Fig. 4

Inflammatory mediators differentially regulate SPARC expression in hCMEC/D3 monolayers. hCMEC/D3 were incubated for 24 and 48 h in replenished media with TNF-α, IFN-γ, or LPS, alone or in combination. a Compared to untreated/control conditions, 10 and 100 U/ml TNF-α treatment alone increased SPARC expression at 24 and 48 h. Bars represent the average relative value of blots from n = 3 independent experiments. Error bars indicate SEM. ANOVA and Tukey’s multiple comparison test, *P < 0.05. b The effect of varied concentrations of IFN-γ (100, 200, 500 U/ml) on SPARC expression in hCMEC/D3 cultures was assessed by Western blot analysis. c LPS treatment enhanced SPARC levels in a dose-dependent manner at 24 and 48 h by Western blotting. Pooled results from two biological replicates are shown. d The effect of inflammatory mediators on SPARC expression in confluent hCMEC/D3 cultures was assessed in parallel by quantitative immunocytochemistry with representative images shown and summary of findings shown e. SPARC expression is shown following 100 /ml TNF-α, 100 U/ml IFN-γ, or 25 ng/ml LPS alone or in combination. Quantification was pooled from 10 images of duplicate wells for each treatment. Untreated confluent cultures showed minimal SPARC staining at 24-h TNF-α and LPS treatment alone increased SPARC levels after 24 h (P < 0.0001). Data represents one of the two experiments with similar results; error bars represent SEM. One-way ANOVA and Tukey’s multiple comparison test vs. untreated controls, *P < 0.05; **P < 0.001; ***P < 0.0001. Scale bar = 30 μm. f RT-PCR was used to quantify SPARC mRNA. 100 U/ml TNF-α treatment significantly (P < 0.05) increased SPARC mRNA expression. Bars represent the average densitometric value from n = 4 experiments; error bars represent SEM. One-way ANOVA and Tukey’s multiple comparison test vs. untreated controls, *P < 0.05