Fig. 4.

KLF7 silencing modulates epithelial-mesenchymal transition (EMT) and ovarian cancer stem cell markers in HGSOC cells. a, b Relative EMT markers expression assessed by RT-qPCR analysis, 72 h post transfection, in OV-90 (a) and PEO1 (b) cells transiently transfected with specific siRNAs (siKLF7) and non-targeting siRNA pool as control (siC). Representative western blot and densitometric analysis of EMT markers expression in OV-90 (a) and PEO1 (b) cells transiently transfected with siKLF7 and with siC, as control. Cells were harvested 72 h post transfection and cell lysates were subjected to western blot with specific antibodies. GAPDH was used as loading control. c Gelatin zymography: activity of MMP2 and MMP9 was assessed in serum-free cell culture media of OV-90 and PEO1 cells transiently transfected with siKLF7 and with siC, as control. (d) Relative cancer stem cell markers expression assessed by RT-qPCR analysis, 72 h post transfection, in OV-90 and PEO1 cells transiently transfected with specific siRNAs (siKLF7) and non-targeting siRNA pool as control (siC). e Representative western blot and densitometric analysis and (f) immunofluorescence analysis of CD44 expression in OV-90 and in PEO1 cells transiently transfected with siKLF7 and with siC, as control. Cells were harvested 72 h post transfection and cell lysates were subjected to western blot with the specific antibody. GAPDH was used as loading control. For immunofluorescence, the images show the merged signal of Alexa Fluor 488-CD44 mouse monoclonal antibody (green) and DAPI (4′,6-Diamidine-2′-phenylindole) (blue) in siC and siKLF7 OV-90 and PEO1, 72 h post transfection (40x, scale bar 50 μm). For (a, b, d) figures, data are presented as fold change, calculated with the ΔΔCt method, using siC as reference sample. Bars and error bars refer to mean and SD (Standard Deviation) of three or more experiments. N.d. = not detectable. To establish statistically significant differences, unpaired t-test was carried out: *p < 0.05, **p < 0.01