Versican G3 Modulates Breast Cancer Cell Apoptosis Figure 1. Versican G3 domain enhanced tumor cell survival in serum free medium. a) G3-transfected and vector-transfected 66c14 (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 cells for 6 days. Cell viability was analyzed by light microscopy. b) After culturing in serum free medium for 2 days, cells were analyzed with Annexin V and propidium iodide staining using flow cytometry. Annexin V and propidium iodide assays confirmed that cell death was apoptosis. c) G3- and vector-transfected 66c14 (26105) were cultured in 10% FBS/DMEM medium in culture dishes for 12 hours. After cell attachment, we changed the medium into serum free DMEM medium and cultured them for 14 days. Cells were harvested and counted under light microscopy every 2 days. Experimental results are compared with vector control group, n = 6, * p,0.05, **p,0.01, analyzed with t-test. d) 16104 G3- and vector-transfected human breast cancer cells MT-1 and MDA-MB-468, mouse mammary tumor cells 66c14, 4T07, and 4T1 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 and cultured them for 8 days. WST-1 Cell Survival Assays were used to test cell viability. Compared with vector control group, n = 6, * p,0.05, **p,0.01, analyzed with t-test. e) Cell cycles were analyzed by flow cytometer. Compared with vector control group, n = 4, * p,0.05, **p,0.01, analyzed with t-test. doi:10.1371/journal.pone.0026396.g001 Reduction of endogenous versican expression by siRNA (or by linking G3 domain with 39UTR) prevented G3 modulated effects on cell apoptosis induced by chemotherapeutic drugs The key functions of the EGF-like motifs of versican G3 domain were well demonstrated by our former study [31,32,46]. Here we found that G3 fragment lacking the EGF-like motifs (G3DEGF) construct transfected 4T07 cells did not show enhanced cell apoptosis when treated with C2- ceramide or Docetaxel, and also did not show enhanced anti- apoptosis when cultured in Doxorubicin or Epirubicin as G3 transfected cells (Fig. 7a, 7b). Immunoblotting indicated that G3DEGF expressing cells did not showed enhanced pERK as G3 expressing cells. G3DEGF expressing cells also did not showed enhanced pJNK when treated with Docetaxel and enhanced GSK-3b(S9P) when cultured in Doxorubicin as G3 expressing cells. PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e26396