Fig. 5

CircHAS2 interaction with USP10 to enhance p53 Ubiquitination. A Schematic diagram of USP10 full-length and truncations. B The RIP assay was performed in SW620 cells using anti-Flag antibodies to assess the expression levels of circHAS2 following transfection with USP10 full-length and truncations. C The RNA pull-down assays were performed using biotin-labeled circHAS2 probes to capture interacting proteins and detect the expression of USP10 full-length and truncations by representative Western blotting images in SW620 cells. D The direct interaction between USP10 and p53 was analyzed by Co-IP assays with USP10 and p53 antibodies in SW620 cells. E The Co-IP assays were conducted under the specified treatment conditions using USP10 and p53 antibodies in SW620 cells. F After indicated treatments were treated with various durations of cycloheximide (0.1 mg/ml) in SW620 cells, and the expression of p53 was detected by Western blotting. GAPDH was employed as an internal control. G CircHAS2 modulated the levels of p53 ubiquitination through the interaction with USP10. SW620 cells were transfected with the indicated constructs and subjected to MG132 (50 mM) for 4 h before harvest to evaluate the ubiquitination levels of p53. H Representative images of the FISH assay revealed the localization of p53 after indicated treatments with the target probe labeled with Cy3 and nuclei stained with DAPI in SW620 cells (scale bar: 20 μm). I Quantification of cells with different p53 subcellular localization after indicated treatments in SW620 cells. Nuc, Nucleus only; Cyto + Nuc, both cytoplasm and nucleus. J SW620 cells were transfected with the indicated constructs and subjected to MG132 treatment to evaluate the ubiquitination levels of cytoplasmic or nuclear p53. K Relative p53 and p21 expressions were verified after indicated treatments in SW620 cells. L Representative Western blotting images with indicated treatments in SW620 cells for Flag, p53, and p21 proteins. GAPDH was used as an internal control. Statistical significance in two-group experiments was assessed using a two-sided Student’s t-test. Data are represented as mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significance