Figure 6.

Overexpression of SIRT3 and supplementation with NAD⁺ decreased proliferation and induced apoptosis in PH-Fibs. (A and B) PH-Fibs from (A) patients with IPAH and (B) 2-week hypoxia-induced neonatal PH calves were seeded in 24-well plates in equal numbers and were transfected with plasmid to overexpress SIRT3 in the presence or absence of NAD⁺ for 72 hours or supplemented with NAD⁺ alone. Plates were collected daily, stored at −80 °C, and analyzed for cell proliferation using CyQUANT methods. Data were analyzed using two-way ANOVA and multiple comparisons. Data are presented as fold change relative to Day 0 and presented as mean ± SEM. The P values for each pair of comparisons are shown in the table below the corresponding graphs. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with untreated within the same day. (C) The apoptosis assay was performed with bovine fibroblast cells and measured using annexin V–FITC/PI labeling in flow cytometry. PH-Fibs have a significantly lower percentage of apoptotic cells compared with CO-Fibs, and overexpressing SIRT3 plus NAD⁺ supplementation synergistically induced cell apoptosis in PH-Fibs. *P < 0.05 and **P < 0.01. PI = propidium iodide.