Fig. 2: The role of CUX1 in regulation of p14ARF, p15INK4b, p16INK4a and ANRIL expression via binding to fSNP rs1537371.
From: Post-GWAS functional analysis identifies CUX1 as a regulator of p16INK4a and cellular senescence
a, ChIP assay demonstrating reduced binding of CUX1 to a DNA fragment containing rs1537371 in CUX1 shRNA knockdown ECs (left), and no specific binding of CUX1 to two randomly selected DNA fragments as controls (con; right). CUX1-Ab, anti-CUX1 antibody; IgG-Ab, anti-IgG antibody as an isotype control; NS, not significant. Data for ChIP assay represent n = 3 biologically independent experiments. b, Sequencing analysis showing significant enrichment of the A allele versus the C allele in ChIP DNA compared to input DNA (n = 3), with P = 0.010. c, AIDP–Wb demonstrating specific binding of CUX1 to rs1537371, with risk allele A binding more CUX1 than nonrisk allele C. T is a very rare allele. Data for AIDP–Wb represent n = 3 biologically independent experiments. d, CUX1-dependent luciferase reporter assay in 293T cells showing luciferase activity in CUX1 shRNA knockdown (left) and CUX1-overexpressed ECs (right). pLVX-CUX1, CUX1 expression vector; rs1537371-A, luciferase reporter construct pGL3 (basic promoter vector, Promega); con, negative control. Data for this assay represent n = 6 biologically independent samples. e, qPCR (left) and immunoblot (right) showing downregulation of CUX1 in human ECs by shRNA knockdown. α-Tubulin was used as a loading control. Data for qPCR analysis represent n = 4 biologically independent samples, each performed in triplicate. Data for immunoblot analysis represent n = 3 biologically independent experiments. f, qPCR showing downregulation of p14ARF, p15INK4b, p16INK4a and ANRIL expression in CUX1 shRNA knockdown human ECs. Data for qPCR analysis represent n = 4 biologically independent samples, each performed in triplicate. g, Immunoblot analysis showing downregulation of p14ARF, p15INK4b and p16INK4a expression in CUX1 shRNA knockdown human ECs. Data for immunoblot analysis represent n = 3 biologically independent experiments. P values were calculated using two-tailed Student’s t-test, and all data are presented as mean ± s.e.).
