Fig. 4
Circ-hnRNPU inhibits interaction of NONO with c-Myc in gastric cancer cells. a, Immunofluorescence assay showing the localization of NONO and c-Myc protein (arrowheads) in AGS cells stably transfected with empty vector (mock), NONO, or c-Myc. Scale bar: 10 μm. b, Schematic diagram, co-IP and western blot assays indicating the interaction between full-length or truncations of recombinant GST-tagged c-Myc and MBP-tagged NONO proteins. c, Co-IP and western blot assays revealing the direct interaction between recombinant MBP-tagged NONO and GST-tagged c-Myc proteins, with or without treatment by in vitro generated circ-hnRNPU. d, BiFC assay showing the interaction between NONO and c-Myc (arrowheads) in AGS cells stably transfected with mock, circ-hnRNPU, scramble shRNA (sh-Scb), or sh-circ-hnRNPU #1. Scale bar: 10 μm. e, Representative images (left panel) and quantification (right panel) of immunofluorescence assay revealing the co-localization of NONO and c-Myc (arrowheads) in AGS cells stably transfected with mock or circ-hnRNPU. Scale bar: 10 μm. f, Immunofluorescence (lower left panel), RIP and western blot (lower right panel) assays showing the localization (arrowheads) and interaction of NONO with circ-hnRNPU or c-Myc protein in AGS cells transfected with Flag-tagged wild-type (WT) or NLS-mutant (mNLS) form of NONO as indicated (upper panel). Student’s t test compared the difference in e. **P < 0.01. Data are representative of three independent experiments in a-f