Fig. 5

Circ-HuR suppresses HuR expression, growth, and invasion of gastric cancer cells via repressing CNBP transactivation. a Dual-luciferase assay revealing the transactivation of CNBP in AGS and MKN-45 cells stably transfected with empty vector (mock) or CNBP, and those co-transfected with circ-Mock or circ-HuR. b ChIP and qPCR assays showing the changes in binding of CNBP to HuR promoter in AGS and MKN-45 cells stably transfected with mock or CNBP, and those co-transfected with circ-Mock or circ-HuR. c and d Dual-luciferase (c) and real-time qRT-PCR (d) assays indicating the activity of HuR promoter with wild type (WT) or mutant (Mut) CNBP binding site and transcript levels (normalized to β-actin, n = 4) of HuR in AGS and MKN-45 cells stably transfected with mock or CNBP, and those co-transfected with circ-Mock or circ-HuR. e Western blot assay showing the expression of CNBP, HuR, CCND2, and CTNNB1 in AGS cells stably transfected with CRISPRi-Scb or CRISPRi-CNBP #1, and those co-transfected with mock, CNBP, circ-Mock, or circ-HuR. f MTT colorimetric assay indicating the viability of AGS and MKN-45 cells stably transfected with mock or CNBP, and those co-transfected with circ-Mock or circ-HuR. g and h Soft agar (g) and matrigel invasion (h) assays showing in vitro growth and invasion of AGS and MKN-45 cells stably transfected with mock or CNBP, and those co-transfected with circ-Mock or circ-HuR. ANOVA analyzed the difference in (a-d and f-h). *P < 0.01 vs. mock+ circ-Mock. Data are shown as mean ± SEM (error bars) and representative of three independent experiments in (a-h)