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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 3. TNF-α treatment promotes the VIC osteoblast-like phenotype and matrix calcification.

Figure 3.

a) Representative images from 3D cryosectioned samples stained with Alizarin Red (top) and Von Kossa (bottom) for untreated conditions and 0.1, 1, and 10 ng/mL TNF-α. Increasing TNF-α treatment leads to an increase in Alizarin Red (red) and Von Kossa (black) staining. Scale bars=100μm. b,c) mRNA gene expression levels relative to L30 for calcific markers b) RUNX2 and c) BMP2. Both RUNX2 and BMP2 gene expression were significantly upregulated relative to untreated with 10 ng/mL of TNF-α. d) Quantification of Caspase 3 fluorescence signal normalized with dsDNA content. Caspase 3 fluorescence shows a significantly increased in VICs treated with TNF-α compared to untreated samples. e) Deposited calcium signal normalized to dsDNA content and expressed as percent of untreated control (0 ng/mL of TNF-α). No trends were observed with TNF-α treatment, although significantly reduced calcium content was observed with 0.1 ng/mL of TNF-α. ***=p<0.001, **=p<0.01 and **=p<0.05 based on one-way ANOVA and t-test.