Fig. 4

Exosomal circLPAR1 regulates the translation of BRD4 by binding to eIF3h. The circLPAR1 and control lentiviral vectors were transfected separately into DLD1 cells and used as the circLPAR1 group and NC group, respectively. Exosomes were isolated from DLD1 cells transfected with circLPAR1 and control lentiviral vector separately and used as the circLPAR1-Exos and NC-Exos group, respectively. A The cellular location of circLPAR1 (red) in HCT116 (left) and DLD1 (right) cells as determined by FISH. Scale bar, 25 μm. Nuclei were stained with DAPI (blue). B Flow chart showing circLPAR1 pull-down results using the MS2-tagging system. C HCT116 cells were transfected separately with circLPAR1, control or MS2-labelled circLPAR1 expression plasmids and called circLPAR1, NC and circLPAR1-MS2, respectively. The relative expression of circLPAR1 as detected by RT-qPCR in NC, circLPAR1 and circLPAR1-MS2 cells. D Co-transfection of circLPAR1-MS2 and MS2-CP into HCT116 cells. Representative fluorescence images of circLPAR1-MS2 (green) and MS2-CP (red). Scale bar, 50 μm. E The enrichment of circLPAR1 in complex with MS2-CP-Flag as measured by RT-qPCR. F Correlation between the level of circLPAR1 and 15 differentially expressed proteins based on 25 pairs of colorectal cancer tissues and NATs. G Immunofluorescence assessment of circLPAR1 (red) and eIF3h (green) colocalization in DLD1 cells. Scale bar, 20 μm. H RIP assay with an anti-eIF3h antibody or control IgG performed on circLPAR1/NC cells (left) or circLPAR1/NC-Exos (right). The expression of circLPAR1 in the immunoprecipitates was detected using RT-qPCR. I Model of the interaction between eIF3h and METTL3 to regulate translation of BRD4. (J) The BRD4 protein in circLPAR1/NC cells or circLPAR1/NC-Exos as determined by Western blotting. Statistical significance was assessed using two-tailed Student’s t-test. The values represent mean ± SD. *P < 0.05