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. 2017 Jan 31;292(10):4280–4292. doi: 10.1074/jbc.M116.733030

FIGURE 2.

FIGURE 2.

Gas6 promoted anti-apoptosis and cell proliferation of MM cell lines RPMI-8226. A, MM cell lines RPMI-8226 were incubated for 24 h in serum-free X-VIVO medium, and then FCM analysis was performed. Representative data are from three independent experiments are shown. B, RPMI-8226 cells were cultured with serum-free X-VIVO medium for 24–48 h, and Gas6 levels of culture supernatants were quantified by ELISA. Representative data are from four independent experiments. Data are expressed as means ± S.D. (error bars) (n = 8, each group). *, p < 0.05. C, MM cells were incubated in serum-free X-VIVO medium with Gas6 (0–200 ng/ml) for 24 h, and cell apoptosis was analyzed by FCM after staining with Annexin V and 7-AAD. Representative data are from four independent experiments. C, untreated RPMI-8226 cells. D, RPMI-8226 cells treated with 100 ng/ml Gas6. E, RPMI-8226 cells treated with 200 ng/ml Gas6. F, the statistical analysis indicated the effect of exogenous Gas6 on the cell apoptosis of the MM cell line RPMI-8226 cells in a dose-dependent manner. Results are shown as mean ± S.D. of statistical analyses from four separate experiments. **, p < 0.01. G, cell apoptosis was analyzed by FCM after staining with Annexin V and 7-AAD. Representative data are from four independent experiments. RPMI-8226 cells were treated with 100 ng/ml Gas6. H, RPMI-8226 cells treated with 100 ng/ml Gas6 plus 20 μg/ml anti-Gas6 antibody. I, RPMI-8226 cells treated with 100 ng/ml Gas6 plus 10 μg/ml anti-IL-6 antibody. J, the statistical analysis indicated the effect of Gas6 and IL-6 inhibition on cell apoptosis reduced by exogenous Gas6 in RPMI-8226 cells. Results are shown as mean ± S.D. of statistical analyses from four separate experiments. *, p < 0.05. K, the cells were preincubated with or without recombinant Gas6 (100–200 nmol/liter) for 48 h at 37 °C in serum-free X-VIVO medium before the MTT assay as described under “Experimental Procedures.” Data are expressed as means ± S.D. (n = 8, each group). *, p < 0.05; **, p < 0.01. L, RPMI-8226 cells were incubated in serum-free X-VIVO medium in the presence of 100 ng/ml Gas6 with or without neutralizing antibodies to Gas6 (20 μg/ml) for 48 h, followed by an MTT assay. Data are expressed as means ± S.D. (n = 8, each group). *, p < 0.05. M, mouse IgG (10 μg/ml) and rabbit IgG (20 μg/ml) isotype control antibodies are used as negative controls in MTT assay. RPMI-8226 cells were incubated and treated with mouse or rabbit IgG isotype control antibody for 48 h in serum-free X-VIVO medium, followed by MTT assay. Data are expressed as means ± S.D. (n = 8, each group). *, p < 0.05. N.S., not significant.