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. 2022 Apr 1;298(5):101887. doi: 10.1016/j.jbc.2022.101887

Figure 2.

Figure 2

PALMD accelerates hVIC in vitro calcification, osteogenic differentiation, and apoptosis. hVICs were transfected with 20 nM siScrambled or siPALMD and treated with control medium (Ctr) or calcifying medium (Cal.) for 48 h or for up to 7 days. A, alizarin red staining for calcium deposition in hVICs at day 7. Plate view (upper) and microscopic view (lower), and the scale bar represents 500 μm. B, quantitative calcium assay showing calcium deposition in hVICs at day 7, n = 5. C–F, RT-qPCR showing MSX2, RUNX2, BMP2, and ALPL mRNA expression in hVICs at 48 h, n = 6. G, representative Western blotting images for PALMD, RUNX2, BMP2, MSX2, Cleaved-caspase3, Caspase3, and β-actin protein expression in hVICs at day 3 and day 7, n = 3 or 5. H, hVICs were infected with Ad-null (MOI = 100) or Ad-PALMD (MOI = 100) and treated with control medium (Ctr) or calcifying medium (Cal.) for up to 7 days. Quantitative calcium assay showing calcium deposition in hVICs at day 3, n = 5. I–K, RT-qPCR showing MSX2, BMP2, and ALPL mRNA expression in hVICs for 48 h, n = 6. L, representative Western blotting images for PALMD, RUNX2, BMP2, MSX2, Cleaved-caspase3, Caspase3, and β-actin protein expression in hVICs at day 3 and day 7, n = 3, 4 or 5. M, hVICs were treated with control medium (Ctr) or calcifying medium (Cal.) in the presence or absence of 20 μM caspase inhibitor ZVAD, 25 ng/ml IL1β, or 10 μg/ml cycloheximide for up to 7 days. Quantitative calcium assay showing calcium deposition at day 7, n = 4. Data are presented as mean ± SEM, and statistical significance was analyzed by a two-tailed unpaired Student’s t test or one-way ANOVA followed by Tukey's multiple comparisons test. hVIC, human valve interstitial cell.