Fig 2: Mechanistic model depicting the effects of SESN2 on the drug resistance of osteosarcoma. After chemotherapy drugs are applied to osteosarcoma cells, SESN2 expression is increased, and the phosphorylation of eIF2a by PERK during ER stress is inhibited. Finally, the expression of CHOP is suppressed, and apoptosis and autophagy are regulated by Bcl-2 and mTOR, respectively, thus causing drug resistance in osteosarcoma cells.

Fig 3: Chemotherapeutic agents increased the expression of SESN2 in osteosarcoma cells (A,B). After treatment with Cis (20 µmol/L), Dox (0.2 µg/mL) or Mtx (50 µmol/L), the expression level of SESN2 in osteosarcoma cells was detected by western blot (A) (n = 3), and the SESN2 mRNA level was detected by quantitative real-time PCR (B) (n = 3). Western blotting was used to detect SESN2 expression in MG-63, HOS, and 143B cells after Cis (20 µmol/L) treatment for 24 to 72 h (C) (n = 3). The data are presented as the mean ± SD. *p < 0.05 vs. the – group.

Fig 4: Sestrin2 inhibition restores mitochondrial function in H9c2 cells. (A) Intracellular calcium was assessed by flow cytometry. (B) The mitochondrial membrane potential was measured by flow cytometry. (C) MPTP opening was detected by flow cytometry. The fluorescence intensity was negatively associated with MPTP opening. (D) The level of ATP was measured by ATP assay kit. The activities of (E) caspase-3 and (F) cytochrome c were measured by ELISA. Data are presented as mean ± SD (n=3). *P<0.05 vs. Control group. dP<0.01 vs. Control group. &P<0.001 vs. Control group. ^P<0.05 vs. HG group. §P<0.01 vs. HG group. #P<0.05 vs. Negative control group. ?P<0.01 vs. Negative control group. Sesn2, Sestrin2; C, control group; G, HG group; NC, negative control; shRNA, small hairpin RNA; HG, high glucose.

Fig 5: SESN2 confers drug resistance to target cells by reducing their sensitivity to chemotherapeutic agents. MG-63 and HOS cells were transfected with control shRNA or SESN2 shRNA, and the knockdown effect was detected by western blot (n = 3) and quantitative real-time PCR (n = 3) (A,B). MG-63 and HOS cells with SESN2 knockdown were treated with Cis (20 µmol/L), Dox (0.2 µg/mL), or Mtx (50 µmol/L) for 24, 48, and 72 h. Cell activity was detected by CCK-8 (C) (n = 3). After treatment for 24 h, apoptotic cells were detected by flow cytometry (D) (n = 3). SESN2 knockdown significantly reduced the number of colony-forming MG-63 cells (E) (n = 3). After MG-63 cells were treated with Cis (20 µmol/L), Dox (0.2 µg/mL) or Mtx (50 µmol/L) for 24 h, the apoptosis of cells transfected with control shRNA or SESN2 shRNA and cultured in the presence or absence of ZVAD-FMK (20 µmol/L) was detected using the Caspase 3 kit (F) (n = 3). The data are presented as the mean ± SD. *p < 0.05 vs. the Control shRNA group. The expression of SESN2 after transfection of a plasmid carrying the SESN2 gene was evaluated by western blot (G) (n = 3). MG-63 and HOS cells overexpressing SESN2 as well as their corredsponding control cells were treated with Cis (20 µmol/L), Dox (0.2 µg/mL), or Mtx (50 µmol/L). Cell viability and apoptosis were detected by CCK-8 (H) (n = 3) and flow cytometry (I) (n = 3), respectively. SESN2 increases the number of colony-forming units of HOS cells (J) (n = 3). Cleaved and total PARP in SESN2-overexpressing and control HOS cells were analysed by western blot (K) (n = 3). The data are presented as the mean ± SD. *p < 0.05 vs. the pHBLV control group.