Fig. 3

ZCRB1 promoted circHEATR5B formation, thereby inhibiting glycolysis and proliferation in GBM cells. A The expression of nascent circHEATR5B in response to ZCRB1 transfection in U251 and U373 cells. Data are presented as the mean ± SD (n = 3, each group). ^***P < 0.001 vs. OV-ZCRB1-NC group; ^#P < 0.05, ^##P < 0.01 vs. sh-ZCRB1-NC group by one-way ANOVA. B Upper panel, primer (a-f) designs for RIP assays. Lower panel, the enrichment of ZCRB1 on circHEATR5B flanking regions was detected by RIP assays. β-actin served as the negative control. Data are presented as the mean ± SD (n = 3, each group). ^***P < 0.001 vs. other anti-ZCRB1 groups except e; ^###P < 0.001 vs. other anti-ZCRB1 groups except b by one-way ANOVA. C Upper panel, biotinylated probe (Bio1–5) designs for RNA pull-down assay. Lower panel, western blot assays showed the level of ZCRB1 pulled down by biotinylated probes. D Schematic of Alu elements in circHEATR5B flanking sequences. Red arrows indicated the polarity of Alu elements. Red arc lines indicated the only pair of inverted repeated Alu elements within 1 kb with their identities and minimum free energies (ΔG) listed below. E Left panel, schematic of plasmids with wild-type Alu elements (Alu-WT) and deletion constructs (Alu-Del-1/2/3). SA, splicing acceptor; SD, splicing donor. Right panel, the expression of circHEATR5B in U251 cells transfected with Alu plasmids and OV-ZCRB1 plasmids. Data are presented as the mean ± SD (n = 3, each group). ^***P < 0.001 vs. Alu-WT in OV-ZCRB1-NC group; ^###P < 0.001 vs. Alu-WT in OV-ZCRB1 group by one-way ANOVA. F and G The effects of ZCRB1 and circHEATR5B on glucose consumption (F) and lactate production (G) in U251 and U373 cells. H The effects of ZCRB1 and circHEATR5B on ECAR in U251 and U373 cells. I The effects of ZCRB1 and circHEATR5B on proliferation in U251 and U373 cells. Data are presented as the mean ± SD (n = 3, each group). ^**P < 0.01, ^***P < 0.001 vs. OV-ZCRB1 + sh-circHEATR5B-NC group by one-way ANOVA