Figure 4. The effect of celastrol on cell size of the SP subpopulation in LOVO/DX cell cultures.

(A) A gating strategy used to analyze only singlet viable cells. The cells were pre-incubated with celastrol for 5-10 minutes with subsequent incubation with DNA-binding dye Hoechst 33342 (Ho.) [5μg/ml] for 90 minutes at 37°C. Then the cells were stained with Annexin V-FITC and PI for exclusion of dead and apoptotic cells from the analysis. The cells-associated fluorescence was evaluated by the means of flow cytometry. SP (Side Population) is defined as subpopulation of cells that show the lowest Ho. content (a low-Ho.fluorescence “tail” in dual wavelength of fluorescence emission: Hoechst red [630nm]and Hoechst blue [455nm]). (B) Representative cytograms of cell size of the SP subpopulation in the presence of celastrol [20μM] or vehicle-DMSO. (C) Cell size of the SP subpopulation after treatment of LOVO/DX cultures with the range of celastrol concentrations. Results are expressed as E/E₀ ratios, where E = % of SP cells in cultures incubated with celastrol and E₀ = % of SP cells in cultures incubated with diluent -DMSO (mean ±SD, n=6 ; ^*p<0.05, ^***p<0.0001).