Figure 4.

Downregulation of MDR1/P-gp by Twist1 silencing in HeLa cells. Two stably transfected clones of HeLa cells, HeLa/sh-NC and HeLa/sh-Twist1, were established as described in Materials and methods. (A) Cell lysates were subjected to SDS-PAGE followed by western blotting using specific antibodies against Twist1 and MDR1/P-gp. GAPDH served as a loading control. HeLa/sh-Twist1 cells showed no expression of Twist1 and decreased expression of MDR1/P-gp. (B) Quantitative analyses for protein levels were shown and the data represent the mean ± SEM of three independent experiments. ^*P<0.05 vs. parental HeLa cells. (C) Immunofluorescence staining of Twist1 and MDR1/P-gp in HeLa/sh-NC and HeLa/sh-Twist1 cells. Cells were double-stained for Twist1 (red) and MDR1/P-gp (green). Nuclei were counterstained blue with DAPI. HeLa/sh-NC cells showed strong cytoplasmic and nuclear staining for Twist1, cytoplasmic and membrane staining for MDR1/P-gp, whereas HeLa/sh-Twist1 cells showed weak Twist1 and MDR1/P-gp staining.