FIGURE 6:

IL-15 regulates proliferation and self-renewal of NSCs. (A) Effect of IL-15 on the proliferation of neurospheres, evaluated by the MTT assay. Cells were cultured in incomplete medium (CTL) or incomplete medium supplemented with IL-15 (5 ng/ml) for 24, 48, or 72 h. Data are expressed as mean ± SEM of optical density (OD) 595 nm. (B) Effect of IL-15 deficiency on the proliferation of neurospheres, evaluated by the MTT assay. WT or IL-15 knockout cells were cultured in complete medium for 24, 48, or 72 h. Data are expressed as mean ± SEM of OD 595 nm. (C) Western blotting analysis of the expression of the cell cycle regulators cyclin D1, cyclin D3, CDK4, CDK6, phospho Rb (pRb), phospho histone H3 (pHH3), p21, p27, and p15, using GAPDH as housekeeping gene. WT or IL-15 knockout (KO) neurospheres were cultured for 12, 24, or 48 h in complete medium to further analyze protein expression. (D) Phase contrast analysis of neurosphere size after a self-renewal assay. WT (black bars) and IL-15 knockout (white bars) cells were cultured in complete medium for 7 d to quantify the number of individual cells able to generate a neurosphere (E) and the size of the generated neurospheres (F). (E) Data are expressed as mean ± SEM of the number of neurospheres/well when 5, 50 or 500 cells/well were initially plated. (F) Data are expressed as mean ± SEM of the neurosphere diameter (μm). Scale bar in D, 50 μm. Statistical differences of CTL vs. IL-15 (A), WT vs. IL15 knockout (B, E, F): *p < 0.05, **p < 0.01, ***p < 0.001. Data were analyzed with an ANOVA and a post hoc Tukey test.