Figure 1.
Protective effects of SERPINA3K in retinal cells.
Y79 (A), R28 (B), RPE (C) and rMC-1 cells (D) were exposed to 400 µM H2O2 in the presence of 1 µM SERPINA3K or BSA for 8 h, or incubated with 1 µM SERPINA3K alone for 8 h. Viable cells were quantified by MTT assay (mean±SEM, n = 3). * P<0.01, versus the H2O2 treated cells. # P<0.01, versus the control cells.
Figure 2.
Protective effects of SERPINA3K on rMC-1 cells against H2O2-induced necrosis.
Müller-derived rMC-1 cells were pre-treated with SERPINA3K or BSA for 1 h and then exposed to 400 µM H2O2 for 8 h (A–C, G) and 4 h (D–F, H, I). The protein concentration in the media was brought to 1 µM in each well by addition of BSA. (A–C) Representative phase contrast images showing cell morphology in untreated control (A), cells treated with 1 µM BSA and H2O2 (B), and cells treated with 1 µM SERPINA3K and H2O2 (C). Scale bar, 50 µm. (G) Viable cells were quantified using the MTT assay (mean±SEM, n = 3). (D–F, H) The viable and necrotic cells were analyzed by flow cytometry following staining with PI and Annexin-V; (D) Untreated control; (E) treated with 1 µM BSA and H2O2; (F) treated with 1 µM SERPINA3K and H2O2; (H) quantification of necrotic cells as percentages of total cells (mean±SEM, n = 3). (I) DNA laddering analysis showed no apparent DNA fragmentation in the H2O2-treated cells. Lane 1, control cells; 2, cells treated with H2O2 for 4 h; 3, cells treated with H2O2 and SERPINA3K, and 4, over-grown cells as positive control. # P<0.001, H2O2 treated cells versus control cells. * P<0.05, ** P<0.01, *** P<0.001, the cells treated by different doses of SERPINA3K versus the cells treated only by BSA.
Figure 3.
Lack of effect of SERPINA3K on the intracellular ROS generation in cells exposed to H2O2.
(A, B) The rMC-1 cells were treated with 400 µM H2O2 (A) or 20 µM TBHP (B) for 1, 2, 4 and 8 h in the absence or presence of 1 µM BSA or SERPINA3K. Viable cells were quantified using MTT assay (mean±SEM, n = 3). (C) The rMC-1 cells were exposed to 400 µM H2O2 or 20 µM TBHP for various durations as indicated, to define the time point for the ROS increase. Note that ROS levels were elevated by H2O2 as early as 15 min, while the ROS generation induced by TBHP occurred at 4 h of the treatment. (D&E) rMC-1 cells were pre-treated with various concentrations of SERPINA3K for 1 h. The medium was supplemented with BSA to the same total protein concentration in each well. The cells were then exposed to 400 µM H2O2 for 15 min (D) or to 20 µM TBHP for 4 h (E). The intracellular ROS generation was measured using CM-H2DCFDA as a probe. Values are arbitrary fluorescence units (mean±SEM, n = 8). SERPINA3K blocked the ROS increase induced by TBHP but not that by H2O2. * P<0.01, the SERPINA3K-treated cells versus the BSA-treated cells. & P<0.01, the BSA-treated cells exposed to H2O2 versus the BSA-treated cells without H2O2 exposure. # P<0.05, the H2O2 or TBHP-treated cells versus control cells.
Figure 4.
SERPINA3K blocks intracellular calcium overload induced by H2O2.
(A) Elevated intracellular [Ca2+] in cells exposed to H2O2. The Müller-derived rMC-1 cells were treated with 400 µM H2O2 for various durations as indicated. TBHP (20 µM) was used as control. Intracellular [Ca2+] was measured by the fluorescence of the probe Fluo-4/AM (mean±SEM, n = 8). (B–E) The cells were pre-treated with 1 µM SERPINA3K or BSA for 1 h followed by the H2O2 exposure. BSA was added to bring the total protein concentration to the same in each well. Representative fluorescence images were captured under a fluorescent microscope from untreated cells (B), cells pre-treated with 1 µM BSA (C) and with 1 µM SERPINA3K (D) followed by a 3-h exposure to H2O2. (E) [Ca2+] in cells pre-treated with various concentrations of SERPINA3K prior to the exposure to H2O2 (mean±SEM, n = 6). (F) The protective effects of 1 µM SERPINA3K and 10 µM BAPTA/AM (a calcium chelator) were quantified using the MTT assay (mean±SEM, n = 3). * P<0.05, ** P<0.01, *** P<0.001, the cells treated by H2O2 versus the control cells. # P<0.01, the SERPINA3K or BAPTA-treated cells versus the BSA-treated cells. Scale bar, 100 µm.
Figure 5.
SERPINA3K does not chelate free calcium.
Free [Ca2+] was measured by the fluorescence of the Fluo-4 (pentapotassium salt). (A) A Ca2+/Fluo-4 binding curve was plotted with different concentrations of CaCl2 and 10 µM Fluo-4. (B) SERPINA3K (10 µM) was incubated with 10 µM CaCl2 or a cell lysate in the presence of 10 µM Fluo-4. EGTA (10 µM) and BSA (10 µM) were used as positive and negative controls, respectively. Free [Ca2+] was measured (mean±SEM, n = 3). * P<0.01, versus control cells.
Figure 6.
SERPINA3K inhibits the H2O2-induced calcium overload and necrosis via PLC but not the ERK or PI3K/Akt pathways.
(A) The rMC-1 cells were treated with 400 µM H2O2 in the presence of 1 µM SERPINA3K, 250 nM U73122 (PLC inhibitor), 10 µM U0126 (ERK inhibitor), 100 nM Wortmannin (PI3K inhibitor) or 10 µM BAPTA. BSA (1 µM) was used as negative control. Intracellular [Ca2+] was measured using the fluorescence of the probe Fluo-4/AM at different intervals of the treatment (mean±SEM, n = 6). (B) The protective effects of the inhibitors and 1 µM SERPINA3K against the H2O2-induced cell death with or without 10 µM m-3M3FBS (PLC activator) were quantified by the MTT assay after exposure to H2O2 for 8 h (mean±SEM, n = 3). (C) The rMC-1 cells were treated with 400 µM H2O2. The cells were harvested at different time points for Western blotting. (D) The cells were exposed to H2O2 for 15 min in the presence of 1 µM SERPINA3K or BSA. The phosphorylated PLC-γ1 (p-PLC), ERK1/2 (p-ERK) and Akt (p-AKT) were blotted with phosphorylation-specific antibodies. The total PLC-γ1 (t-PLC), ERK1/2 (t-ERK) and Akt (t-AKT) were blotted with antibodies for total proteins. (E) Protein levels was quantified using densitometry of the Western blotting results (mean±SEM, n = 3). (F) The reduced phosphorylation level of each kinase was shown as percentages of the maximum phosphorylation levels at 15 min exposure (mean±SEM, n = 3). $ P<0.01, under H2O2 exposure from 1.5 h to 4 h; the U73122 and SERPINA3K-treated cells versus the BSA-treated cells. * P<0.01, versus control cells. # P<0.01, the cells treated with H2O2, m-3M3FBS and SERPINA3K versus the cells treated with H2O2 and SERPINA3K. & P<0.01, versus control cells exposed to H2O2 for 0 min.
Figure 7.
SERPINA3K binds to Müller-derived rMC-1 cells.
(A) The rMC-1 cells were incubated with increasing concentrations of FITC-SERPINA3K or FITC-BSA for 1 h. (B) The rMC-1 cells were incubated with 400 nM FITC-SERPINA3K in the presence of different concentrations of unlabeled SERPINA3K or BSA for 1 h. Then the cells were washed three times with PBS. The fluorescence in the cells was measured by a fluorometer using 485/530 nm filter (mean±SEM, n = 8).