Fig. 5: YBX1-K26R overexpression partially rescued the inhibitory effect of SENP1-knockdown-mediated CRC cell proliferation, migration and tumorigenesis.
A SENP1 could specifically remove SUMOylation modification at the K26 site of the YBX1 protein. And IP assays indicated the SUMOylation level of Flag-YBX1 in HEK293T cells transfected with indicated plasmids. B Flag-YBX1 or Flag-YBX1K26R overexpression had influence on p-AKT level in CRC cells stably transfecting with sh-SENP1. C SENP1 knockdown had influence on the interaction between cellular endogenous DDX5 and YBX1 in CRC cells. D SENP1 knockdown had influence on the interaction between DDX5 and Flag-YBX1 or Flag-YBX1K26R in CRC cells. E Cell proliferation capacity was compared in Flag-YBX1-expressing or Flag-YBX1K26R-expressing CRC cells under the endogenous SENP1 knockdown. All data are presented as the means ± SD of three independent experiments. F Cell proliferation capacity was compared in Flag-YBX1-expressing or Flag-YBX1K26R-expressing CRC cells under the endogenous SENP1 knockdown. Representative images of the colony formation of the indicated cells in top and quantification of clone numbers in bottom. All data are presented as the means ± SD of three independent experiments. G Cell migration capacity was compared in Flag-YBX1-expressing or Flag-YBX1K26R-expressing CRC cells under the endogenous SENP1 knockdown. Representative images of the wound healing of the indicated cells in left and quantification of clone numbers in right. All data are presented as the means ± SD of three independent experiments. Scale bar: 100 μm. H–J The subcutaneous tumor capacity was compared in Flag-YBX1-expressing or Flag-YBX1K26R-expressing CRC cells under the endogenous SENP1 knockdown. Tumor images (H), volume (I), and weight (J) were shown. Data were presented as the mean ± SD (n = 5 mice). ns no statistical significance, *p < 0.05, **p < 0.01. S-Flag-YBX1 SUMOylated Flag-tagging YBX1, S-YBX1 SUMOylated YBX1, IP immunoprecipitation, Input Same account of cell lysate to load, sh-Control short hairpin RNA targeting to empty vector, sh-SENP1 short hairpin RNA targeting to SENP1, Control Flag-vector.
