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 (2×10⁵) were inoculated in 6 well culture dishes. After cultured for 12 hours, all samples were treated with 0, 10, 40, or 80 µM C2-ceramide for 24 hours. Analysis by light microscopy revealed that treatment with a dose of 40 or 80 µM C2-ceramide induced significant cell death in G3-transfected cells. b) After culture in 40 µM 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 (1×10⁴) 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 µM 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 µM 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 µM C2-ceramide for 6 hours, harvested and subjected to immunoblotting with antibodies to pSAPK/JNK, SAPK/JNK, ERK2, pERK, Caspase-3, and β-actin.