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. Author manuscript; available in PMC: 2022 Mar 1.
Published in final edited form as: FASEB J. 2021 Mar;35(3):e21382. doi: 10.1096/fj.202002013RR

Figure 2. Encapsulated VICs respond to TNF-α treatment with a decrease in expression of fibrotic markers.

Figure 2.

a) Schematic of 3D hydrogel culture platform. 8-arm 40kDa PEG-norbornene is reacted with an MMP-degradable dithiol crosslinker and RGD adhesive peptide is added to allow for VIC-matrix interactions. b) Representative immunostaining images of VICs treated with growth media (untreated) and 0.1, 1, or 10 ng/mL of TNF-α treatment. αSMA stress fibers (green) formation decreases with increasing TNF-α treatment concentration. Nucleus (blue) and αSMA (green). Scale bars=20μm. c) αSMA mean intensity analysis with respect to cell number for VICs in untreated and 0.1, 1, or 10 ng/mL TNF-α treatment conditions. TNF-α treatment resulted in a significant decrease in αSMA mean intensity at higher doses. d) VIC αSMA protein expression normalized to untreated controls (dotted line) and measured via western blot. A representative western blot used for analysis (top) where all lanes are from the same blot. αSMA protein expression significantly decreases with increasing TNF-α concentration. e-g) mRNA gene expression levels relative to L30 for the fibrotic markers e) αSMA f) COL1A1 and the fibroblast marker g) CTGF. Increasing treatment with TNF-α results in significant downregulation in αSMA gene expression, while COL1A1 gene expression shows a decreasing trend, albeit not statistically significant. CTGF gene expression was slightly increased with TNF-α treatment, but no significant differences were observed. ***=p<0.001 and **=p<0.01, based on one-way ANOVA.