Figure 3

hBMVEC IL-6 and IL-1β enhance C6 glioma sCp gene expression. (A) qPCR of C6 glioma sCp transcript from total RNA isolated from C6 cells incubated with either nothing, IL-1β (10 ng/mL), IL-6 (10 ng/mL), or both for 24 h. (B) SB203580 and IRAP, which inhibit the action of hBMVEC-secreted IL-1β, repress the ability of hBMVEC-secreted IL-1β to enhance C6 glioma sCp gene expression. Relative C6 glioma sCp transcript abundance grown either alone (C6), or distal to hBMVEC (EC/-/C6) with or without 20 μM SB203580 or 1 μg/mL IRAP for 24 h. (C) Relative abundance of C6 sCp from total RNA of C6 glioma cells seeded alone with or without the addition of basal hBMVEC-conditioned media (Basal ECM) and the IL-1β inhibitor IRAP (1 μg/mL) or the IL-6 inhibitor SC144 (20 μM). Drugs were added to C6 glioma cells for 1 h prior to the addition of Basal ECM for an additional 1 h before total RNA was isolated. (D) Immunoblot probing for Cp in C6-conditioned media in which the cells had been incubated with either nothing, IRAP (1 μg/mL), SC144 (20 μM), or both for 1 h prior to the addition of both IL-1β (10 ng/mL) and IL-6 (10 ng/mL), or nothing for an additional 6 h. Purified human sCp (20 ng) is used as a positive control in the top blot. (E) Total RNA isolated from C6 glioma cells treated as described in (D) were analyzed for sCp transcript abundance by qPCR. One-way ANOVA analyses of the variance parameters were used to determine the significance of the data. *P < 0.05, **P < 0.01, ***P < 0.001, ns is not significant. Data are represented as means ± s.d. (n = 3, technical replicates).