Versican G3 Modulates Breast Cancer Cell Apoptosis Figure 2. Versican G3 domain enhanced tumor cell survival in serum free medium by up-regulating pERK. a) G3- and vector- transfected 66c14 (26105) 26105 were cultured in 10% FBS/DMEM medium in culture dishes for 12 hours. After cell attachment, we changed the medium to serum free DMEM and cultured them for 6 days. Cell lysates were prepared and subjected to immunoblotting with antibodies to ERK2, pERK, GSK-3b (S9P), GSK-3b, CDK2, CDK6, CDC2P34, and b-actin. 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 changed the medium to serum free DMEM, with EGF (20 ng/ml), selective EGFR inhibitor AG 1478 (2.0 mM), selective MEK (ERK kinase) inhibitor PD 98059 (50 mM), or selective JNK inhibitor SP 600125 (100 nM) cultured for 6 days with medium changed every 2 days. WST-1 Cell Survival Assays were used to analyze cell viability. Compared with vector control group, n = 6, * p,0.05, **p,0.01, analyzed with t-test. c) After culture in serum free medium for 6 days, cell lysates were prepared and subjected to immunoblotting with antibodies to ERK2, pERK, GSK-3b (S9P), GSK-3b, and b-actin. doi:10.1371/journal.pone.0026396.g002 Immunoblotting and RT-PCR showed that versican V1 isoform expressed differently in the four human breast cell lines. It was expressed highly in MT-1, MDA-MB231 and MDA-MB-468 cells, and low levels were observed in MCF-7 cells (Fig S1a). The anti- versican siRNA that has been confirmed to be able to silence vesicant expression [47] was used to transfect MT-1 cells, and it revealed significant versican V1 mRNA and protein down- regulation through RT-PCR and immunoblotting (Fig S1b). The PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e26396