Fig. 4: circABCC4 facilitates PKM2 nuclear translocation in CAFs. | Cell Death & Disease

Fig. 4: circABCC4 facilitates PKM2 nuclear translocation in CAFs.

From: Glycolysis reprogramming in CAFs promotes oxaliplatin resistance in pancreatic cancer through circABCC4 mediated PKM2 nuclear translocation

Fig. 4

A Silver staining for RNA pull-down assay with the specific biotin-labeled circABCC4 probe in CAFs lysates. Red arrows indicate the unique differential band precipitated by the circABCC4 probe. B, C Mass spectrometry (B) and western blot (C) analysis of proteins in unique differential bands. PKM2 was identified as a candidate protein interacting with circABCC4. D RNA immunoprecipitation (RIP) assays in CAFs using IgG and PKM2 antibodies. The relative enrichment of circABCC4 was calculated by qRT-PCR. E Dual RNA-FISH and immunofluorescence staining assay indicating the colocalization of circABCC4 (red) and PKM2 (green), with nuclear staining with DAPI (blue). Scale bars, 40 μm. F Western blot of the expression kinetics of PKM2 in CAFs transfected with empty vector, circABCC4, lenti-NC-shRNA, or lenti-circABCC4-shRNA. G Dual RNA-FISH and immunofluorescence staining assay indicating the colocalization of circABCC4 (red) and PKM2 (green), with nuclear staining with DAPI (blue) in CAFs stably transfected with empty vector, circABCC4, lenti-NC-shRNA, or lenti-circABCC4-shRNA for 3 days. Scale bars, 40 μm. H Western blot of the expression kinetics of PKM2 in CAFs’ nucleus and cytoplasm, transfected with empty vector, circABCC4, lenti-NC-shRNA, or lenti-circABCC4-shRNA. I, J Mass spectrometry (I) and western blot (J) analysis of proteins in unique differential bands. KPNA2 (Importin α-1) was identified as a candidate protein interacting with circABCC4. K RNA immunoprecipitation (RIP) assays in CAFs using IgG and KPNA2 antibodies. The relative enrichment of circABCC4 was calculated by qRT-PCR. L Immunofluorescence staining assay indicating the localization of PKM2 (green) with nuclear staining with DAPI (blue) in CAFs. Scale bars, 40 μm. M Western blot of the expression kinetics of KPNA2 in CAFs transfected with empty vector, circABCC4, lenti-NC-shRNA, or lenti-circABCC4-shRNA. N Western blot of the expression kinetics of KPNA2 binding to PKM2 in CAFs in CAFs transfected with empty vector, circABCC4, lenti-NC-shRNA, or lenti-circABCC4-shRNA by immunoprecipitation assay.

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