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. Author manuscript; available in PMC: 2023 Dec 6.
Published in final edited form as: Sci Signal. 2022 Dec 6;15(763):eabn2743. doi: 10.1126/scisignal.abn2743

Figure 3. Reduced TSC2 abundance in PAVSMCs promotes YAP/TAZ accumulation, mTOR activity, and proliferation of non-diseased pulmonary vascular cells through ECM remodeling.

Figure 3.

a, b. Human control and PAH PAVSMCs (from 5 subjects/group) were immunoblotted for fibronectin (FN) and collagen 1A1 (Col1A1). Data are means±SE. PAH was normalized to Cont, which was set at 1. P values for PAH compared to Cont were determined by Mann Whitney U test.

c, d. Human control PAVSMCs were transfected with siRNA directed against TSC2 (siTSC2) or control siRNA GLO (siCont) and immunoblotted for FN and Col1A1. Data are means±SE from n=3 subjects/group. siTSC2 was normalized to siCont, which was set at 1. P values for siTSC2 compared to siCont were determined by Mann Whitney U test.

e-o. Pre-confluent control PAVSMCs were infected with adenoviruses encoding control shRNA (shCont) or shRNA directed against TSC2 (shTSC2) for six days (e). Cells were removed and an equal amount of non-diseased (control) untreated PAVSMCs (f-l), PAVSMCs treated with diluent, 10μM ATN161 (α5β1 integrin inhibitor), or 10μM BTT3033 (α2β1 integrin inhibitor) (m), control PAAFs (n), or control PAECs (o) were plated on the matrices. Four days after plating, immunoblot (f-j) and proliferation analyses as measured by Ki67 staining (k) or cell counting (l-o) were performed. Data are means±SE from n=3 subjects/group. P values for siTSC2 compared to siCont were determined by Mann Whitney U test (significance) (g-l, n, o) and by Kruskal-Wallis test with Dunn’s pairwise comparison (m).