Figure 6

Pro-inflammatory cytokines favor EIIIA^pos over EIIIB^pos splicing in primary rat astrocytes. (a) Schematic representation of astrocyte treatment and analysis. Primary rat grey matter (gm) and white matter (wm) astrocytes were pre-incubated for 24 hours with a mixture of IFNγ (500 units/mL), IL1β (10 ng/mL) and TNFα (10 ng/mL), and left untreated or treated with Poly(I:C) (50 μg/mL) for 24 hours. (b–d) Western blot analysis of EIIIA^pos-fibronectin and total fibronectin in total cell lysates. Representative blots of 4 independent experiments are shown in (b), quantification of EIIIA^pos-fibronectin of total fibronectin in (c) and total fibronectin in (d). Actin serves as a loading control. (e–h) RT-PCR analysis of EIIIA^pos-, EIIIA^neg-, EIIIB^pos- and EIIIB^neg-Fn mRNA. Representative agarose gels of 4–5 independent experiments are shown in e, quantification of EIIIA^pos/total Fn, EIIIB^pos/total Fn, and EIIIA^pos/EIIIB^pos-Fn mRNA ratios in (f,g,h) respectively. Note that EIIIB^pos-Fn mRNA, but not EIIIA^pos-Fn mRNA levels are decreased in cytokine pre-treated astrocytes (wm + cytokines p = 0.043; wm + cytokines + Poly(I:C) p = 0.031; gm + cytokines p = 0.003; gm + cytokines + Poly(I:C) p = 0.094, ns), resulting in an enhanced EIIIA^pos/EIIIB^pos-Fn mRNA ratio (wm + cytokines p = 0.104, ns; wm + cytokines + Poly(I:C) p = 0.044; gm + cytokines p = 0.075, ns; gm + cytokines + Poly(I:C) p = 0.102, ns). Bars represent mean relative to their respective control astrocytes (ctrl), which was set at 1 for each independent experiment (horizontal line). Error bars show the standard error of the mean. Statistical analyses were performed using a one-sample t-test to test for differences with their respective control astrocytes (*p < 0.05, **p < 0.01).