Fig. 2: Co-existence of EMT and early-apoptosis in same population of Camptothecin treated cells.

a Cells were treated with CPT for 0, 12, 24, 36, and 48 h, tagged with AnnexinV-FITC, propidium iodide and analyzed through flow cytometry for onset of apoptosis. Bar graph showing quantification of cells in various phases of apoptosis (n = 3, error bars ± s.d.). b HCT-116 cells treated with CPT (250 nM) for 0, 12, 24, 36, and 48 h, and subjected to FACS analysis for identification of disruption of mitochondrial membrane potential by TMRE staining. Bar graph depicting loss of TMRE fluorescence indicating loss of mitochondrial membrane potential. c Western blot analysis was performed to study the expression pattern of Bid, Bcl₂, Bax by exposing HCT-116 cells to 250 nM CPT for 0, 12, 24, 36, and 48 h. β-actin was used as loading control. d Western blot analysis of Apc floxed intestinal tissue treated with CPT (0.4, 0.8, and 1.2 mg/kg) for 24 and 48 h was performed to analyze the expression of Twist-1, Vimentin, Bax, and Bcl₂. β-actin was used as loading control. e HCT-116 treated with 250 nM CPT for 0, 12, 24, 36, and 48 h was subjected to immunoblot analysis to check the expression of Caspase-3 and PARP-1. f HCT-116 cells were treated with CPT (250 nM) for 0, 12, 24, 36, and 48 h, stained with AnnexinV-FITC and PI and sorted using MoFlo cell sorter for the early apoptotic and viable cells (schematic). The sorted cells were then analyzed for the expression of Vimentin by immunoblot analysis; β-actin was used as loading control. Bar graph represents the densiometric analysis of expression of Vimentin in early apoptotic and viable population of sorted cells; (n = 3, error bars ± s.d.). g HCT-116 cells were transfected with N3-secAnnexinV-mVenus construct and then treated with 250 nM of CPT for 24, 36, and 48 h. Images were taken at ×20 magnification. Red arrows indicate cells with fluorescence bordering the cells and yellow arrows indicate cells having sufficient fluorescence throughout the cells. Bar graph depicting number of PS (Phosphatidyl serine) flipped cells vs condensed cells showing bright fluorescence. Cell counting was performed in Image j software (n = 3, error bars ± s.d). h HCT-116 cells transfected with N3-secAnnexinV-mVenus construct were subjected to transwell migration assay and treatment was given with 250 nM of CPT for 36 h. The migrating cells were processed further and observed under fluorescence microscope for detecting the fluorescence from chimeric AnnexinV-mVenus protein