Fig. 3

p113 physically interacts with ZRF1 and BRD4 in NB cells. a Volcano plots showing differentially expressed genes (fold change> 1.5, P < 0.05) in SH-SY5Y cells stably transfected with empty vector (mock) or ecircCUX1. b Coomassie blue staining (left panel) and Venn diagram (right panel) revealing identification of p113-interacting proteins pulled down by p113 or Flag-tag antibody in SH-SY5Y cells stably transfected with 3Flag-tagged p113, and those overlapped with transcription factors (TF) or epigenetic factors derived from ChIP-X and EpiFactors databases. c Co-IP and western blot assays indicating the interaction among p113, ZRF1, and BRD4 in SH-SY5Y and BE(2)-C cells stably transfected with mock, ecircCUX1, scramble shRNA (sh-Scb), or sh-ecircCUX1. d Secondary co-IP assays showing protein interaction among p113, ZRF1, and BRD4 in SH-SY5Y cells stably transfected with HA-tagged p113, Flag-tagged ZRF1, and His-tagged BRD4. e BiFC assay revealing the interaction of p113 with ZRF1 or BRD4 (arrowheads) in SH-SY5Y cells stably transfected with indicated constructs, with nuclei stained by DAPI. Scale bars: 10 μm. f and g Western blot assay (g) validating the knockdown of ZRF1 or BRD4 in SH-SY5Y cells stably transfected with scramble (Scb) or specific sgRNA for CRISPR interference (CRISPRi, f). Wild type (WT) cells were taken as negative controls. h Co-IP and western blot assays indicating the interaction of p113 with ZRF1 or BRD4 in SH-SY5Y cells stably transfected with mock or ecircCUX1, and those co-transfected with CRISPRi sgRNA specific against ZRF1 or BRD4. i Schematic illustration of protein interaction among p113, ZRF1, and BRD4. j Dual-luciferase assay showing the activity of ZRF1 in NB cells stably transfected with mock, ecircCUX1, ecircCUX1 Mut, p113, sh-Scb, or sh-ecircCUX1 (n = 5). Fisher’s exact test for overlapping analysis in b. ANOVA compared the difference in j. *P < 0.05 vs. mock or sh-Scb. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in c-e, g, h and j