Fig. 4

CircHEATR5B encoded a novel protein termed HEATR5B-881aa. A The cytoplasmic and nuclear distribution of circHEATR5B in U251 and U373 cells. β-actin and U6 served as cytoplasmic and nuclear RNA markers, respectively. B The localization of circHEATR5B in U251 and U373 cells by FISH assays. Red, circHEATR5B; blue, DAPI nuclear staining. Scale bars, 10 μm. C Schematic of putative protein encoded by circHEATR5B. Red arrow, the junction site; black arrow, the ORF sequences; red sequence, the unique amino acid encoded by the junction-spanning ORF; blue sequences, the putative IRES sequences. D Upper panel, schematic of plasmids with wild-type, mutated, or truncated IRES inserted between the Rluc stop codon and Fluc start codon. Lower panel, the luciferase activity of Fluc/Rluc was detected by dual-luciferase reporter assays. Data are presented as the mean ± SD (n = 3, each group). ^***P < 0.001 vs. #2 group by one-way ANOVA. E Left panel, plasmid constructions for detection of the coding ability of circHEATR5B-ORF. Right panel, FLAG tag and HEATR5B-881aa antibodies were used to detect HEATR5B-881aa expression in U251 cells transfected with plasmids. F The expression of HEATR5B-881aa in response to circHEATR5B transfection in U251 and U373 cells. G The expressions of HEATR5B protein and HEATR5B-881aa in response to ZCRB1 transfection in U251 and U373 cells. H The expression of HEATR5B-881aa in NBTs, LGGTs, and HGGTs groups. Data are presented as the mean ± SD (n = 9, each group). ^***P < 0.001 vs. NBTs group; ^###P < 0.001 vs. LGGTs group by one-way ANOVA. The expression of HEATR5B-881aa in NHA, U251, and U373 cells. Data are presented as the mean ± SD (n = 3, each group). ^ΔΔP < 0.01 vs. NHA group by one-way ANOVA. I Mass spectrometry analysis of HEATR5B-881aa in GBM cells