Fig. 3
CircCDKN2B-AS1 cooperates with IMP3 to promote glycolysis in cervical cancer. a The localization of circCDKN2B-AS1 in cytoplasm in SiHa and CaSki cells by RNA-FISH assays. CircCDKN2B-AS1 was labeled with a specific probe (red) and the nucleus was stained with DAPI (blue). b Left: differential proteins pulled down by the circCDKN2B-AS1 or the oligo probe in SiHa cells. The red box indicating the differential proteins; Right: their overlap with those proteins obtained from the RBPmap analysis. c Western blot after RNA pull-down assays showing the IMP3 protein pulled down by biotin-labeled circCDKN2B-AS1 probes from the lysates of SiHa cells. d qRT-PCR after RIP assays showing circCDKN2B-AS1 recruited by the IMP3 protein from the lysates of SiHa cells (mean ± SEM, n = 3, unpaired Student’s t-test). e Upper: the ECAR in SiHa and CaSki cells transfected with two siRNAs or a negative control siRNA with Seahorse XFe assays. Lower: quantification of basal glycolysis and compensatory glycolysis in two cells (mean ± SEM, n = 6, one-way ANOVA). f Quantification of the %PER from the glycolysis of SiHa and CaSki cells transfected with two siRNAs or a negative control siRNA (mean ± SEM, n = 6, unpaired Student’s t-test). g-i The growth curve (g), apoptosis level (h), migration and invasion (i) of SiHa and CaSki cells after addition of 2-DG or PBS (mean ± SEM, n = 3, unpaired Student’s t-test). j Western blot assay showing the level of IMP3 protein pulled down by biotin-labeled circCDKN2B-AS1 probes from lysates of SiHa cells with or without stable circCDKN2B-AS1 knockdown. k Western blot assay showing the level of IMP3 protein pulled down by biotin-labeled circCDKN2B-AS1 probes from the lysates of HEK293 cells following transfection with the WT/mutant circCDKN2B-AS1 overexpression vector or the negative control vector. l Left: the ECAR in SiHa and CaSki cells transfected with the negative control plasmid, the circCDKN2B-AS1-overexpressing plasmid, and the mutant circCDKN2B-AS1-overexpressing plasmid with Seahorse XFe assays. Right: quantification of basal glycolysis and compensatory glycolysis in SiHa and CaSki cells (mean ± SEM, n = 6, unpaired Student’s t-test). *P < 0.05, **P < 0.01, ***P < 0.001