Figure 5. Depletion of CAXII does not affect proliferation and survival of chemoresistant cells.

HT29/dx cells were cultured for 48 h with fresh medium (CTRL), treated with a non targeting scrambled siRNA (scr) or with a CAXII-targeting specific siRNA pool (siCAXII). HT29 cells were included as control. (A) The expression of CAXII was measured in whole cell lysates by Western blotting. The β-tubulin expression was used as a control of equal protein loading. The figure is representative of three experiments with similar results. (B) Confocal microscope analysis of cells stained for the proliferation marker Ki67. The samples were analyzed by laser scanning confocal microscopy for Ki67 protein signal (green fluorescence) or for PI (red fluorescence), used to visualize nuclei. Magnification: 60 × objective; 10 × ocular lens. Bar = 20 μm. (C) The percentage of cells positive to annexin V-FITC, as index of early apoptosis, and to PI, as index of late apoptosis, was measured by flow cytometry. Percentages indicate annexin V-positive cells (lower right quadrant), PI-positive cells (upper left quadrant), annexin V/PI-positive cells (upper right quadrant). The figures are representative of three experiments with similar results. (D) Western blot analysis of the autophagy markers beclin, ATG12 and LC3B. The β-tubulin expression was used as a control of equal protein loading. The figure is representative of three experiments with similar results. (E) Cells were fixed and stained for β-galactosidase activity, then examined by fluorescence microscopy. Magnification: 20 × objective; 10 × ocular lens. Bar = 100 μm.