Figure 2. Knockdown of autocrine/paracrine EPO expression by siRNA inhibits cell signaling but not cell proliferation of SKBR3 cells. (A) SKBR3 cells were subjected to knockdown of EPO expression with three different siRNAs or control siRNA for 48 h. The cells were then cultured for an additional 40 h. The conditioned medium was harvested for detection of EPO as described in Figure 1. (B) SKBR3 cells were subjected to knockdown of EPO as described in (A). The cells were then harvested and lysed for western blotting analysis with the antibodies shown. Lysates from separate dishes of SKBR3 cells treated with rHuEPO (40 unit/mL) or insulin (10 nM) for 30 min prior to cell lysis were used as positive controls. (C) Parental SKBR3 cells and the SKBR3 cells with and without EPO knockdown were cultured for additional 5 d in normoxia, and then subjected to an MTT assay as described in Materials and Methods.