Fig. 4: p16INK4a is the downstream mediator of CUX1-regulated senescence.
From: Post-GWAS functional analysis identifies CUX1 as a regulator of p16INK4a and cellular senescence
a, Immunoblot and qPCR analysis demonstrating that overexpression of CUX1 results in increased expression of p16INK4a (middle) in ECs. Increased p16INK4a expression was repressed by p16INK4a shRNA knockdown (right). Data for immunoblot analysis represent n = 3 biologically independent experiments; data for qPCR analysis represent n = 3 biologically independent samples, each performed in duplicate. b, SA-β-gal (top) and γ-H2AX (bottom) staining showing that overexpression of CUX1-induced cellular senescence in human ECs (middle versus left). Downregulation of p16INK4a by shRNA in CUX1-overexpressed human ECs rescued senescent phenotypes (right; n = 3). Quantitative plots for both β-gal+ cells (%) in SA-β-gal staining and γ-H2AX foci/cells (%) in γ-H2AX staining are shown. c, qPCR showing increased expression of SASP genes IL-6, IL-1β and ICAM1 in CUX1-overexpressed ECs (middle versus left). Increased expression of SASP genes IL-6, IL-1β and ICAM1 remained unchanged in CUX1-overexpressed and p16INK4a shRNA downregulated human ECs (right). Data for SA-β-gal and γ-H2AX staining represent n = 3 biologically independent experiments; data for qPCR analysis represent n = 3 biologically independent samples, each performed in duplicate. d,e, Decrease in BrdU incorporation (d) and percentage of S/G2/M cell numbers (e) in CUX1-overexpressed human ECs (middle) demonstrated an increase in cellular senescence. Knockdown of p16INK4a by shRNA in CUX1-overexpressed human ECs (right) resulted in recovery from decreased BrdU incorporation and reduced percentage of S/G2/M cell numbers, indicating blockage of cellular senescence in CUX1-overexpressed and p16INK4a-downregulated human ECs. Data for BrdU incorporation represent n = 12 biologically independent samples; data for cell cycle analysis represent n = 3 biologically independent samples. P values were calculated using two-tailed Student’s t-test, and all data are presented as mean ± s.e.
