Extended Data Fig. 6: Regulation of H2O2-induced premature senescence by CUX1.
From: Post-GWAS functional analysis identifies CUX1 as a regulator of p16INK4a and cellular senescence
A. Western blots and qPCR analysis showing an induction in CUX1 and p16INK4a expression in response to 200 µM H2O2 exposure for 4 hrs in human ECs (middle lane). p53 expression was also induced by H2O2 activation, but in a post-transcriptional fashion. A downregulation of CUX1 by shRNA in human ECs blocks the induction of p16INK4a in response to the H2O2 treatment, but not affect the p53 expression (right lane). Data for Western blot analysis represents three biologically independent experiments (n=3). Data for qPCR analysis represents three biologically independent experiments (n=3), each performed in duplicate. B. SA-β-gal (upper) and γ-H2AX staining (lower) showing an increase in cellular senescence in H2O2-treated ECs (middle panel) and the restoration of senescence following a CUX1 knockdown (right panel) by comparing to the scrambled control (left panel). Quantitative plots for both β-gal positive cells (%) in SA-β-gal staining (upper) and γ-H2AX foci/cells (%) in γ-H2AX staining (lower) are presented on the right side of the panel. Data for SA-β-gal staining and γ-H2AX staining represents three biologically independent experiments (n=3). C. qPCR analysis showing an increased expression of the SASP genes IL-6 and IL-1β in the H2O2-treated ECs (middle lane) and the restoration of the expression of these genes upon a CUX1 shRNA knockdown (right lane) by comparing to the scrambled control (left lane). Data for qPCR analysis represents three biologically independent experiments (n=3), each performed in duplicate.
