Fig. 3: CUX1 regulates replicative senescence in ECs.
From: Post-GWAS functional analysis identifies CUX1 as a regulator of p16INK4a and cellular senescence
a, SA-β-gal (top) and γ-H2AX staining (bottom) showing an increase in replicative senescence from p5 ECs (left) to p10 ECs (middle), and a reduction in replicative senescence in CUX1 shRNA knockdown p10 ECs (right) compared to scrambled p10 ECs (middle). Right, quantitative plots are shown for both β-gal+ cells (%) in SA-β-gal staining (top) and γ-H2AX foci/cells (%) with γ-H2AX staining (bottom). Data for SA-β-gal and γ-H2AX staining represent n = 3 biologically independent experiments. b, Immunoblots and qPCR showing increased expression of CUX1 and p16INK4a in p10 ECs compared to p5 ECs. Data for immunoblot analysis represent n = 3 biologically independent experiments; data for qPCR analysis represent n = 3 biologically independent samples, each performed in duplicate. c, PCR-based analysis showing significant decrease in telomeric length from p5 to p10 ECs. Data for PCR analysis represent n = 4 biologically independent samples. d, Immunoblots and qPCR showing that shRNA knockdown of CUX1 resulted in decreased expression of p16INK4a in p10 human ECs. Data for immunoblot analysis represent n = 3 biologically independent experiments; data for qPCR analysis represent n = 3 biologically independent samples, each performed in duplicate. e, qPCR showing significant downregulation of SASP genes, IL-6, IL-1β and ICAM1 in CUX1 shRNA knockdown p10 ECs. Data for qPCR analysis represent n = 3 biologically independent samples, each performed in duplicate. f,g, Decrease in both BrdU incorporation (f) and percentage of S/G2/M cell numbers (g) in p10 ECs (middle) versus p5 ECs (left) indicated an increase in replicative senescence. Knockdown of CUX1 in p10 ECs (right) resulted in recovery from both decreased BrdU incorporation and reduced percentage of S/G2/M cell numbers, indicating a blockage in cellular senescence in CUX1 shRNA knockdown p10 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.).
