FIGURE 1.

Nucleotide‐binding domain and leucine‐rich‐repeat‐containing protein X1 deficiency accelerates cellular senescence in mouse embryonic fibroblasts. Primary mouse embryonic fibroblasts (MEFs) from wild‐type (WT) or NLRX1 knockout (KO) mice were subcultured continuously for induction of replicative senescence. (a) Senescence‐associated beta‐galactosidase (SA‐β‐gal) staining to examine the population of senescent cells. Scale bars, 200 μm. (b) Graphs showing the percentage of SA‐β‐gal‐positive cell at each passage (P; n = 4). (c) Relative growth rate at each passage (n = 3). (d) MTT assay to investigate cell proliferation in passages 2 and 6 (n = 3). (e) Immunoblot and (f) Densitometry evaluation to detect p53, mTOR, and phosphorylation of mTOR at serine 2448 (p‐mTOR^S2448) in WT or NLRX1 KO MEFs at passage 4. β‐actin used as loading control. (g) SA‐β‐gal fluorescence staining using SPiDER‐β‐gal to examine the population of senescent cells measured by confocal microscopy. Hoechst 33342 was used for nucleus staining. Scale bars, 50 μm. WT or NLRX1 KO MEFs at passage 4 were treated Nigericin for 1 h after primed LPS for 4 h for activation of inflammasome. (h) ELISA assay to detect IL‐1β and (i) IL‐6 secretion in supernatants from inflammasome‐induced WT or NLRX1 KO MEFs (n = 3). Error bars indicate means ± SD. Data were analyzed by two‐tailed unpaired t test (f) or two‐way ANOVA followed by Tukey's multiple comparisons test. ***p < 0.005, **p < 0.01, *p < 0.05