Fig 1: Overexpression of PGC-1α improved PAEC function and inhibits calcium release in smooth muscle cells (SMCs). Nanoparticles containing control vector or PGC1-α expression plasmid (OE:PGC1-α) were injected to mice via tail vein. Mice were exposed to normoxia (Ctrl) or hypoxia (10% O2) for 4 weeks. A-B, Endothelial nitric oxide synthase (eNOS) expression in lung tissues, as detected by Western blotting (A) and normalized to GAPDH. *P < 0.01 vs. Ctrl; #P < 0.01 vs. Vector group; n = 6. C-D, eNOS protein expression. Human PAECs were transfected with empty vector (Ctrl) or PGC-1α expression plasmid and then treated with vehicle or TGF-β (5 ng/ml) for 3 days. eNOS expression was detected by Western blotting (C) and normalized to GAPDH (D). *P < 0.05 vs. vehicle-treated cells; #P < 0.01 vs. Ctrl vector-transfected cells with TGF-β; n = 6. E, Nitric oxide (NO) release assay. PAECs were treated with nanoparticles containing empty Vector (Ctrl) or PGC-1α expression plasmid (PGC-1α). One day later, cells were treated with vehicle or TGF-β for 20 min. NO generated by PAECs was measured using a Nitric Oxide Assay Kit (Abcam, ab65327). The relative NO levels were quantified by normalizing to the vehicle-treated cells, which was set as 1. *P < 0.01 vs. vehicle-treated cells; #P < 0.01 vs. Ctrl vector nanoparticle-treated cells with TGF-β; n = 6. F-G, Calcium flux assay. PAECs were transfected with empty vector (Ctrl) or PGC-1α expression plasmid and cocultured with SMCs in a transwell plate. PACEs in the upper chamber were treated with vehicle (Ctrl) or TGF-β (5 ng/ml) for 3 days. Then SMCs in the lower chamber were incubated with Fluo-4 AM at 37 °C and room temperature each for 45 min. Calcium flux signal (green) was detected with a Nikon fluorescence microscope using FITC channel (F) and normalized to control (G). *P < 0.05 vs. vehicle-treated cells; #P < 0.01 vs. Ctrl vector-transfected cells with TGF-β; n = 6. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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