells were seeded (Figure 4). The protocol we present requires seeding a low number of cells, i.e., 3,000 when cells are cultured for 3 days and 5,000 when cells are cultured for 2 days (Figure 1). Another limitation is that the concentration of H2O2 is depleted with time; Kaczara et al.39 have shown depletion of H2O2 over a few hours in ARPE-19 cell cultures, which affects the development of chronic oxidative stress models. These investigators have proposed an alternative method for sustained H2O2 treatment, specifically continuously generating H2O2 from glucose in the medium using the glucose oxidase, but a standardized concentration of H2O2 cannot be guaranteed. On the other hand, the protocol we established with delivery of the oxidant agent in one single pulse, has the advantage of being faster and simpler to perform compared with chronic models in which the H2O2 treatment has to be repeated for several days38.
The ability of cells to counteract the oxidative damage is determined by the balance between ROS production and the capacity to generate antioxidants. In the cell, the tripeptide glutathione (GSH) is the predominant reducing agent, which can be oxidized to glutathione disulfide (GSSG) and regenerated by glutathione reductase utilizing NADPH40. In healthy cells, more than 90% of the total glutathione pool is present in the reduced form. When cells are exposed to an increased level of oxidative stress, GSSG accumulates and the ratio of GSSG to GSH increases. Consequently, monitoring the glutathione redox state in biological samples is essential for the evaluation of the detoxification status of cells and tissues from free radicals generated during oxidative stress and cell injury. The protocol detailed here for the quantification of glutathione is sensitive enough to detect the antioxidant effect of PEDF and GM-CSF expressed by RPE cells genetically modified.
Since oxidative stress affects mitochondrial activities40, it is particularly interesting that the control of ROS levels by PEDF is related to the regulation of the mitochondrial uncoupling protein 2 (UCP2), and PEDF attenuates the effects of oxidative stress by increasing UCP2 expression11,41. The main function of UCP2 is controlling mitochondria-derived ROS and acting as a sensor of mitochondrial oxidative stress41,42. Here, in addition to examining the effect of PEDF and GM-CSF on glutathione levels, we have the gene expression of UCP2 tend to increase (Figure 7); additional studies are necessary to establish the role of PEDF and GM-CSF on UCP2 gene expression.
Overall, the present H2O2-model offers a comprehensive approach to investigate the beneficial effect of transposon-based gene therapies that aim to deliver antioxidant therapeutic genes to the patient's cells to treat neurodegenerative disease as AMD.
The authors have nothing to disclose.
The authors would like to thank Gregg Sealy and Alain Conti for excellent technical assistance and Prof. Zsuzsanna Izsvák from the Max-Delbrück Center in Berlin for kindly providing the pSB100X and pT2-CAGGS-Venus plasmids. This work was supported by the Swiss National Sciences Foundation and the European Commission in the context of the Seventh Framework Programme. Z.I was funded by European Research Council, ERC Advanced [ERC-2011-ADG 294742].
24-well plates | Corning | 353047 | |
6-well plates | Greiner | 7657160 | |
96-well culture plate white with clear flat bottom | Costar | 3610 | Allows to check the cells before measuring the luminescence (GSH-Glo Assay) |
96-well plates | Corning | 353072 | |
Acrylamid 40% | Biorad | 161-0144 | |
Amphotericin B | AMIMED | 4-05F00-H | |
Antibody anti-GMCSF | ThermoFisher Scientific | PA5-24184 | |
Antibody anti-mouse IgG/IgA/IgM | Agilent | P0260 | |
Antibody anti-PEDF | Santa Cruz Biotechnology Inc | sc-390172 | |
Antibody anti-penta-His | Qiagen | 34660 | |
Antibody anti-phospho-Akt | Cell Signaling Technology | 9271 | |
Antibody anti-rabbit IgG H&L-HRP | Abcam | ab6721 | |
Antibody donkey anti-rabbit Alexa Fluor 594 | ThermoFisher Scientific | A11034 | |
Antibody goat anti-mouse Alexa 488 | ThermoFisher Scientific | A-11029 | |
ARPE-19 cell line | ATCC | CRL-2302 | |
BSA | Sigma-Aldrich | A9418-500G | |
chamber culture glass slides | Corning | 354118 | |
CytoTox-Glo Cytotoxicity Assay | Promega | G9291 | |
DAPI | Sigma-Aldrich | D9542-5MG | |
DMEM/Ham`s F12 | Sigma-Aldrich | D8062 | |
Duo Set ELISA kit | R&D Systems | DY215-05 | |
EDTA | ThermoFisher Scientific | 78440 | |
ELISAquant kit | BioProducts MD | PED613-10-Human | |
Eyes (human) | Lions Gift of Sight Eye Bank (Saint Paul, MN) | ||
FBS | Brunschwig | P40-37500 | |
Fluoromount Aqueous Mounting Medium | Sigma-Aldrich | F4680-25ML | |
FLUOstar Omega plate reader | BMG Labtech | ||
GraphPad Prism software (version 8.0) | GraphPad Software, Inc. | ||
GSH-Glo Glutathione Assay | Promega | V6912 | |
hydrogen peroxide (H2O2) | Merck | 107209 | |
ImageJ software (image processing program) | W.S. Rasband, NIH, Bethesda, MD, USA; https://imagej.nih.gov/ij/; 1997–2014 | ||
Imidazol | Axonlab | A1378.0010 | |
Leica DMI4000B microscope | Leica Microsystems | ||
LightCycler 480 Instrument II | Roche Molecular Systems | ||
LightCycler 480 SW1.5.1 software | Roche Molecular Systems | ||
NaCl | Sigma-Aldrich | 71376-1000 | |
NaH2PO4 | Axonlab | 3468.1000 | |
Neon Transfection System | ThermoFisher Scientific | MPK5000 | |
Neon Transfection System 10 µL Kit | ThermoFisher Scientific | MPK1096 | |
Neubauer chamber | Marienfeld-superior | 640010 | |
Ni-NTA superflow | Qiagen | 30410 | |
Nitrocellulose | VWR | 732-3197 | |
Omega Lum G Gel Imaging System | Aplegen Life Science | ||
PBS 1X | Sigma-Aldrich | D8537 | |
Penicillin/Streptomycin | Sigma-Aldrich | P0781-100 | |
PerfeCTa SYBR Green FastMix | Quantabio | 95072-012 | |
PFA | Sigma-Aldrich | 158127-100G | |
Pierce BCA Protein Assay Kit | ThermoFisher Scientific | 23227 | |
Primers | Invitrogen | See Table 1 in Supplementary Materials | |
pSB100X (250 ng/µL) | Mátés et al., 2009. Provide by Prof. Zsuzsanna Izsvak | ||
pT2-CMV-GMCSF-His plasmid DNA (250 ng/µL) | Constructed using the existing pT2-CMV-PEDF-EGFP plasmid reported in Johnen, S. et al. (2012) IOVS, 53 (8), 4787-4796. | ||
pT2-CMV-PEDF-His plasmid DNA (250 ng/µL) | Constructed using the existing pT2-CMV-PEDF-EGFP plasmid reported in Johnen, S. et al. (2012) IOVS, 53 (8), 4787-4796. | ||
QIAamp DNA Mini Kit | QIAGEN | 51304 | |
recombinant hGM-CSF | Peprotech | 100-11 | |
recombinant hPEDF | BioProductsMD | 004-096 | |
ReliaPrep RNA Cell Miniprep System | Promega | Z6011 | |
RIPA buffer | ThermoFisher Scientific | 89901 | |
RNase-free DNase Set | QIAGEN | 79254 | |
RNeasy Mini Kit | QIAGEN | 74204 | |
SDS | Applichem | A2572 | |
Semi-dry transfer system for WB | Bio-Rad | ||
SuperMix qScript | Quantabio | 95048-025 | |
Tris-buffered saline (TBS) | ThermoFisher Scientific | 15504020 | |
Triton X-100 | AppliChem | A4975 | |
Trypsin/EDTA | Sigma-Aldrich | T4174 | |
Tween | AppliChem | A1390 | |
Urea | ThermoFisher Scientific | 29700 | |
WesternBright ECL HRP substrate | Advansta | K-12045-D50 | |
Whatman nitrocellulose membrane | Chemie Brunschwig | MNSC04530301 |