Figure 5. SRT2104 restores functional TSC2, inhibits proliferation, and induces apoptosis in human PAH PAVSMCs.

a-g. Human PAH PAVSMCs were treated with diluent (0) or the indicated concentrations of SRT2104 and immunoblotted for the indicated proteins. Data are means±SE from three experiments, each performed on the cells from a different subject. P values for SRT2104 treatment compared to diluent (0) were determined by Kruskal-Wallis rank test with Dunn’s pairwise comparison.

h. Immunocytochemical analysis to detect fibronectin produced by human non-diseased (control) and PAH PAVSMCs treated with diluent or 10 μM SRT2104 for 48 hr. Scale bar, 100 μm.

i-k. Human PAH PAVSMCs were treated with diluent (−) or 10 μM SRT2104 (+) for 48 hr. Proliferation assessed by cell counts (i) or BrdU incorporation (j) and apoptosis as determined by TUNEL staining (k) analyses were performed. Data are means±SE from three experiments, each performed on the cells from different subject. P values for SRT2104 treatment compared to diluent (0) were determined by Mann Whitney U test.

l. Equal numbers of human control PAVSMCs were seeded on hydrogels with 25 kPa stiffness, treated with diluent (−) or 10 μM SRT2104 (+) for 48hr, and counted. Data are means±SE from three experiments, each performed on the cells from a different subject. P value for SRT2104 treatment compared to diluent (0) was determined by Mann Whitney U test.

m-q. Immunoblot (m-n), proliferation (as measured by cell counting or BrdU incorporation) (o-p) and apoptosis (as measured by TUNEL staining) (q) analyses were performed on PAH PAVSMCs transfected with siContr or siTSC2 and treated with 10μM SRT2104 or diluent for 48hr. Data are means±SE from three experiments, each performed on the cells from a different subject. P values for SRT2104 and/or siTSC2 compared to siContr (Scr) and diluent were determined by Kruskal-Wallis rank test with Dunn’s pairwise comparison.