Versican G3 Modulates Breast Cancer Cell Apoptosis Figure 4. Versican G3 domain enhanced tumor cell apoptosis induced by C2-ceramide by up-regulating the EGFR/JNK pathway. a) 16104 G3-transfected and vector-transfected MT-1, MDA-MB-468, 66c14, 4T07, and 4T1 cells were inoculated and cultured in 10% FBS/DMEM medium in 96 well culture dishes for 12 hours. After cell attachment, cells were treated with 40 mM for 24 hours. WST-1 assays were used to analyze cell viability. b) G3-transfected and vector-transfected 66c14 cells (16104) were inoculated and cultured in 10% FBS/DMEM medium in 96 well culture dishes for 12 hours. After cell attachment, we added C2-ceramide (40 mM), and EGF (20 ng/ml), AG 1478 (2.0 mM), PD 98059 (50 mM), or SP 600125 (100 nM) cultured for 24 hours. WST-1 Cell Survival Assays were performed. Compared with vector control group, n = 8, * p,0.05, **p,0.01, analyzed with t-test. c) Treated with C2-ceramide (40 mM) and EGF (20 ng/ml), AG 1478 (2.0 mM), PD 98059 (50 mM), or SP 600125 (100 nM) for 6 hours, cells were harvested and subjected to immunoblotting with antibodies to pSAPK/JNK, SAPK/JNK, ERK2, pERK, Caspase-3, and b-actin. doi:10.1371/journal.pone.0026396.g004 S1c). WST-1 assays showed that versican G3 promoted cell apoptosis induced by C2-ceramide and Docetaxel, whereas cell apoptosis induced by Doxorubicin and Epirubicin was reduced. While the anti-versican siRNA- transfected cells showed a reduction in the extent of cell apoptosis induced by C2-ceramide, we observed enhanced effects on cell apoptosis induced by Doxorubicin and Epirubicin when compared with G3-transfected and vector-transfected cells (Fig S1d). In order to further confirm the role of G3 in apoptosis, we linked the G3 domain with versican 39-UTR (Fig S2a). Our previous research indicated that G3-39 UTR transfected cells expressed lower G3 protein compared to G3 expressing cells [48]. So we can use the G-UTR construct to observe the effect of decreasing expression of G3 in G3 expressing cells. Immunoblot- ting demonstrated that G3-39 UTR stably transfected 66c14 cells expressed much lower levels of G3 protein than the G3 transfected cells (Fig S2b). The microscopic morphology of G3 transfected cells was quite different from the vector control cells. The G3 expressing cells spread evenly on the culture dishes, while the vector control cells were prone to cell aggregation. The G3-39 PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e26396