Fig. 3: Depletion of MTHFD2 enhances ROS levels and induces cellular senescence.

A RAS-induced senescence (OIS) in HFF cells (P18). Left: oncogene RAS-induced cell senescence schematic. Right: SA-β-gal staining in oncogene RAS-induced cell senescence. B qPCR (Left) and Immunoblots (Right) of MTHFD2 and p21 in oncogene RAS-induced cell senescence. C Immunoblots of MTHFD2 and p21 in replicative cell senescence models. D Immunofluorescence of p21 (Left), and immunoblot of MTHFD2 and p21 (Right) in H₂O₂-induced senescence model. HFF (P18) were treated with 100 μM H₂O₂ for 1 h. E Expression of the indicated proteins was determined by immunoblot in IMR90 cells transfected with vehicle control or ER: RAS to induce senescence with or without simultaneous treatment of Lys05 (5 μM). F Expression of the indicated proteins was determined by immunoblot in IMR90 cells with or without ATG5 knockdown. G, H Immunofluorescence of MTHFD2 co-localization with specific for autophage (LC3I/II) and lysosomal marker LAMP1. I IMR90 cells were transfected with control or MTHFD2 siRNAs, the cell cycle progression was detected by flow cytometry analysis. J Effect of MTHFD2 silencing (siMTHFD2) and siControl on cellular senescence. HFF (P18) were cultured in medium and then transfected with siMTHFD2 and siControl, respectively. The cellular senescence was indicated by SA-β-gal staining. K Effect of MTHFD2 overexpression (3xFlag-MTHFD2) and vector on cellular senescence. HFF (P40) were cultured in medium and then transfected with 3xFlag-MTHFD2 and 3xFlag-Vector, respectively. The cellular senescence was indicated by SA-β-gal staining. L Protein expression in HFF cells (P18) treated with control siRNA (−) or increasing amounts of MTHFD2 siRNA for 48 h was analyzed by Western blot using the indicated antibodies. Data are representative of three independent experiments. M HFF cells (P18) were transfected with control or MTHFD2 siRNAs, the NADPH levels was detected. N HFF cells (P18) transfected with MTHFD2 siRNA or control siRNA, the relative ROS level was determined by immunofluorescence. O Protein expression in HFF cells (P18) treated with control siRNA (−) or MTHFD2 siRNA for 48 h was analyzed by Western blot using the indicated antibodies. Data are representative of three independent experiments. P The activation status of p53 and AMPK was evaluated in IMR90 cells transfected with either control siRNA or MTHFD2-targeting siRNA, in the presence or absence of the AMPK inhibitor Compound C (10 μM). Q Cellular senescence was assessed by SA-β-gal staining in IMR90 cells with or without MTHFD2 silencing, in the presence or absence of exogenous NADPH (10 μM). R The protein expression levels of p53 and its downstream target p21 were examined in IMR90 cells treated with or without NAC (2 mM), NADPH (10 μM), or Formate (1 mM), in the presence or absence of MTHFD2 knockdown.