Fig 1.
H2O2 exposure increases autophagic flux and p62 expression in RPE cells.
(A) ARPE-19 cells were exposed to 400μM H2O2 and autophagy was monitored on western blots of protein lysates following treatment at 1, 3, 6, 12 and 24h by LC3 II/I conversion and p62/SQSTM1 levels using anti-LC3 and anti-p62/SQSTM1 antibodies respectively. β-actin was used as an internal control (upper panel). p62 levels were analyzed by densitometry as described in Experimental Procedures (lower panel). (B) ARPE-19 cells were exposed to 200 or 400μM H2O2 for 24h and p62 protein levels were monitored by Western blot and analyzed by densitometry. β-actin was used as an internal control. Control (Ctrl) is H2O2 (0μM) without Bafilomycin A1. ‘Short’ and ‘Long’ Exposure indicate time of exposure of immunoblot image. (C) Confocal images of control ARPE-19 cell cultures and ARPE-19 cells following 6h exposure to 400μM H2O2 labelled with anti-p62 (red) and nuclear-stained with DAPI (blue). (D) ARPE-19 cells were treated with 200 or 400μM H2O2 for 24h and mRNA extracted. p62 mRNA level was determined by qRT-PCR with p62 specific primers. (E) Primary human fetal RPE cells were exposed to H2O2 (400μM) and and p62/SQSTM1 levels were monitored by Western blot of protein lysates following treatment at 1, 3, 6, 12 and 24h using anti-p62 antibody. β-actin was used as an internal control (upper panel). Densitometric analyses were plotted in the lower panel. (F) Primary human fetal RPE cells were treated with H2O2 for 24h and then p62 mRNA level was examined by relative quantitative real-time qRT-PCR with p62 specific primers. Statistical significance between untreated control and H2O2-exposed cells was determined by Mann-Whitney U-test, p < 0.05.
Fig 2.
p62 expression is enhanced in AMD mice model.
APOE4 mice at the age of 18–20 months were fed a high fat, cholesterol enriched diet (HFC) for 2 months and control age-matched APOE4 mice were kept on a normal diet (ND), (A) Paraffin embedded mouse retina sections from each group (n = 6, disease and control groups) were incubated with monoclonal p62 antibody followed by secondary antibodies conjugated with AlexaFluor® 594 dye. Sections were covered with Vectashield mounting medium/DAPI. Photographs were taken by Zeiss AX10.1 Observer fluorescent microscope. (B) Protein lysates from RPE/Choroid layers (pooled left and right eye cup for each mouse) were analyzed by Western blot against p62 and LC3. 10 mice were used per group. β-actin was used as an internal control. Lane numbers denote the animal number in each group. Densitometric quantification of p62 band intensity was analyzed. Statistical significance between the normal diet (n = 10) and high-fat diet-fed groups (n = 10) was determined by ANOVA, p<0.05. (C) Protein lysates from the neural retina (pooled left and right retina for each mouse) were analyzed by Western blot against p62 and LC3. 10 mice were used per group. β-actin was used as an internal control. Densitometric quantification of p62, LC3 (II/I) ratio and total LC3 were plotted. Statistical significance between the normal diet (n = 10) and high-fat diet-fed groups (n = 10) was determined by ANOVA, p<0.05. (D) Real Time PCR analyses for Lc3 and p62 in mouse RPE/Choroid was performed with Gapdh as internal control.
Fig 3.
H2O2 inhibits proteasome activity and proteasome inhibitor lactacystin induces p62 up-regulation.
(A) ARPE-19 cells were treated with 200 or 400μM H2O2 and then chymotrypsin-like proteasome activity was measured at 24h post-exposure using a fluorogenic substrate. Control (Ctrl) = No H2O2 (B) Chymotrypsin-like proteasome activity was detected in RPE cells treated with the proteasome specific inhibitor lactacystin (4nM and 25μM, 12h). Ctrl = DMSO control (C) qRT-PCR was used to measure the p62 mRNA level following lactacystin treatment (25μM, 12h). Ctrl = DMSO control (D) p62 protein level was examined by western blot after proteasome specific inhibitor lactacystin (25μM, 12h) exposure. Ctrl = DMSO control. Data represent the mean + S.E.M. for three samples per group. Differences in means were considered statistically significant when p<0.05.
Fig 4.
p62 expression under oxidative stress is dependent on NFκB p65 Ser536 phosphorylation.
(A) ARPE-19 cells were treated with H2O2 (400μM) for 3, 6 and 12 hours and p62, NFκB p65 ser 536 phosphorylation, and total NFκB p65 protein levels were measured by western blot. β-actin was used as an internal control. Representative western blot from atleast 3 independent experiments has been shown. (B) NFκB p65 nuclear translocation was determined after treating ARPE-19 cells with H2O2 (400μM) for 3, 6 and 12 hours and western blotting on separated nuclear and cytosolic fraction lyastes. Purity of cytosolic and nuclear fractions were determined by blotting for tubulin and Histone (H3) respectively. Representative Western blot from at least 3 independent experiments are shown. (C) p62 mRNA levels were measured by qRT-PCR after pretreating the ARPE-19 cells with IKKβ inhibitor SC-514 (100μM) for 1 hour and subsequently co-treating with H2O2 (400μM) for 3 or 24 hours. (D) ARPE-19 cells were pretreated with IKKβ inhibitor SC-514 for 1 hour and subsequently co-treated with H2O2 (400μM) for 6 or 24 hours. p62, LC3, NFκB p65 ser 536 phosphorylation, and total NFκB p65 protein levels were measured by western blot. β-actin was used as an internal control. Representative Western blot from at least 3 independent experiments is shown. (E) ARPE-19 cells were either treated alone or co-treated with competing NFκB Ser529, 536 short peptide or co-treated with H2O2 (400μM) for 3 hours. p62 mRNA levels were measured by qRT-PCR. (F) ARPE-19 cells were either treated alone or co-treated with competing NFκB Ser529, 536 short peptide or co-treated with H2O2 (400μM) for 3 or 24 hours. p62 and NFκB p65 ser 536 phosphorylation protein levels were measured by western blotting. Ctrl = Untreated control; Pep = NFκB Ser529, 536 short peptide treated. (G) Empty pcDNA3.1 vector, p65-WT, dominant negative mutant p65-S536A and dominant positive mutant S536D were transiently overexpressed in ARPE-19 cells for 24 hours and cell lysates were analyzed for NFκB p65 ser 536 phosphorylation and total NFκB p65 protein levels by western blotting. (H) ARPE-19 cells transfected with respective plasmids for 24 hours were either left untreated or treated with H2O2 (400μM) for an additional 3 hours and p62 mRNA levels were measured by qRT-PCR. Data represent the mean +/- S.E.M. for three samples per group. Differences in means were considered statistically significant when p<0.05. (I) p62, NFκB p65 ser 536 phosphorylation and total NFκB p65 protein levels were measured by Western blot. β-actin was used as an internal control. Representative western blot from at least 3 independent experiments is shown.
Fig 5.
Inhibition of cellular ROS but not mitochondrial superoxide alone can attenuate p62 expression in RPE.
(A) ARPE-19 cells were pre-treated with, and subsequently challenged with, H2O2 (400μM) for 6 hours or with Lactacystin (25μM for 12 hours) and cellular ROS was measured by CM-H2DCFDA assay. The mean from 3 independent experiments is plotted as Mean +/- SEM. Differences in means was considered significant when p<0.05 by analysis of variance. (B) p62 protein level in ARPE-19 cells after pre-treatment with NACA (1mM) for 1 hour and subsequent challenge with H2O2 (400μM) for 6 hours or with Lactacystin (25μM for 12 hours) was determined by Western blotting. Representative Western blot from 3 independent experiments are shown. Densitometric data was plotted as Mean +/- SEM for the three experiments. β-actin was used as internal control. (C) p62 mRNA levels were measured by qRT-PCR after pre-treating the ARPE-19 cells with NACA (1mM) (100μM) for 1 hour and subsequently treating with H2O2 (400μM) for 6 hours or Lactacysin for 12 hours. (D) ARPE-19 cells were pre-treated with NACA (1mM) for 1 hour and subsequently treated with H2O2 (200 and 400μM) for 6 hours. p62, NFκB p65 ser 536 phosphorylation, and total NFκB p65 protein levels were measured by Western blot. β-actin was used as an internal control. Representative Western blot from 3 independent experiments is shown. (E) ARPE-19 cells were pre-treated with MitoTEMPO (250μM) for 1 hour. Mitochondrial superoxide was measured by MitoSOX Red smitochondrial superoxide sensor. (F) ARPE-19 cells were either treated alone or pre-treated with MitoTEMPO (250 μM) for 1 hour and subsequently treated with H2O2 (400μM) for 6 hours or Lactacystin for 12 hours. A representative Western blot of p62 protein levels is shown. (G) ARPE-19 cells were either treated alone or pre-treated with MitoTEMPO (250 μM) for 1 hour and subsequently treated with rotenone (5 μM) for 3, 6 and 24 hours. A representative Western blot of p62 protein levels is shown.
Fig 6.
Knockdown of p62 renders RPE cells more susceptible to oxidative stress.
ARPE-19 cells were transfected with p62 or scrambled siRNA for 24h and then treated with 800μM H2O2 for 6h. (A) Mitochondrial activity in p62 or scrambled siRNA-transfected cells was assessed using MTT assay. (B) Cell viability was assayed using crystal violet assay. Statistical significance was calculated by ANOVA and differences were considered statistically significant when p<0.05. (C) Primary fetal RPE cells were transfected with p62 or scrambled siRNA for 48h and then treated with varying concentrations of H2O2 for 24h followed by MTT assay. Statistical significance was calculated by ANOVA and differences were considered statistically significant when p<0.05.
Fig 7.
p62 positively regulates autophagic flux.
LC3II/I ratio and p62 protein levels in cell lysates were examined by Western blot for (A) ARPE-19 cells, transfected with p62 specific siRNA or scrambled siRNA (10nM) for 24h and treated with 400μM H2O2 for additional 24 h. For assessment of autophagic flux, Bafilomycin A1 (75nM) was added 3h prior to harvesting in one set of treatments per experiment. (B) RPE cells transfected with p62 construct or empty vector for 24h and treated with 400μM H2O2 for an additional 24 h. β-actin was used as an internal control. (C) Densitometric analyses of LC3 II/I ratio in (A = endogenous p62 knock-down and B = ectopic overexpression of p62) were plotted as mean + S.E.M. of three independent experiments. Mean differences were considered statistically significant when p<0.05 by Mann-Whitney U-test. (D) Autophagosome numbers were determined in RPE cells co-transfected with GFP-LC3 and p62 siRNA or scrambled siRNA (10nM), after H2O2 treatment. 50 cells per condition per experiment were analyzed for autophagosome numbers. Differences between mean counts from 3 independent experiments were considered statistically significant when p<0.05 by Mann-Whitney U-test. (E) RPE cells transfected with GFP-LC3 together with p62 siRNA or scrambled siRNA for 24h and treated with 400μM H2O2 for additional 24h were fixed and immunostained for p62. AlexaFluor® 594-conjugated goat anti-mouse secondary antibody was used for visualization. White arrows represent the co-localization of p62 (red) and GFP-LC3 (green).
Fig 8.
ATG10 is involved in p62-regulated autophagy.
(A) Scatter plots comparing the normalized, relative expression of autophagy-related proteins in ARPE-19 cells transfected with p62 specific or scrambled siRNA (10nM) for 24 h. mRNA expression was detected using autophagy qRT-PCR array from SA Biosciences each. The 'fold-change boundary' denoted by the parallel lines flanking the median line was set at 2 and segregates the genes up or down regulated. Dots represent genes plotted on the basis of Log 10(2-ΔΔCt). Blue circles denote the genes ATG10 and p62 with -2.44 and -6.77 fold changes respectively. (B) p62 mRNA level was examined by qRT-PCR to confirm the knockdown of p62 transcription and mRNA level of autophagic protein ATG10 was assessed in p62-knockdown cells. (C) RPE cells were treated by 400μM H2O2 and ATG10 mRNA level was measured by qRT-PCR. Mean+ S.E.M. from three independent experiments were plotted. (D) RPE cells were transfected with p62 or scrambled siRNA for 24h and then exposed with 400 H2O2 for 1, 3, 6, 12h respectively. Whole-cells lysates were analyzed by Western blot with anti-p62, ATG10, or β–actin antibodies. Differences between means were considered statistically significant when p<0.05 by Mann-Whitney U-test.
Table 1.
Differentially expressed genes after p62 knockdown.
Fig 9.
Knockdown of ATG10 makes ARPE-19 cells more sensitive to H2O2.
(A) ARPE-19 cells were transfected with pooled ATG10 specific siRNAs or scrambled siRNA (30nM) for 24h. ATG10 and ATG5 levels were assessed in protein lysates by Western Blot. (B) RNA isolated from cells was analyzed for ATG10 mRNA expression by qRT- PCR. Differences between means were considered statistically significant when p<0.05 by Mann-Whitney U-test. (C) Lysates of ARPE-19 cells transfected with siRNAs for 24 hours were assessed for p62 and LC3 levels by Western blot. One set of wells per experiment was exposed to Bafilomycin A1 (75nM) for 3h prior to harvest. β-actin was used as an internal control. ‘Short’ and ‘Long’ Exposure indicate time of exposure of immunoblot image. (D) 24h after transfection with respective siRNAs, ARPE-19 were subjected to oxidative stress with 800μM H2O2 and incubated at 37°C for 6h. Total cell numbers were determined by trypsinization and counting on TC10® cell counter. (E) Primary fetal RPE cells were transfected with scrambled or pooled ATG10 specific siRNAs and 48 hours after transfection, were subjected to H2O2 for 24 hours and MTT assay was performed. Differences between means were considered statistically significant when p<0.05 by Student’s t-test.