Effect of Deguelin on the growth and expression of signaling molecules in TNBC cells.

<p>A. Effect of deguelin on MDA-MB 231 cells in the presence of EGF (1 ng or 20 ng) alone, Deguelin alone (31–500 nM)or Deguelin+EGF. The cells were plated in six well plates, allowed to attach overnight, then treated with indicated test compound/(s) (in serum free media) and incubated for 72 h at 37°C. Number of cells was counted in each well using coulter counter. Data represent % cells in the respective control treatment. * indicates significant difference as compared to respective control, ** indicates significant difference (p<0.05) between the groups shown. B. Effect of Deguelin on total (after permeabilization)and cell surface (without permeabilization) EGFR expression in the presence/absence of indicated concentration of EGF. The cells plated on glass coverslips were incubated for 10 min-24 h at 37°C in the serum free media containing vehicle only, Deguelin (250 nM) or Deguelin+EGF as indicated. At the end of incubation cells were fixed in 4% buffered formalin, permeabilized and then processed for immunofluorescence examination of EGFR (red) and mounted in DAPI (blue) containing media. Representative photographs captured using fluorescence microscope equipped with appropriate filters are shown. Red = EGFR expression, Blue = DAPI staining of nuclei. Data are shown for 2 and 24 h post treatments. C. Effect of Deguelin, EGF and EGF+Deguelin on EGFR/1045-phospho EGFR and 1068-Phospho EGFR levels in MDA-MB-231 cells. MDA-MB-231 cells were treated for 10 min–24 h with indicated treatments in serum free media as described above. At the end of incubation cells lysates were prepared and subjected to western blot analysis. For loading control appropriate control proteins were developed using appropriate antibodies. Lanes 1 = control, 2 = Deguelin 250 nM, 3 = EGF1 ng 4 = EGF 1 ng +Deguelin 250 nM, 5 = EGF 20 ng 6 = EGF20 ng+Deguelin 250 nM. Number under each lane indicate % of normalized (protein intensity/control protein intensity) specific band intensity in relation to control treatment. Control was considered as 100%. D. Western blot and Immunofluorescence analyses of p-ERK and Survivin in MDA-MB-231 cells treated for 24 h with vehicle only, Deguelin (250 nM) in the presence or absence of EGF. Serum free media was used for this study. Lane 1 = Control, 2 = Deguelin 250 nM, 3 = 1 ng EGF, 4 = 1 ngRGF+Deguelin250 nM, 5 = 20 ng EGF, 6 = 20 ng EGF+Deguelin. For western blot number under each band indicates % of intensity of normalized values as described above. In immunofluorescence images, green fluorescence indicates p-ERK, red immunofluorescence indicates Survivin and Blue staining is due to nuclear counterstaining with DAPI. Representative images are shown. E. Western blot analysis of various cell signaling molecules (p-ERK, ERK, p-STAT-3, c-Myc) in MDA-MB-231 cells treated for 24 h with varying concentration of Deguelin in serum free media. Intensity of specific band is normalized in relation to loading control protein intensity, ratio in vehicle treated control with housekeeping protein control was considered as 100%. Number under each band indicate % of control intensity.