FIGURE 4.

HK2 regulated EMT and oxaliplatin resistance via Twist1 in CRC cells. (A) RKO cells were transfected with Flag‐tagged HK2 plasmid or HA‐tagged Twist1 for 48 h and then lysed and analysed by immunoprecipitation using anti‐Flag or anti‐HA magnet beads, followed by immunoblotting with the indicated antibody. (B) Immunofluorescence was performed to detect HK2 and Twist1 in RKO cells. Scale bar = 25 μm. (C) RKO cells were treated with 100 μM MG132 for 4, 6, 8, 10 and 12 h, and Western blot was performed to detect the expression of Twist1. (D) Western blot was performed to detect the expression of Twist1 after treatment with HK2 after CHX (50 μg/ml) for 4, 6, 8 and 12 h in RKO cells. (E) Coimmunoprecipitation was performed to detect endogenous ubiquitin levels in HK2 knockdown RKO cells by using anti‐Twist1 antibody, and RKO cells were cotransfected with the indicated plasmids and then lysed and analysed by immunoprecipitation using anti‐Flag magnet beads followed by immunoblotting with anti‐Flag or anti‐HA antibody. (F) HK2 knockdown RKO cells were transfected with Twist1 or control plasmid for 24 h and collected for cell lysates. The expression levels of EMT‐related TJP1, E‐cadherin, vimentin, and Twist1 were determined by Western blot. (G) HK2 knockdown RKO cells were transfected with Twist1 or control plasmid for 24 h and treated with oxaliplatin (50 μM) for 48 h. Flow cytometry was performed to analyse the apoptotic rate in the indicated RKO cells. **0.001 < p < 0.01; ***p < 0.001. Data are represented as the mean ± SEM