Fig. 4
CircCDKN2B-AS1 regulates the stability of HK2 mRNA by recruiting IMP3. a qRT-PCR after RIP assays showing the 3’UTR of HK2 mRNA recruited by the IMP3 protein from the lysates of SiHa cells (mean ± SEM, n = 3, unpaired Student’s t-test). b The levels of HK2 mRNA expression following IMP3 knockdown using two siRNAs or a negative control siRNA in SiHa cells (mean ± SEM, n = 5, one-way ANOVA). c Left: the levels of HK2 mRNA expression after transfection with two circCDKN2B-AS1 siRNAs or a negative control siRNA in SiHa and CaSki cells (mean ± SEM, nSiHa = 5, nCaSki = 3, one-way ANOVA). Right: the levels of HK2 protein expression in SiHa and CaSki cells. d Actinomycin D was added to block RNA synthesis. The levels of HK2 mRNA in SiHa and CaSki cells with circCDKN2B-AS1 knockdown or not (mean ± SEM, n = 4, unpaired Student’s t-test). e Left: the ECAR in SiHa cells with stably transfected sh-circCDKN2B-AS1, or a negative control shRNA, or sh-circCDKN2B-AS1 plus HK2 overexpression plasmids, using Seahorse XFe assays. Middle: basal glycolysis and compensatory glycolysis. Right: Quantification of the %PER. (mean ± SEM, n = 6, one-way ANOVA). f Left: the ECAR in SiHa cells with lentivirus stably overexpressing circCDKN2B-AS1, or a negative control lentivirus, or lentivirus stably overexpressing circCDKN2B-AS1 plus HK2 siRNA, using Seahorse XFe assays. Middle: basal glycolysis and compensatory glycolysis. Right: Quantification of the %PER. (mean ± SEM, n = 3, one-way ANOVA). g qRT-PCR following RIP assays showing the 3’UTR of HK2 mRNA recruited by the IMP3 protein from the lysates of SiHa cells with or without stable circCDKN2B-AS1 knockdown (mean ± SEM, n = 3, unpaired Student’s t-test). h qRT-PCR following RIP assays showing the 3’UTR of HK2 mRNA recruited by the IMP3 protein from the lysates of HEK293 cells following transfection with the WT/mutant circCDKN2B-AS1 overexpression or the negative control vector (mean ± SEM, n = 3, unpaired Student’s t-test). *P < 0.05, **P < 0.01, ***P < 0.001