Figure 4.

Effect of α-amanitin on DD1-mediated apoptosis and knockdown of DIF-1 by siRNA leads to apoptosis of breast cancer cells. A, T-47D cells were incubated with or without α-amanitin (2.5 μmol/L) for 3 h and then transfected with vector (100 ng) to express GFP-DD1. Twenty hours later, the cells were analyzed for GFP-DD1 expression and apoptosis by TUNEL assay. Shown are representative fields. No apoptosis was identified in cells preincubated with α-amanitin and the levels of GFPDD1 were similar under both conditions. B, siRNA (25 nmol/L) was used to knockdown DIF-1 expression in five different breast cancer cell lines (SKBR3, MCF-7, T-47D, MDA-435, and MDA-231) or cells of other origin (U2OS, human osteosarcoma; 293, human kidney epithelium; UOK-145, kidney carcinoma; HepG2, human hepatoma; HeLa, human cervical epithelium). A control siRNA (25 nmol/L) contained four base changes (mut siRNA). Thirty hours later, cells were examined for apoptosis by TUNEL assay. All the breast cancer cell lines exhibited apoptosis, whereas the other cells did not. Shown are representative results from the study with SKBR3 breast cancer cells and HeLa cells. Supplementary Fig. S1 shows results with the other cell lines. Similar results were found with four different siRNAs that targeted DIF-1 mRNA. C, expression of DD1 (GFP-DD1) does not lead to apoptosis of MCF10A cells, a cell line derived from normal breast epithelial cells. D, DIF-1 siRNA does not lead to apoptosis of MCF10A cells.