Fig 1: NEAT1 delivered by BMSC-EVs induces proliferation and autophagy of chondrocytes but represses their apoptosis via the miR-122-5p/Sesn2 axis. (a) Sesn2 mRNA expression in chondrocytes treated with Lv-NEAT1-BMSC-EVs or combined with sh-Sesn2 detected by RT-qPCR. (b) Proliferation of chondrocytes treated with Lv-NEAT1-BMSC-EVs or combined with sh-Sesn2 detected by CCK-8. (c) Apoptosis of chondrocytes treated with Lv-NEAT1-BMSC-EVs or combined with sh-Sesn2 measured by flow cytometry. (d) Autophagy of chondrocytes treated with Lv-NEAT1-BMSC-EVs or combined with sh-Sesn2 measured by MDC staining. (e) Formation of autophagosomes and autolysosome in chondrocytes treated with Lv-NEAT1-BMSC-EVs or combined with sh-Sesn2 analyzed by mRFP-GFP-LC3 double fluorescence assay. (f) Beclin-1 and LC3-II/I mRNA expression in chondrocytes treated with Lv-NEAT1-BMSC-EVs or combined with sh-Sesn2 analyzed by RT-qPCR. (g) Beclin-1 protein expression and the ratio of LC3-II/I in chondrocytes treated with Lv-NEAT1-BMSC-EVs or combined with sh-Sesn2 analyzed by Western blot analysis. *p < 0.05. Measurement data were expressed as mean ± standard deviation. Data among groups were compared by one-way ANOVA followed by Tukey's post hoc test. Data at different time points were compared using repeated-measures ANOVA followed by Bonferroni's post hoc test. The cell experiment was run in triplicate independently.
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 against muscle atrophy and myofiber type transition after denervation. A Western blot analysis of SESN2 expression in GAS post-denervation. B Western blot analysis confirming the expression of SESN2 and MHC. C Appearance and wet weight ratio of GAS in different groups. D Quantification of muscle fiber diameter by immunofluorescence staining of WGA. Scale bar, 50 µm. E Western blot analysis of proteins related to muscle fiber type. F Immunofluorescence analysis of fiber type composition in denervated GAS. Scale bar, 50 µm. G, H Relative ATP content and metabolic enzyme activities (LDH and SDH) in GAS. Data presented as mean ± SD. n = 6. *P < 0.05 versus control (week 0 or sham operation group). #P < 0.05 versus denervation group. Den denervation, Con control
Fig 4: The protective effect of SESN2 on DCs in CLP-induced septic mice.Mice underwent a sham procedure or CLP. a SESN2 expression was determined by Western blotting at 24 h after after CLP. b PE-Annexin-V and 7-AAD were used to stain DCs and subjected to flow cytometry to assess cell apoptosis at 24 h after CLP in vivo. c Expressions of cleaved-caspase-3, Bcl-2, and Bax were measured as described in the section of methods after CLP procedure. d In the sesn2-/- CLP group, 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 WT CLP group (×1200). ER was detected with ER-tracker red. Levels of GRP78, ATF4, and CHOP were assessed by Western blotting. ß-actin served as the internal standard. e The 24-h survival rate of mice in the sesn2-/- CLP group was markedly lower than that in the WT CLP group. Data of three independent experiments were presented as the mean ± SD, n = 6 per sham group, n = 11–13 per CLP group. Statistical significance: *P < 0.05 for in vivo comparison of the sesn2-/- CLP group versus the WT CLP group.
Fig 5: Protective effect of SESN2 on DC2.4 cells under HMGB1 stimulation.a, b Lentiviral vectors (SESN2 knockdown and SESN2 over-expression) were transduced DC2.4 cells, and scramble cells were transduced with blank vector. Transduced cells were analyzed by fluorescence microscopy (×400), scale bar = 200 µm, and then protein levels of SESN2 were determined by Western blotting. DC2.4 cells underwent SESN2-knockdown or SESN2-overexpression were incubated with 100 ng/ml HMGB1 for 48 h, and DC2.4 cells underwent blank vector treated with 100 ng/ml HMGB1 for 48 h served as the scramble group. Apoptotic rates of cells were measured with PE-Annexin-7-AAD by flow cytometry (c, d), and cell apoptosis was analyzed by Hoechst 33342 dye (blue) to observe nuclear condensation (×400, e, f), scale bar = 200 µm. Levels of SESN2, cleaved-caspase-3, cleaved-caspase-9, Bcl-2, and Bax were assessed by Western blotting (g, h), respectively. ß-actin served as the internal standard. Values were represented as mean ± SD obtained from three independent experiments, n = 3 per group. Statistical significance: *P < 0.05 versus the scramble group; #P < 0.05 versus the scramble group treated with HMGB1.
Supplier Page from Abcam for Anti-SESN2/Sestrin-2 antibody [EPR18907]