Fig. 3. TRIM50 exerted its antitumor effect through directly targeting SNAIL and reversing EMT.

a BEL7402 and HepG2 cells were transfected with TRIM50 plasmid or mock control, the binding between TRIM50 and SNAIL protein were detected by immunoprecipitation. b HCC cells were cultured for 24 h before immunofluorescence assay to detect the expression and colocalization status of TRIM50 (green) and SNAIL (red). c TRIM50 and SNAIL proteins were separately expressed by the in vitro transcription and translation system, and the direct binding between TRIM50 and SNAIL were analyzed by co-IP assay. d, e BEL7402 cells were transfected with TRIM50 plasmid or mock control, and HepG2 cells were transfected Si-TRIM50 or Si-NC, the transfected cells were further cultured for 24 h. e The protein level of SNAIL was detected by western blot and further quantitatively analyzed (d); and the mRNA level of SNAIL was detected by qRT-PCR (e). f BEL7402 cells were transfected with TRIM50 plasmid or mock control and further cultured for 24 h before cyclohexamide (CHX) was put into the transfected cells. The cells were further cultured for 0 h, 2 h, and 4 h before being harvested for western blot assay of SNAIL expression. The band intensities were further quantitatively analyzed (right panel). g BEL7402 cells and HUH7 cells were transfected with TRIM50 plasmid or mock control, and further cultured for 24 h. Transfected cells were treated with MG132 (10 μM) for 4 h before protein lysates were isolated to detect the expression level of TRIM50 and SNAIL by western blot. h Wild-type Myc-tagged TRIM50 or its RING domain deleted mutant(△RING) was co-transfected with SNAIL into HUH cells, and further cultured for 24 h before being harvested for western blot assay of SNAIL. i BEL7402 cells were transfected with TRIM50 plasmid or mock control, and further cultured for 24 h. The expression of E-cadherin, vimentin, and SNAIL was detected by western blot and quantitatively analyzed. j HepG2 cells were transfected with Si-TRIM50 or Si-NC, and further cultured for 24 h. The expression of E-cadherin, vimentin, and SNAIL was detected by western blot and quantitatively analyzed. k BEL7402 cells were transfected with TRIM50 plasmid or mock control, and the cells were further cultured for 24 h before immunofluorescence assay to detect the expression of E-cadherin, β-catenin, N-cadherin, and SNAIL. Similar results were obtained in at least three independent experiments. **P < 0.01 and ***P < 0.001 for statistical analysis of the indicated groups