Fig. 4. Induction of Vimentin hinders the progression through apoptosis.

a HCT-116 cells were either treated with vehicle, CPT (250 nM) and CPT (250 nM) + DIM (25 µM) for indicated time points; whole cell lysates were prepared and subjected to western blot analysis of Vimentin and PARP-1 protein. b Cells were either transfected with vehicle, CPT (250 nM), SCR + CPT (250 nM), si-Vimentin plus CPT (250 nM) and si-Vimentin for 24 h; whole cell lysates were employed for western blot analysis of Vimentin, PARP-1, NFκB, and cFLIP. c Immunocytochemistry was performed for the indicated treatment conditions to examine the expression and trafficking of FAS ligand in HCT-116 cells. Images were taken by using Floid Cell Imaging Station; magnification ×20. d Similar set of treatment conditions were used to check the cells capability for gelatin matrix degradation through FITC-gelatin degradation assay. Imaging was performed using Floid Cell Imaging Station at ×20 magnification. The images were further analyzed and quantified for threshold area of degradation by Image j software and represented in bar graphs (n = 3, error bars ± s.d.); ***p < 0.0004. e HCT-116 cells were either treated with vehicle, CPT (250 nM), AT7867 (1 µM) and combination of both for 24 h; whole cell lysates were subjected to western blot analysis for the expression of p^ser38-Vimentin, Vimentin, p-AKT, AKT, NFκB, and cFLIP_L. f HCT-116 cells were either transfected with vector, NFκB-luc alone and/or treated with NFκB-luc + CPT (250 nM), NFκB-luc + CPT (250 nM) + SCR, NFκB-luc + si-Vimentin + CPT (250 nM), NFκB-luc + si-Vimentin, and NFκB-luc + SCR in 96 well plates for 24 h; luciferase activity was measured using Dual-Glo Luciferase Assay system (Promega). Normalization was done with luciferase activity of vector (n = 3, error bars ± SD). ***p < 0.001