Fig. 7: TRIM65 functions as an oncoprotein through K41 ubiquitination of BTG3.

A Western blotting analysis of 3 groups for transfection into ACHN cells. B CCK8 assay. Cells were transfected with the indicated vectors, then subjected to CCK8 analysis. C Low serum assay. At the indicated times, cells were trypsinized and counted. Data are presented as mean ± SEM (n = 3). D Colony formation assay. After 10–14 days, cells grown in 6-well plates were fixed and stained with 2% crystal violet. The number of cell colonies were calculated. E Soft agar colony formation assay. 3 groups of ACHN cells were seeded in 6-well plates. 15–20 days later, cells were imaged, colonies larger than 50 μm were counted, and calculated the area. F The number of cell colonies were calculated in eight random selected views. Data are presented as mean ± SEM (n = 8). G The average area of colonies was calculated in eight random selected views. H Cell cycle analysis of indicated ACHN cells via flow cytometry after PI staining. Data are presented as mean ± SEM (n = 8). n.s. p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t-test.