Fig. 5

Circ-hnRNPU inhibits dual NONO activity in regulating c-Myc transactivation and mRNA stabilization. a and b, Dual-luciferase assay using a reporter containing three canonical c-Myc binding sites (a), ChIP and qPCR (b, normalized to input) assays indicating c-Myc transactivation and enrichment on target gene promoters in MKN-45, AGS, and HeLa cells stably transfected with empty vector (mock) or circ-hnRNPU, and those co-transfected with NONO or c-Myc (n = 5). c and d, Dual-luciferase assay (c), western blot (d, left panel), and real-time qRT-PCR (d, normalized to β-actin, right panel) assays showing promoter activity or expression of GALNT6 and MGAT1, as well as circ-hnRNPU or downstream targets (FN1 and GLUT1) levels in AGS cells stably transfected with mock or circ-hnRNPU, and those co-transfected with NONO or c-Myc. e, Venn diagram (left pane) and CLIP-seq peak (middle panel) indicating comprehensive analysis of 112 altered genes in RNA-seq and NONO-binding targets (GSE90650). RIP and real-time qRT-PCR assays (right panel) revealing endogenous NONO binding to 3'-UTR of hnRNPU, GALNT2, and GALNT6 in AGS cells. f and g, Dual-luciferase assay using a 3'-UTR reporter containing four canonical NONO binding sites (f), RIP and real-time qRT-PCR (g) assays showing the activity and enrichment of NONO on 3'-UTR of hnRNPU, GALNT2, and GALNT6 in MKN-45, AGS, and HeLa cells stably transfected with mock or circ-hnRNPU, and those co-transfected with NLS-mutant (mNLS) form of NONO (n = 5). h, Real-time qRT-PCR (normalized to β-actin, n = 5) assays showing the mRNA stability of hnRNPU, GALNT2, and GALNT6 in AGS cells stably transfected with mock or circ-hnRNPU, and those co-transfected with mNLS form of NONO or treated with actinomycin D (5 μg/ml) as indicated. i, Western blot assay indicating the expression of hnRNPU, GALNT2, and GALNT6 in AGS cells stably transfected with mock or circ-hnRNPU, and those co-transfected with mNLS form of NONO. Student’s t test or analysis of variance compared the difference in a-h. *P < 0.05, **P < 0.01. Data are shown as mean ± s.e.m. (error bars) or representative of three independent experiments in a-i