Fig 2: Influence of silencing of KLF4 on activation of A1/A2 astrocytes under OGD/R conditions. A Representative images of western blot for KLF4, C3, S100A10, TNF-a, iNOS, p-NF-kB in astrocytes transfected with the negative control siRNA (si-Ctl) or siRNA-KLF4 (si-KLF4) at 48 h restoration from OGD. The NO-OGD/R si-Ctl treated cells served as a control. B–G Bar graphs show the quantitative analyses of western blots as ratios of KLF4/ß-actin (B), C3/ß-actin (C), S100A10/ß-actin (D), TNF-a/ß-actin (E), iNOS/ß-actin (F) and p-NF-kB/total NF-kB (G), and the data were analyzed by two-way ANOVA (n = 4 per experimental group). Note that the protein levels of C3, TNF-a, iNOS and the phosphorylation of NF-?B were significantly elevated relative to the si-Ctl-treated group at 48 h restoration from OGD, but the levels of KLF4 and S100A10 in the si-KLF4-treated astrocytes were markedly reduced. *P < 0.05, **P < 0.01, ***P < 0.001. H, I Representative images of immunofluorescent staining for C3/S100A10, GFAP, and DAPI in astrocytes after indicated treatments. Scale bar = 100 µm. J–O mRNA level of pro- or anti-inflammatory genes was determined by qPCR in the astrocytes of si-Ctl and si-KLF4 group at 48 h restoration of OGD or NO-OGD/R, and the data were analyzed by two-way ANOVA (n = 4 per experimental group). NO-OGD/R si-Ctl-treated cells served as control. Note that the mRNA levels of pro-inflammatory genes including IL-1ß (J), TNF-a (K) and iNOS (L) were shown to significantly increase, but anti-inflammatory genes including IL-1ra (M), IL-10 (N), and Arg1 (O) were found to remarkably decrease in astrocytes after OGD/R injury. Furthermore, OGD/R-induced expression of pro-inflammatory genes was exaggerated by diminishing the expression of KLF4 in astrocytes; likewise, the decreased expression of anti-inflammatory genes caused by OGD/R was further augmented by silencing the levels of KLF4 in the astrocytes. *P < 0.05, **P < 0.01, ***P < 0.001; ns not significant

Fig 3: Impact of overexpression of KLF4 on activation of A1/A2 astrocytes under OGD/R conditions. A Representative images of western blot for KLF4, C3, S100A10, TNF-a, iNOS, p-NF-kB in astrocytes in astrocytes transfected with control plasmid (mock) or KLF4 overexpression plasmid at 48 h restoration from OGD. The NO-OGD/R mock treated cells served as a control. B–G Bar graphs show the quantitative analyses of western blots as ratios of KLF4/ß-actin (B), C3/ß-actin (C), S100A10/ß-actin (D), TNF-a/ß-actin (E), iNOS/ß-actin (F), and p-NF-kB/total NF-kB (G), and the data were analyzed by two-way ANOVA (n = 4 per experimental group). Note that compared to the mock-transfected group, the protein levels of C3, TNF-a, iNOS and the phosphorylation of NF-?B were markedly reduced, but the levels of KLF4 and S100A10 were significantly elevated in astrocytes transfected with KLF4 after 48 h restoration from OGD. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. H, I Representative images of immunofluorescent staining for C3/S100A10, GFAP and DAPI in astrocytes transfected with mock or KLF4 overexpression plasmid after OGD/R treatment. Scale bar = 100 µm. J–O mRNA level of pro- or anti-inflammatory genes in the astrocytes were determined by qPCR in the mock or KLF4 overexpression group at 48 h restoration of OGD or NO-OGD/R, and the data were analyzed by two-way ANOVA (n = 4 per experimental group). NO-OGD/R mock-treated cells served as control. Note that compared to the mock-transfected group, overexpression of KLF4 decreased the expression of IL-1ß (J), TNF-a (K), and iNOS (L), but increased the levels of IL-1ra (M), IL-10 (N), and Arg1 (O) in astrocytes under OGD/R conditions. *P < 0.05, **P < 0.01, ***P < 0.001; ns not significant