Fig. 3

Circ-hnRNPU interacts with and induces cytoplasmic retention of NONO in gastric cancer cells. a, Coomassie bright blue staining (left panel) and Venn diagram (right panel) showing the differential proteins pulled down by biotin-labeled sense (S) or antisense (AS) probe targeting junction sites of circ-hnRNPU from the lysates of AGS cells, and overlapping analysis of proteins identified by mass spectrometry (MS) with established RBP from RBPDB (http://rbpdb.ccbr.utoronto.ca) and c-Myc-binding proteins derived from BioGRID and IntAct (https://www.ebi.ac.uk/intact) databases. b, Western blot (upper panel) and RT-PCR (lower panel) assays indicating the NONO protein or circ-hnRNPU pulled down by biotin-labeled S or AS probes targeting junction site of endogenous circ-hnRNPU from lysates of AGS cells. c, RIP (upper panel) and western blot (lower panel) assays using NONO antibody showing the interaction of NONO with circ-hnRNPU or hnRNPU mRNA in MKN-45 and AGS cells stably transfected with empty vector (mock) or circ-hnRNPU. d, RNA EMSA determining the interaction between recombinant GST-tagged NONO protein and biotin-labeled circular probe of circ-hnRNPU, with or without competition using an excess of unlabeled circular probe. e, Schematic diagram of NONO truncations (left panel) and in vitro binding assay (middle and right panels) depicting the recovered circ-hnRNPU levels detected by RT-PCR after incubation with full-length or truncation forms of GST-tagged or Flag-tagged recombinant NONO protein validated by western blot. f–h, Representative images and quantification of dual RNA-FISH and immunofluorescence (f), real-time qRT-PCR (g, normalized to β-actin), and western blot (h) assays indicated the localization of circ-hnRNPU and NONO (arrowheads), transcript and protein levels of NONO in MKN-45 and AGS cells stably transfected with mock, circ-hnRNPU, lin-hnRNPU, scramble shRNA (sh-Scb), or sh-circ-hnRNPU. Scale bar: 10 μm. Student’s t test compared the difference in f and g. **P < 0.01. Data are representative of three independent experiments in b-h