Fig. 5: ATM-mediated control of Vimentin and its downstream responses.
a HCT-116 cells were treated with vehicle, CPT (250 nM), KU60019 (3 µM), KU60019 (3 µM) + 250 nM CPT for 36 h; whole cell lysates were prepared and western blotting was performed to determine the expression of pser38 Vimentin and total Vimentin. b Cells were either treated with Vehicle, CPT, SCR + CPT (250 nM), si-Vimentin + CPT (250 nM), and si-Vimentin for 36 h; western blotting was performed to analyze the expression of Vimentin, ATM, and E-cadherin. c HCT-116 cells treated with Vector, Flag-His-ATM wt + CPT (250 nM), CPT (250 nM) and Flag-His-ATM wt for 24 h and subjected to western blot analysis to evaluate the expression of ATM, pser38Vimentin, and total Vimentin. β-actin was used as loading control. d Western blot analysis was performed after HCT cells were treated with vehicle, CPT (250 nM), CPT (250 nM) + AT 7867 (1 μM), CPT (250 nM) + KU60019 (3 μM), CPT (250 nM) + AT 7867 (1 μM) + KU60019 (3 μM), AT 7867 (1 μM), and KU60019 (3 μM). The expression of pser38Vimentin, total Vimentin, p-AKT, AKT, NFκB were analyzed keeping β-actin as loading control. e HCT-116 cells were treated with Vector, CPT (250 nM), CPT (250 nM) + GFP-Vimentin, CPT (250 nM) + GFP-Vimentin + KU60019 (3 µM), CPT (250 nM) + KU60019 (3 µM), GFP-Vimentin + KU60019 (3 µM), and KU60019 (3 µM) for 48 and 72 h and subjected to western blot analysis to understand the expression of Caspase-3 and Vimentin, β-actin was used as loading control. f HCT-116 cells (upper panel) were seeded onto gelatin-FITC coated glass coverslips and treated with Vehicle, CPT (250 nM), CPT (250 nM) + GFP-Vimentin, CPT (250 nM) + GFP-Vimentin + KU60019 (3 µM); lower panel: GFP-Vimentin, CPT (250 nM) + KU60019 (3 µM), GFP-Vimentin + KU60019 (3 µM), and KU60019 (3 µM) for 36 h and the slides were observed in Floid imaging station at ×20 magnification. Bar graph describes the percentage threshold area of degradation analyzed by Image j software. (n = 3, error bars ± SD); ***p < 0.001. g Immunoprecipitation analysis of ATM with Vimentin after HCT-116 cells were treated with Vehicle and CPT (250 nM) for 36 h
