Figure 5

CSF-1R inhibits apoptosis and promotes proliferation in canine mammary cancer cells. The effect of csf-1r specific siRNA and CSF-1 treatment of canine mammary cancer cell lines (CMT-U27, CMT-U309, P114, CMT-W1 and CMT-W2) on apoptosis (A). The number of apoptotic cells represented as a percentages of Annexin-V-positive cells obtained with FACS Aria II (Becton Dickinson) is significantly increased after csf-1r silencing. Treatment of cells neither with non-coding siRNA with transfection reagent nor with CSF-1 cause any significant effect comparing to the control. Similarly, cell culture neither in serum-starved conditions (RPMI-1640 without FBS) nor in serum-starved conditions with CSF-1 caused any effect. Error bars refer to S.D. p<0.05 was marked as *, p<0.01 was marked as ** and p<0.001 was marked as ***. One-way ANOVA followed by Tukey HSD post-hoc test were applied. (B) The representative histograms and cytograms of CMT-U27 cell line double stained with AnnexinV-FITC and PI. Cells located on the right side of the histograms represented apoptotic cells. On the cytograms are showed normal, early apoptotic, late apoptotic and necrotic cells. Left bottom quadrant shows normal cells, top left quadrant shows necrotic cells (damaged cell membrane but no phosphatydilserine exposure), right bottom quadrant shows the early apoptotic cells (intact cell membrane) and top right quadrant shows cells in late stage of apoptosis. The effect of csf-1r gene silencing and CSF-1 (25 ng/ml) treatment on proliferation (C) of canine mammary cancer cells was assessed by Ki67 test (BD Bioscience, USA). The number of cycling cells represented as a percentage of Ki67-positive cells is significantly decreases after the transfection whereas significantly increased after CSF-1 treatment. Error bars refer to S.D. The statistical analysis was performed using Prism version 5.00 software (GraphPad Software, USA). One-way ANOVA followed by Tukey HSD post-hoc test were applied. p<0.05 was marked as *, p<0.01 was marked as ** and p<0.001 was marked as ***.