Fig 2: Evaluation of the efficiency of short hairpin RNA (shRNA) against sulfiredoxin 1 (Srxn1) following exposure to oxygen-glucose deprivation (OGD) or hydrogen peroxide (H2O2). (A and B) Srxn1 protein expression was measured by western blot analysis. (C) Srxn1 mRNA expression was determined by RT-qPCR. The results showed that Srxn1 protein and mRNA expression were decreased by shRNA Srxn1 knockdown and that sensitivity to OGD or H2O2 was increased. The exposure to astrocytes to OGD or H2O2 resulted in a significant increase in Srxn1 protein and mRNA expression, and Srxn1 knockdown prevented this increase. Data are the means ± SEM. *P<0.05 and **P<0.01 vs. controls (untreated cells); #P<0.05 and ##P<0.01 vs. control + OGD (or H2O2) group; n=4. NC, negative control.

Fig 3: Knockdown of sulfiredoxin 1 (Srxn1) increases apoptosis following exposure to oxygen-glucose deprivation (OGD) or hydrogen peroxide (H2O2). (A and B) Apoptosis was assessed by Hoechst 33342 staining and visualized by fluorescence. Exposure to OGD or H2O2 increased cell apoptosis. Following exposure to OGD or H2O2, the knockdown of Srxn1 increased apoptosis increased more significantly. Scale bar, 20 µm. Results are expressed as the means ± SEM; **P<0.01 vs. controls (untreated cells); ##P<0.01 vs. control + OGD (or H2O2) group, n=4. (C and D) Flow cytometric analysis of apoptosis and necrosis. (C) Typical representative dot plots of the distribution of normal (Annexin V-/PI-), early apoptotic (Annexin V+/PI-), late apoptotic (Annexin V+/PI+) and necrotic (Annexin V-/PI+) cells. (D) Late-apoptotic and early-apoptotic astrocytes were calculated from the total astrocytes. Following exposure to OGD or H2O2, the knockdown of Srxn1 markedly increased apoptosis. Data are the means ± SEM; **P<0.01 vs. controls (untreated cells); ##P<0.01 vs. control + OGD (or H2O2) group, n=4. NC, negative control (unreated cells).

Fig 4: Changes in mitochondrial membrane potential (??m) following exposure to oxygen-glucose deprivation (OGD) or hydrogen peroxide (H2O2) and the knockdown of sulfiredoxin 1 (Srxn1) based on JC-1 staining. (A) (Panels a and b) Laser scanning confocal microscopic images revealed that both green and red fluorescence were detected in the controls (untreated cells) and that the red fluorescence was stronger than the green fluorescence. (Panel c) The green fluorescence was stronger and the red fluorescence was weaker in the sh-Srxn1 group than the control group. (Panels f and i) After the knockdown of Srxn1 and exposure to OGD or H2O2, the green fluorescence became much stronger than that in the control + OGD (or H2O2) group. Scale bar, 100 µm. (B) The JC-1 fluorescence ratio was calculated by the average optical fluorescence density ratio of red/green in each group. The JC-1 fluorescence ratio decreased by ~38% in the sh-Srxn1 group compared with that in the control group. The JC-1 fluorescence ratio in the sh-Srxn1 + OGD (or H2O2) group declined more significantly compared with that in the control + OGD (or H2O2) group. Data are the means ± SEM; *P<0.05 vs. controls (untreated cells); #P<0.05 vs. control + OGD (or H2O2) group, n=4. (NC, negative control).

Fig 5: Effect of the knockdown of sulfiredoxin 1 (Srxn1) on endoplasmic reticulum stress pathway-mediated apoptosis. (A and B) Intracellular calcium ([Ca2+]i) levels were measured by laser scanning confocal microscopy. Images showed that green fluorescence had a fluo-3 fluorescence intensity. The stronger the fluorescence for [Ca2+]i, the higher the expression. Compared with the controls (untreated cells), the level of [Ca2+]i markedly increased in the control + oxygen-glucose deprivation (OGD) [or hydrogen peroxide (H2O2)] group, and the level of [Ca2+]i did not change significantly after the knockdown of Srxn1. Data are the means ± SEM; *P<0.05 vs. controls (untreated cells), n=4; (C and D) Western blot analysis for caspase-12. Exposure to OGD or H2O2 increased the level of caspase-12, but it did not affect caspase-12 expression after the knockdown of Srxn1. Data are the means ± SEM; *P<0.05 vs. controls (untreated cells), n=4.