Extended Data Fig. 9: Accumulated DNA damage by Bmal1 depletion triggers senescence and inflammation.
a, Clonogenic survival experiments showing the colony formation capacity of NIH3T3 cells expressing shNC or shBmal1 after treatment with the indicated doses of CPT with or without dNTPs. b, Representative images and quantification of IHC staining for the indicated markers of livers from 6-month-old WT and whole-body Bmal1 knockout (BKO) mice. More than three fields for each of three samples were counted. c, Representative IF images of primary MEFs at passage 5 stained with the anti-γH2ax antibody and quantification of the number of γH2ax foci in individual cells. n = 53 cells counted for each condition taken from three independent experiments. Scale bar, 15 μm. d, qRT-PCR showing the expression of Bmal1, G6pdx, and the senescent markers P21 and P16, in shNC or shBmal1 MEFs at passage 5. e, Representative IHC images in mouse liver sections stained with anti-F4/80 antibody and optical density-based quantification. WT and LBKO mice with AAV8-GFP or AAV8-G6pdx were subjected to paraquat (20 mg kg−1) treatment and euthanized after 5 days. More than three fields for each of three independent samples were counted. Scale bar, 100 μm. f, qRT-PCR showing the expression of the indicated SASP genes in shNC- or shBmal1-expressing primary MEFs at passage 5. g, Immunoblotting showing the indicated protein levels in the liver lysates of WT or LBKO mice treated with 20 mg kg−1 paraquat and then with 0 or 5 mg kg−1 SN-011 treatment for 2 days before sacrifice. h-j, qRT-PCR showing the expression of the indicated genes (h,j) and representative images of IHC staining with the anti-F4/80 antibody (i) of livers from mice as treated in g. Scale bar, 100 μm. For a,d,f, n = 3 independent biological samples. For h,j, n = 3 mice per group. Data are mean ± s.e.m. P-values were calculated using RM two-way ANOVA (a), unpaired two-tailed Student’s t-test (b), two-way ANOVA with Sidak’s test (d,f,h,j) and one-way ANOVA with Dunnett’s test (e).
