Fig. 5

SLUG stabilizes SOX2, leading to ALDH activation and anoikis resistance. A, B Cells were transfected with SLUG expression vector or siRNA specific to SLUG for 48 h, and then whole-cell lysates were prepared for immunoblot analysis. Anti-myc antibody was used to detect myc-tagged SLUG (A right). Arrow indicates endogenous SLUG (B). C Cells were transfected for 48 h with a SLUG expression vector or siRNA specific to SLUG, and then lysed for real-time qPCR analysis. Values represent mean ± SD. ***P < 0.001. D PC3 cells were co-transfected with SLUG expression vector and siRNA specific to TWIST1 for 48 h, and then lysed for real-time qPCR analysis of SOX2 expression. Values represent mean ± SD. **P < 0.01; ***P < 0.001 compared with siControl + Vector; ^§§§P < 0.001 compared with siControl + SLUG. E PC3 cells were transfected with siRNA for 42 h and then treated with MG132 or vehicle control (0.1% DMSO) for 6 h before lysates preparation for immunoblot analysis. Values represent mean ± SD. *P < 0.05 compared with siControl + DMSO; ^§P < 0.05 compared with siSLUG + DMSO. Densitometric quantification was performed on the immunoblot using GAPDH as a loading control. The mean relative density from three independent experiments is shown (A, B, E). F Co-immunoprecipitation analysis of the interaction of SLUG with SOX2 in HEK293E cells transfected with SLUG