Fig. 2: ATF6-deficient hMSCs exhibit phenotypes associated with premature cellular senescence.

a Growth curve showing the cumulative population doublings of WT and ATF6-deficient hMSCs over passages. b Immunostaining of Ki67 showing decreased cell proliferation of ATF6-deficient hMSCs compared to WT control; the percentages of Ki67 positive cells were shown in the bottom panel. Scale bar, 20 μm. Data were presented as the mean ± SEM, n = 3, ***P < 0.001. c Cell cycle profiles showing the decreased S-phase cells in ATF6-deficient hMSCs compared to WT control. Data were presented as the mean ± SEM, n = 3, ***P < 0.001. d SA-β-Gal staining of WT and ATF6-deficient hMSCs at passages 5 and 11, respectively. The percentages of SA-β-Gal-positive cells are shown in the bottom panel. Data were presented as mean ± SEM, n = 3, ns not significant, ***P < 0.001. Scale bar, 20 μm. e The heatmap showing the qRT-PCR analysis of the expression of senescence marker genes in WT and ATF6-deficient P10 hMSCs. Transcript levels were normalized to the WT control. f Western blotting analysis of P16, LAP2 and Lamin B1 in WT and ATF6-deficient hMSCs. β-Actin was used as the loading control. g Analysis of luciferase activity by imaging system (IVIS) showing premature attrition of ATF6-deficient hMSCs in vivo. WT (1 × 10⁶, left) and ATF6-deficient (1 × 10⁶, right) hMSCs (Passage 10) infected with luciferase lentivirus were transplanted into the tibialis anterior (TA) muscles of four immunodeficient mice. Luciferase activities were imaged and quantified at days 0, 2, 4, and 7 after transplantation. Data were presented as the ratios of ATF6^−/− to ATF6^+/+ (fold), mean ± SD, n = 4, **P < 0.01. See also Supplementary Figure S3