SP promotes autophagy and increases GAS5 in PAECs. (A) Abundance of LC3B in SP (10 and 100 µM)-treated PAECs, as assessed by immunofluorescence assays (scale bar, 25 µm; magnification, ×20). (B) Autophagic vacuoles in the cellular cytoplasm of SP (10 and 100 µM)-treated PAECs, as evaluated by transmission electron microscopy (scale bar, 2 µm; magnification, ×10,000). Red arrows indicate the autophagic vacuoles. (C) Protein expression of LC3B, Beclin-1 and ATG7 in SP (10 and 100 µM)-treated PAECs were assessed by Western blotting. (D) Expression of GAS5 in PAECs treated with SP (10 and 100 µM). (E) Sequence information of GAS5. (F) Prediction of protein-coding potential of GAS5. (G) Prediction of subcellular location of GAS5. (H) The subcellular location of GAS5 was assessed by nuclear/cytoplasmic RNA separation assays. (I) Effect of SP (10 µM) on the subcellular location of GAS5. *P<0.05; **P<0.01; ***P<0.001. There were at least three replicates in each group available for analysis. SP, spermidine; GAS5, growth arrest-specific transcript 5; PAECs, pulmonary artery endothelial cells.