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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 5. Myofibroblasts treated with TNF-α have a calcific response whereas fibroblasts decrease expression of calcific markers.

Figure 5.

a) Experimental set up to investigate the effects of TNF-α (10 ng/mL) on defined fibroblast and activated myofibroblast populations to assess calcific markers. b) Quantification of deposited calcium signal normalized to dsDNA content and expressed as percent of untreated fibroblast control (0 ng/mL of TNF-α). A significant decrease of deposited calcium was observed with fibroblasts (red) treated with TNF-α (patterned), while the opposite trend was observed with activated myofibroblast (green) resulting in a significant increase of calcium with TNF-α treatment. c) Representative images from cryosectioned samples stained with Alizarin Red (top) and Von Kossa (bottom) for activated myofibroblast and fibroblast populations in the presence or absence of TNF-α. Fibroblasts had no positive stain for either calcific marker and treatment with TNF-α had no effect on either stain. The activated myofibroblast phenotype presented minimal positive Von Kossa and Alizarin Red stain but addition of TNF-α treatment resulted in exacerbated increase of positive staining for both stains. Scale bars=100μm. d) RUNX2 and e) BMP2 mRNA gene expression levels relative to L30. Both calcific markers, RUNX2 and BMP2, gene expression was downregulated on fibroblasts treated with TNF-α. Activated myofibroblast treated with TNF-α resulted in significant upregulation of both RUNX2 and BMP2 gene expression. f) Caspase 3 fluorescence signal normalized to dsDNA content. Activated myofibroblast treated with TNF-α had a significant increase in Caspase 3 fluorescence, while fibroblasts resulted in a significant decrease of Caspase 3 in the presence of TNF-α. ****=p<0.0001, ***=p<0.001, **=p<0.01 and **=p<0.05 based on t-test.