Fig. 6
T-type Cav3.2 Ca2+ channels are up-regulated by cytokines in a NF-κB-dependent manner. a Representative confocal images of primary astrocytes co-stained with specific antibodies for Cav3.2 and GFAP. b qPCR measurement of CACNA1H mRNA levels following treatment with TNFα, ***P = 0.0003, IL-1β, **P = 0.0042 and IFNγ, ***P = 0.0005 (n ≥ 8 per condition). c CACNA1C mRNA levels assessed as above (TNFα; P = 0.2536, IL-1β; P = 0.8093, IFNγ; P = 0.0537) (n ≥ 8 per condition). Statistics in (b, c) by one-way ANOVA followed by Dunnett’s multiple comparisons test. d–f Representative western blots showing Cav3.2 and p-NF-κB levels following treatment with TNFα, IL-1β or IFNγ for the indicated time points. g, h Fold induction of pNFκB (g, n ≥ 3 independent replicates per treatment) and Cav3.2 (h, n = 4 independent replicates per treatment) expression levels relative to untreated controls following cytokine treatment for 5 min. i Experimental timeline explaining the treatment of primary astrocytes with IL-1β in the absence or presence of the NF-κB pathway inhibitor, BAY 11-7083. j Representative western blot showing the levels of Cav3.2, p-NF-κB and p-IκB in the absence or presence of IL-1β and BAY 11-7083. k Fold induction of Cav3.2 expression levels following treatment with IL-1β in the absence or presence of BAY 11-7083 for 5 min (*P = 0.0354) and 60 min (**P = 0.0060). Values are estimated relative to untreated from three independent biological replicates. Statistics by unpaired Student’s t test. l–n Representative plots from live calcium imaging of primary astrocytes upon exposure to IL-1β with or without pre-treatment with the Cav3.2 inhibitor, NiCl2. o Quantification of F350/380 peak ratios from primary astrocytes after addition of IL-1β alone (*P = 0.0431 vs control) or IL-1β and NiCl2 (*P = 0.0364 vs IL-1β alone). Statistics were performed by one-way ANOVA followed by Tukey’s multiple comparisons test (n ≥ 12 images per condition in triplicates)