Extended Data Fig. 1: Characterization of CCF and the roles of ESCRT-III in mediating CCF nuclear egress.

a, IMR90 cells were subjected to IR (20 Gy) and harvested at indicated time points for immunoblotting. b, Quantification of CCFs. Data presented are mean values with s.d. from four randomly selected fields with over 200 cells. c, Related to Fig. 1b, additional images of CCF and nuclear membrane blebs. Scale bar: 1 μm. d, Quantification of the length of nuclear membrane blebs, with mean values labeled. Results are from three independent experiments. e, Related to Fig. 1d, additional images of CHMP4B and ALIX localization at the nuclear membrane blebs of senescent cells. Scale bar: 1 μm. f, Related to Fig. 1d, antibody validation using KO cells. CHMP4B or ALIX-deficient proliferating IMR90 cells were stained with CHMP4B or ALIX antibody and imaged under a confocal microscopy. Scale bar: 1 μm. g, CHMP4B and ALIX are not enriched at the CCFs of senescent cells. Scale bar: 1 μm. Representative images from three independent experiments are shown. h, IMR90 cells were stably infected with construct expressing shRNA against non-targeting control (NTC), cGAS, or STING. The cells were analyzed by immunoblotting. i, Cells as in h were subjected to etoposide-induced senescence, followed by RT-qPCR analyses. Results shown are mean values with s.d., normalized to Lamin A/C. j, IMR90 cells were subjected to IR-induced senescence. H151, a STING inhibitor, was added on day 6 and the cells were harvested on day 14 and analyzed by RT-qPCR. Results shown are mean values with s.d., normalized to Lamin A/C. P values in this figure were calculated with one-way ANOVA coupled with Tukey’s post hoc test. Results in this figure were representative of at least three independent biological replicates.

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