Fig 1: Hypoxia-induced changes of IL-17A and ER stress markers in retinas and macrophages. a Western blotting analysis of GRP78, ATF-4 and IL-17A protein levels at P12, P15, P17, P21 in retinas of OIR mice compared with their age-matched controls. b Immunofluorescent staining of F4/80 (red, specific expressed by macrophage) and ATF-4 (green), GRP78 (green), IL-17A (green) on representative sections of retinas from P17 OIR mice and normal controls. Boxed areas are magnified in the images. Yellow represents co-localization of GRP78, ATF-4 or IL-17A and macrophages infltration. Scale bars, 100 μm. c, d Analysis of mRNA (c) and protein (d) levels of GRP78, ATF-4 and IL-17A in macrophages exposed to hypoxia for 0, 4, 6, 8, 12 or 24 h. e Immunofluorescent staining of F4/80 (red) and ATF-4 (green), GRP78 (green), IL-17A (green) in macrophages cultured under hypoxic conditions for 0 and 24 h. Yellow represents cellular localization of GRP78, ATF-4 or IL-17A. Scale bars, 50 μm. β-actin served as an endogenous reference for normalization. Data are shown as mean ± SEM, n = 6–8 per group for Real-time RT-PCR, n = 3 per group for western blotting. Each experiment repeated three times. ns, no significance. * P < 0.05 and **P < 0.01 compared with control groups. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer
Fig 2: Neutralization or knockout of IL-17A dampened ER stress in macrophages cultured under hypoxic conditions. a, d Real-time RT-PCR and western blotting analysis of GRP78, ATF-4 and CHOP mRNA (a) and protein (d) levels in macrophages pretreated with rmIL-17A for 24 h at different concentrations (0, 5, 10, 25, 50, 100 ng/ml). b, e Real-time RT-PCR and western blotting analysis of GRP78, ATF-4, CHOP and IL17A mRNA (b) and protein (e) levels in macrophages exposed to hypoxia for 12 h pretreated with IL-17ANab at different concentrations (0, 0.5, 1.0 µg/ml). c Changes of GRP78, ATF-4 and CHOP mRNA levels in macrophages from WT mice and IL-17A KO mice cultured under hypoxic conditions for 12 h. ß-actin served as an endogenous reference for normalization. Data are shown as mean ± SEM, n = 6–8 per group for Real-time RT-PCR, n = 3 per group for western blotting. Each experiment repeated three times. ns, no significance. * P < 0.05 and **P < 0.01 compared with control groups
Fig 3: Neutralization or knockout of IL-17A inhibited ER stress in OIR retinas. a Effects of rmIL-17A (100 ng/µl) stimulation on the protein levels of GRP78, ATF-4 and CHOP in retinas from WT normal mice. b Effects of different concentrations of IL-17ANab (0, 0.5, 1.0 µg/µl) on GRP78, ATF-4 and IL-17A protein levels in retinas from WT-OIR mice. c Immunofluorescent staining of F4/80 (red) and ATF-4 (green), GRP78 (green) on representative sections of retinas from normal WT mice with PBS or rmIL-17A stimulation. d Immunofluorescent staining of F4/80 (red) and ATF-4 (green), GRP78 (green) in retinas of OIR mice from WT ones and IL-17A KO ones. Boxed areas are magnified in the images. Yellow represents co-localization of GRP78 or ATF-4 and macrophages infltration. Scale bars, 100 µm. e, f Changes of retinal GRP78, ATF-4 and CHOP protein (e) and mRNA (f) levels in OIR mice from WT ones and IL-17A KO ones. ß-actin served as an endogenous reference for normalization. Data are shown as mean ± SEM, n = 6–8 per group for Real-time RT-PCR, n = 3 per group for western blotting. Each experiment repeated three times. ns, no significance. * P < 0.05 and **P < 0.01 compared with control groups, ##P < 0.01 compared with each other. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer
Fig 4: ER stress promoted production of IL-17A in macrophages exposed to hypoxia both in vivo and in vitro. a, b Effects of different concentrations of TM (0, 0.1, 0.5, 1.0 µg/µl) on GRP78, ATF-4, CHOP, IL-17A mRNA (a) and protein (b) expression levels in normal retinas. c, d Effects of different concentrations of 4-PBA (0, 0.1, 1, 10 nmol/µl) on GRP78, ATF-4, IL-17A mRNA (c) and protein (d) expression levels in OIR retinas. e Effects of different concentrations of TM (0, 0.1, 1, 2, 4, 6 µg/ml) on GRP78, ATF-4 and IL-17A protein expression levels in macrophages cultured under normoxic conditions. f, g Effects of different concentrations of 4-PBA (0, 0.1, 1, 5, 10 nmol/ml) on GRP78, ATF-4, CHOP, IL-17A mRNA (f) and protein (g) expression levels in macrophages cultured under hypoxic conditions. ß-actin served as an endogenous reference for normalization. Data are shown as mean ± SEM, n = 6–8 per group for Real-time RT-PCR, n = 3 per group for western blotting. Each experiment repeated three times. ns, no significance. * P < 0.05 and **P < 0.01 compared with control groups
Fig 5: Effects of IL-17ANab or 4-PBA on ER stress markers and IL-17A production in OIR retinas. a Western blotting analysis of GRP78, ATF-4, CHOP and IL-17A protein levels in normal retinas and OIR retinas pretreated with 4-PBA (10 nmol/µl) or IL-17ANab (1.0 µg/µl) or vehicle. b–d Immunofluorescent staining of F4/80 (red) and ATF-4 (b, green), GRP78 (c, green), IL-17A (d, green) in OIR retinas treated with 4-PBA or IL-17NAab or vehicle. Boxed areas are magnified in the images. Yellow represents co-localization of ATF-4 or GRP78 and macrophages infltration. Scale bars, 100 µm. ß-actin served as an endogenous reference for normalization. Data are shown as mean ± SEM, n = 6–8 per group for Real-time RT-PCR, n = 3 per group for western blotting. Each experiment repeated three times. * P < 0.05 and **P < 0.01 compared with control groups or each other. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer
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