Fig 1: PARP Inhibitors Decrease Calcification of hVSMCs In Vitro(A) qRT-PCR analysis of hVSMCs and MC3T3 cells in vitro shows expression of PARP1 and PARP2 enzymes in control cultures. hVSMC cultures under calcifying conditions show increased expression of PARP2 (n = 3).(B) Slot blot of hVSMC and MC3T3 cells under calcifying conditions showing increased PAR.(C) The o-cresolphthalein assay (n = 3) showed a time-dependent increase in mineralization, which was inhibited in a dose-dependent manner by the PARP inhibitor PJ-34 (0.5, 1.5, and 10 uM).(D) PARP activity was reduced by PJ34 treatment in a dose-dependent manner (n = 4) in hVSMC cultures. See Figures S6A and S6B for data on bVSMC model.(E) The PARG inhibitor DEA (0.1 mM) increased calcification of hVSMCs treated with high Ca/P media quantified by o-cresolphthalein. (n = 6). Mean ± SEM, Student’s t test, **p < 0.01, ****p < 0.0001.(F) PARP inhibitors can block calcification of hVSMCs (n = 3) and MC3T3 (n = 6) cells. PJ = PJ-34, Min, minocycline; Ola, Olaparib; Ruc, Rucaparib; Nir, Niraparib; Vel, Veliparib; all are at 3 µM. See Table S2 for enzyme inhibitor assay details and Figure S6C for details on minocycline as a PARP1/2 inhibitor.Graphs in (A), (C), (D), and (F) show mean ± SEM. Statistical significance was tested with one-way ANOVA with Dunnett’s post hoc tests. *p between p < 0.05, **p < 0.01, ****p < 0.0001.(G) Alizarin Red S staining of mineral in hVSMC cultures in the absence and presence of PARP inhibitors PJ-34 (3 µM) and minocycline (3 µM).(H) Slot blot showing increased PAR under calcifying conditions and its inhibition by minocycline (3 µ?).