Fig. 2: Effect of the interaction of TSPY1 with TSPYL5 on cell proliferation associated with the ubiquitin-mediated degradation of p53.

a, b CCK-8 assays showed that the proliferation levels of p53-expressed HEK293 (a) and HepG2 (b) cells that overexpressed TSPY1 and TSPYL5 together were significantly higher than those in cells that overexpressed TSPY1 or TSPYL5 alone. Cell proliferation was analyzed at 24, 36, 48, and 72 h after transfection. The total amount of the TSPY1 and TSPYL5 plasmids used for co-transfection was equal to the individual amount of the TSPY1 or TSPYL5 plasmid used when they were transfected separately. The relative proliferation was presented as the fold change, which was calculated based on the absorbance and was normalized to a control value. c CCK-8 assays showed that the proliferation of p53-null PC-3 cells was not altered after transfecting the plasmids expressing TSPY1 and/or TSPYL5. d–g Immunoblotting assays showed that the overexpression of TSPY1 (d) or TSPYL5 (e) could downregulate p53 level, and the overexpression of both TSPY1 and TSPYL5 (f) induced a more significant reduction in the level of p53 in HEK293 cells. Columns (g) showing the difference of p53 level in HEK293 cells with different conditions (overexpression of TSPY1 or TSPYL5 vs. overexpression of both proteins). The cells were lysed at 48 h after transfection. Data are presented as the mean ± S.D. (n = 3, *p < 0.05). h The overexpression of both TSPY1 and TSPYL5 induced a more significant increase in p53 ubiquitylation relative to the overexpression of TSPY1 or TSPYL5 alone in HEK293 cells. The cells were lysed after 10 μM MG132 treatment for 6 h. i, j Two-step Co-IP assays showed the absence of the TSPYL5-USP7-p53 complex (i), while TSPY1, USP7 and p53 could form a complex in HEK293 cells (j). k Schematic of the USP7 domains and the binding tests of the domains to TSPY1. Co-IP assays showed that TSPY1 could interact with non-p53-specific binding domains of USP7, including the core and C-terminal domains in HEK293 cells