Figure 1

TLR3 agonists induce extracellular fibronectin aggregate formation by astrocytes. Western blot analysis of fibronectin (Fn) in DOC-soluble (sol, fibronectin dimers) and DOC-insoluble (ins, fibronectin aggregates) extracellular deposits (a,b,d,e) and Fn, GFAP and iNOS in total cell lysates (a,c,d,f) of primary rat astrocytes treated for 48 hours with pro-inflammatory cytokines IFNγ (500 units/mL), IL1β (10 ng/mL) or TNFα (10 ng/mL), or with TLR2 agonists zymosan (zym, 10 μg/mL), TLR3 agonists Poly(I:C) (50 μg/mL), Poly(A:U) (50 μg/mL), stathmin (0.5 μg/mL) and TLR4 agonist LPS (200 ng/mL). Representative blots of 3–16 independent experiments are shown in (a,d) quantification of DOC-insoluble fibronectin aggregates in deposits in b,e, and quantification of fibronectin in total cell lysates in (c,f). Actin serves as a loading control for cell lysates; equal amounts of protein (12 μg) are subjected to DOC-(in)solubility analysis. Note that TLR3 agonists, and to a lesser extent TLR4 agonist LPS, induce fibronectin aggregation (Poly(I:C) p < 0.001; Poly(A:U) p = 0.114, ns; stathmin p = 0.018, LPS p = 0.016). Bars represent mean relative to their respective untreated control (ctrl), which was set at 1 in each independent experiment (horizontal line). Error bars show the standard error of the mean. Statistical analyses were performed using a Wilcoxon Signed Rank Test (Poly(I:C) and LPS in (a), failed Shapiro-Wilk normality test) or a one-sample t-test to test for differences between treatments and their respective control (*p < 0.05, **p < 0.01).