SUSD3-knockdown MCF7 cells show cell cycle defects. (a) Cell counts of control (siCTL) or SUSD3 siRNA-transfected (oligo 4) MCF7 cells (siSUSD3) were performed at 24, 48, and 72 hours post-transfection using a hemocytometer. **, p< 0.01. (b) MCF7 cells were transfected with control or SUSD3 siRNA (oligo 4) for 72 hours and the percent of cells in the indicated phases of the cell cycle were determined by FACS. E2 treatment led to a decrease in the percentage of cells in the G0/G1 phase (83.37% vs. 80.97, vehicle vs. E2, p=0.0533). (c) E2 treatment caused a significant increase in control cells entering S phase (6.09% vs. 13.8%, vehicle vs. E2, p<0.001). siRNA knockdown of SUSD3 abrogated the effect of E2 treatment (6.9% vs. 7.89%; vehicle vs. E2, p=0.36). (d) E2 significantly increased the number of control cells in G2 phase, an effect that was blunted after SUSD3 knockdown. (e) E2 robustly increased the M-phase fraction, whereas SUSD3 knockdown significantly decreased the fraction of cells in the M phase, from 0.6% to 0.35% (p=0.012). The percentage of E2-treated SUSD3-ablated cells entering M phase was significantly lower than E2-treated control cells (2.13% vs. 0.55%; p<0.0001). Results are reported as mean ± SD from triplicate experiments.