Versican G3 Modulates Breast Cancer Cell Apoptosis Figure 3. Versican G3 enhanced cell apoptosis induced by C2-ceramide by expressing pSAPK/JNK and caspase-3. a) G3- and vector- transfected 66c14 cells (26105) were inoculated in 6 well culture dishes. After cultured for 12 hours, all samples were treated with 0, 10, 40, or 80 mM C2-ceramide for 24 hours. Analysis by light microscopy revealed that treatment with a dose of 40 or 80 mM C2-ceramide induced significant cell death in G3-transfected cells. b) After culture in 40 mM C2-ceramide serum free medium for 4 hours, cells were analyzed with Annexin V and propidium iodide staining using flow cytometry. Annexin V and propidium iodide assays confirmed that cell death occurred through apoptosis. c) 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, all samples were treated with 0, 10, 40, or 80 mM C2-ceramide for 24 hours. Lower cell viability was observed for the G3 experimental group as compared with the control group. Compared with vector control group, n = 6, * p,0.05, **p,0.01, analyzed with t-test. d) Cells were also treated with 40 mM C2-ceramide for 6, 12, 24 hours. WST-1 assays were performed. Compared with vector control group, n = 6, * p,0.05, **p,0.01, analyzed with t-test. e) Cells were also treated with 40 mM C2-ceramide for 6 hours, 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.g003 western blot results presented here are obtained using the antibody from abcam (ab19345) which is indicated suitable for detection of versican V1 isoform, and shows only one band -versican V1, 250– 300 kDa. We then examined the expression of pERK, ERK, pSAPK/JNK, SAPK/JNK in anti-versican siRNA expressing MT-1 cells treated with Docetaxel, Doxorubicin, or Epirubicin. Immunoblotting showed that the expression of pERK V1 was down-regulated in the anti-versican siRNA expressing MT-1 cell, irrespective of whether or not it was chemically treated, and there was no significant change in the expression of pSAPK/JNK (Fig PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e26396