Upregulation of the Piezo1 channel and CaSR and Notch receptors in proliferative pulmonary artery smooth muscle cells (PASMCs) during the contractile-to-proliferative phenotypical transition. A: freshly isolated pulmonary artery (PA) branches from the rat lung (left) and primary cultured PASMCs derived from the same rat in medium containing serum and growth factors (right). A phase-contrast image and a fluorescent image showing DAPI (4′,6-diamidino-2-phenylindole, in blue) and α-smooth muscle actin (SMA, in green) in primary cultured PASMCs are shown. B: Western blot analyses on the contractile smooth muscle cell (SMC) markers, myosin heavy chain 11 (MHC11), SMα2-actin (ACTA2) and SM22-α or transgelin (TAGLN), and the cell proliferation marker, proliferation cell nuclear antigen (PCNA), as well as Piezo1 in freshly isolated PA (mainly containing contractile PASMC) and primary cultured PASMCs (mainly containing proliferative PASMCs). C: summarized data (means ± SE, n = 6–14 in each group) showing protein levels of MHC11, ACTA2, TAGLN, and PCNA (left) and Piezo1 (right) in freshly isolated PA [PA (contractile PASMCs)] and primary cultured PASMCs [PASMCs (proliferative PASMCs)]. Data are shown as arbitrary units (top) and values normalized to PA tissue (bottom). The horizontal dotted line in the lower panel of the Piezo1 graph depicts the level of protein in the PA. **P < 0.01, ***P < 0.001 vs. PA (contractile PASMCs) (unpaired t test). D: Western blot analyses on CaSR, Notch-2, and Notch3, as well as MHC11, ACTA2, and PCNA in freshly isolated PA (mainly containing contractile PASMCs) and primary cultured PASMCs (mainly containing proliferative PASMCs). E: summarized data (means ± SE) showing protein expression level of CaSR (n = 7), Notch2 (n = 6), and Notch3 (n = 6) in the PA and in the PASMCs. Data are presented as arbitrary units (top) or values normalized to PA tissue (bottom). The horizontal broken lines indicate the level of the CaSR, Notch2, and Notch3 proteins in the PA. **P < 0.01, ***P < 0.001 vs. PA (contractile PASMC) (unpaired t test).