Fig. 3

CircDIDO1 encodes a novel 529 aa protein. a) The predicted putative open reading frame (ORF) in circDIDO1. b) Detection of the expression of nascent protein encoded by circDIDO1 overexpressing vector with Flag tag (circDIDO1-Flag) by Western blot with Flag antibody. c) The nascent protein was purified from circDIDO1-Flag transfected 293 T cells by Co-IP with Flag antibody and separated by SDS-PAGE. The image of Coomassie brilliant blue staining for the gel was shown. The nascent protein was harvested for LC–MS/MS analysis to identify its peptide sequence. The front amino acid sequence (upper right) and the end amino acid sequence (lower right) were shown. d) Schematic diagram of domains of DIDO1 protein isoforms and DIDO1-529aa. The subcellular distribution of DIDO1-529aa in GC cells was examined by immunofluorescence. Scale bars = 25 μm. e–i) Cell growth curve (e), colony formation (f), transwell migration (g), matrigel invasion assays (h) and flow cytometry analyses of apoptosis (i) in control or linear form vector of DIDO1-529aa (DIDO1-529aa-Flag) overexpression GC cells. Data are shown as means ± SD (n = 3, **P < 0.01, *P < 0.05)