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. 2020 Apr 6;117(16):8900–8911. doi: 10.1073/pnas.1913788117

Fig. 1.

Fig. 1.

HF inhibits multiple cytokine responses in a range of primary cell types. (A, Left two panels) HF inhibition of MMP13 induction by TNF-α or IL1-β in human FLS (RA-FLS). Human RA-FLS were pretreated with 200 nM HF and/or proline (2 mM) overnight and treated with 10 ng/mL TNF-α for 6 h and mRNA analyzed for MMP13 expression by qRT-PCR. (Right two panels) HUVEC were treated and analyzed for gene expression as described for FLS, above; human LL29 lung fibroblasts were pretreated with HF and/or proline for 6 h and treated with TGF-β for 24 h and analyzed for gene expression, as above. Results are representative of three independent experiments (means ± SD, n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. (B) RNA-seq analysis of HF effects on TNF-α responses. (B, Left) Selective regulation of TNF-α–inducible proinflammatory gene expression in FLS, determined by RNA-seq after 24-h culture in media alone, TNF-α, or TNF-α plus HF. Limma-voom derived log2-fold transformed gene-expression changes for two comparisons are shown; treatment with TNF-α (x axis) as compared to untreated (x axis) or combined TNF-α and HF treatment as compared to TNF-α treatment (y axis). Selected genes are highlighted in the figure with lines connecting to their respective data points. Only genes with real reads per kilobase of transcript per million mapped reads (RPKM) values (>0) are shown (n = 12,973). (Right) Representation of fold-changes following treatment with TNF-α or TNF-α and HF in specific genes referred to in the text. Log2-fold transformed gene-expression changes and their respective 95% confidence intervals are shown for TNF-α versus untreated (UT, Left) and TNF-α and HF treatment versus TNF-α alone treatment (Right) for genes of interest. The respective false-discovery rate-adjusted P values for the gene-expression changes are shown via shading of the plotted values. The vertical black line represents no change (log2 fold-change = 0, or fold-change = 1) with the dotted lines representing twofold expression change in either direction of the comparison.