Fig. 2: Function of GPR153 in cultured human SMCs.

A–E The expression of marker genes for SMC differentiation was determined in hAoSMCs treated with control esiRNA (esiContr) or GPR153-specific esiRNA (esiGPR153) by qRT-PCR (A, D, E; data normalized to GAPDH and control set to 1, n = 6–12) or immunoblotting (B representative immunoblots; C densitometric analysis of signal strength normalized to loading control GAPDH, n = 7–8). F Cell number changes were determined at the indicated times after seeding of equal numbers of control and GPR153 knockdown hAoSMCs; Cells were cultured in growth medium (GM) with or without 10 ng/ml PDGF-BB (n = 9–10). G The percentage of EdU-positive cells was determined by flow cytometry 24 h after the addition of 10 µM EdU (n = 3, cells cultured in growth medium). H The percentage of annexin V-positive (AnnV⁺) cells was determined by flow cytometry (n = 5–7). I Viability of cells was determined by measuring live-cell protease activity (n = 5–7). J, K Gene expression was determined by qRT-PCR in hCASMCs after esiRNA-mediated (J) or siRNA-mediated (K) knockdown of GPR153 (data normalized to GAPDH, control set to 1, n = 7–10). L Cell numbers were determined at the indicated time points after seeding equal numbers of control and GPR153 knockdown hCASMCs (n = 3). If not otherwise indicated, experiments were performed after 48 h of serum starvation to induce a differentiated state. Data are means ± SEM; differences between genotypes were analyzed using two-way ANOVA and Sidak’s post hoc test (A, C–F, J–L), unpaired two-sided t test (G–I). FC fold change; n number of independent knockdown experiments; RLU relative luminescence units; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.