Fig 2: Effects of HG on mitochondrial dysfunction in cultured podocytes. Cells were divided into 3 groups: i) the control (CTL), where cells were cultured in 5 mM glucose for 24 h; ii) the MA group, where cells were cultured in 5 mM glucose and 30 mM mannitol for 24 h; iii) the HG group, where cells were cultured in 35 mM glucose for 24 h. (A and B) Flow cytometric analysis of apoptosis in cultured podocytes in different groups and quantification of the results (*P<0.05 vs. CTL, n=3). (C) Representative images of ultrastructure of cultured podocyte mitochondria (indicated by arrows) by transmission electron microscopy in different groups. (D and E) Representative images of ROS production in cultured podocytes in different groups. ROS production was assessed by the detection of DCFH-DA fluorescence (green) (*P<0.05 vs. CTL, n=3). (F and G) Representative images of mitochondrial membrane potential in podocytes by JC-1 staining in different groups and quantification of the results (*P<0.05 vs. CTL, n=3). sesn2, sestrin-2; AMPK, AMP-activated protein kinase; HG, high glucose; ns, not significant.

Fig 3: SESN2 protected DC2.4 cells against ERS-related apoptosis triggered by TM.DC2.4 cells were treated with various dosages of TM (0.25, 0.5, 1, and 100 ng/ml) for 48 h. Cells cultured for 48 h without TM were used as the controls. a PE-Annexin-V and 7-AAD were used to stain the treated DC2.4 cells and subjected to flow cytometry to assess cell apoptosis with different dosages of TM for 48 h. b SESN2 expression was determined by Western blotting. c After modulated SESN2 expression, PE-Annexin V and 7-AAD were used to stain the treated DC2.4 cells and flow cytometry was used to assess cell apoptosis after stimulated with 2 µg/ml TM for 48 h. d Expressions of SESN2 and CHOP were measured as described in the section of methods following treatment with 2 µg/ml TM for 48 h. ß-actin served as the internal standard. Data of three independent experiments were presented as the mean ± SD. Statistical significance: *P < 0.05 versus the scramble group; #P < 0.05 versus the scramble group treated with HMGB1.

Fig 4: SESN2 protected DC2.4 cells against excessive ERS response induced by HMGB1.a Expressions of GRP78, p-PERK, PERK, ATF4, CHOP, Bcl-2, and Bax were determined by Western blotting after treatment with 100 ng/ml HMGB1 for 48 h. b, c Lentiviral vectors (SESN2 knockdown and SESN2 overexpression) were transduced DC2.4 cells, and scramble cells were transduced with blank vectors. After treated with 100 ng/ml HMGB1 for 48 h, expressions of GRP78, p-PERK, PERK, ATF4, and CHOP were determined by Western blot analysis. d The morphologic alteration of ER in each cell was evaluated by immunofluorescence staining and the extent of the ER morphologic change was compared with the scramble controls (×600, ×1200), scale bar = 25 µm, scale bar = 10 µm, n = 4 per group. ER was detected with ER-tracker red. ß-actin served as the internal standard. Data were presented as the mean ± SD, n = 3 per group. Statistical significance: *P < 0.05 versus the control group.

Fig 5: Co-localization of SESN2 and ER in DC2.4 cells (×600, ×1200).a Laser scanning confocal microscopy was used to observe SESN2 and ER in DC2.4 cells. It was shown by anti-SESN2 antibodies (green) and ER tracker (red) using indirect immunofluorescence and confocal microscopy. b It was observed by anti-SESN2 antibodies (red) and ATF4 (green) using indirect immunofluorescence and confocal microscopy. c It was shown by anti-SESN2 antibodies (red) and GRP78 (green) using immunofluorescence and confocal microscopy. d Immunoblot analysis of ATF4 in SESN2 was immunoprecipitated in total cell lysate. Negative control was using control agarose resin. Scale bar = 25 µm, scale bar = 10 µm, n = 3 per group. Its co-localization was shown in saffron yellow (DAPI stained nucleus, blue).