Fig. 4.

Downregulation of PRKD1 mediated THZ1-induced stabilization of Snail. a HCT-116 cells were treated with 100 nM THZ1 for the indicated times. Phosphorylated and total expression of GSK-3β, p65 and Akt were detected; b HCT116 cells treated with or without 100 nM THZ1 for 4 h, and Snail was immunoprecipitated for detection of GSK-3β, CSN2, and CDK7; c HCT-116 cells were pretreated with inhibitors of NF-κB (10 μM BAY 11-7082, BAY), GSK-3β (10 μM LiCl), PI3K/Akt (10 μM, LY294002, LY), ERK1/2 (10 μM PD98059, PD), EGFR (10 μM AG1478, AG) and p38 MAPK (10 μM SB203580, SB) for 90 min, respectively, followed by 4 h incubation of 100 nM THZ1; d HCT-116 cells treated with or without 100 nm THZ1 for 4 h, and mRNA expression levels of Snail stability-related factors were analyzed by qRT-PCR; e HCT-116 and SW480 cells were treated with or without 100 nm THZ1 for 4 h. Protein levels of PKD1 were analyzed by western blot analysis; f IHC (Snail)-stained paraffin-embedded sections obtained from xenografts based on HCT-116 cells. Red bar = 50 μm; g After 24 h transfection of empty vector or pcDNA/PKD1, HCT-116 or SW480 cells were treated with or without 100 nm THZ1 for 4 h. Protein levels of Snail and PKD1 were analyzed by western blot analysis; h After 24 h transfection of empty vector or pcDNA/PKD1, HCT-116 cells were treated with 50 μg/ml CHX for the indicated times. Protein levels of Snail were analyzed by western blot analysis; i HCT-116 cells were treated as indicated, and further treated with MG132 for 6 h before co-IP experiments. Ser11 phosphorylation of immunoprecipitated Snail protein were detected; j After transfection of pcDNA/Snail WT, pcDNA/Snail S11A, or pcDNA/Snail 2SA for 24 h, HCT-116 cells were further treated with or without 100 nM THZ1 for 4 h. Expression levels of Snail were analyzed by western blot analysis; k After transfection of pcDNA/Snail S11A for 24 h, HCT-116 cells were further treated with 50 μg/ml CHX for the indicated times. Expression levels of Snail were analyzed by western blot analysis. Data were presented as means ± SD from three independent experiments. **p < 0.01 compared with control