Extended Data Fig. 6: Effects of glucose limitation and metformin on ESCRT proteins.
From: Metformin inhibits nuclear egress of chromatin fragments in senescence and aging
a, Related to Fig. 3a, western blotting of proteins involved in nuclear egress upon glucose starvation. b, Related to Figs. 3b and 3c, the cells were analyzed by RT-qPCR. Data shown are mean values with s.d. P values were calculated with one-way ANOVA coupled with Tukey’s post hoc test. c, Analyses of AMPK subunit expression levels from mass spectrometry or RNA-Seq. Results shown are mean values with s.d. and are from three biological replicates. P values were from unpaired two-tailed Student’s t-test. d, Cells with indicated genotypes and treatments were lysed in 1% SDS buffer followed by 95 °C boiling. SDS was then diluted to 0.1%, followed by immunoprecipitation with p-AMPK substrate motif antibody and immunoblotting. This denaturing IP condition ensures that protein-protein interactions were disrupted and thus the ALIX brought down was a direct consequence of AMPK phosphorylation. See Methods for details. e, ALIX harbors five putative Rxx(pS/pT) motifs typical for AMPK substrates. f, IMR90 cells were cultured in 5 mM glucose media in the presence of 1 mM AICAR for indicated days, and were analyzed by immunoblotting. g, Metformin-treated IMR90 cells were analyzed for the expression levels of components of ESCRT-I, II, and III. h, IMR90 cells stably expressing Flag-ALIX were left untreated or treated with metformin or compound 991. The cells were subjected to denaturing IP using the condition of d, followed by immunoblotting. i, IMR90 cells stably expressing Flag-ALIX were treated with metformin and harvested at indicated days to be analyzed by immunoblotting. Results shown in this figure are representative of at least three independent experiments.
