FIGURE 8.

Multiple assays for validation. (A) MTT assay. The cell proliferation MTT assay for the control and MFG-E8 KD BuMEC cells were performed. All experiments were performed six times points at 12-h intervals up to 72 h of culture. The values were calculated using a non-parametric t-test, and the error bar is shown with a standard error of the mean (SEM). **p < 0.01, ***p < 0.001. (B) BrdU assay. The cell proliferation BrdU assay was assessed for the proliferation of the control and MFG-E8 KD BuMEC cells on every 12-h interval up to 72 h of culture by BrdU labeling. The values were calculated using a non-parametric t-test, and the error bar is shown with the standard error of the mean (SEM). **p < 0.01, ***p < 0.001. (C) Caspase-3/7 assay. Caspase-3/7 activities of the control and MFG-E8 KD BuMEC cells were measured by Caspase-Glo 3/7 activity assay kit. The cells were seeded into 96-well plates and incubated for 12 to 72 h with an interval of 12 h each. The Caspase-Glo 3/7 reagent was added and incubated for another 3 h. Fluorescence intensities of the wells were then measured using excitation wavelength 492 nm and emission wavelength 535 nm. The caspase activities of the normal WT BuMEC cells were compared to transfected KD BuMEC cells. All experiments were performed six times with almost similar results, and values were calculated using a non-parametric t-test, and the error bar is shown with the standard error of the mean (SEM). *p < 0.05, **p < 0.005, ***p < 0.001 and ns: non-significant. (D) Western blot analysis of scrambled shRNA WT and KD cells for AKT, STAT3 and STAT5 proteins with respective phosphoprotein antibody counterpart. Bar graph represent ± SD from three replicate. *p < 0.05, **p < 0.005, ***p < 0.001 and ns, non-significant. (E) Schematic representation for the signaling regulated through MFGE8 protein in mammary epithelial cells.