Fig 1: Inhibition of mTOR with rapamycin downregulates protein expression of CALHM1/2 in proliferating PASMCs. (A): Representative Western blot analyses on phosphorylated (p) AKT (pAKT), AKT, pmTOR, mTOR, and PCNA as well as CALHM1 and CALHM2 in primary cultured PASMCs treated with vehicle (Control) or rapamycin (Rapamycin, 10 nM) for 24 h. (B): Summarized data (mean ± SE, n = 5–6 experiments) showing levels of pAKT, AKT, pmTOR, and mTOR in control and rapamycin-treated proliferating PASMCs. (C): Summarized data (mean ± SE, n = 6 experiments) showing the ratio of pAKT/AKT and pmTOR/mTOR in control and rapamycin-treated proliferating PASMCs. (D): Summarized data (mean ± SE, n = 6) showing the level of PCNA, a cell proliferation marker, in control and rapamycin-treated proliferating PASMCs. (E): Summarized data (mean ± SE, n = 6) showing protein expression levels of CALHM1 and CALHM2 in control and rapamycin-treated proliferating PASMCs. Data are presented as arbitrary units as well as normalized to PA tissue. *p < 0.05 vs. PA (Contractile PASMC).
Fig 2: Upregulated calcium homeostasis modulator (CALHM1 and CALHM2) protein expression in proliferative PASMCs. (A): Western blot analyses on MHC11, TAGLN, and PCNA as well as CALHM1 and CALHM2 in freshly isolated PA (mainly containing contractile PASMCs) and primary cultured PASMCs (mainly containing proliferative PASMCs). (B): Summarized data (mean ± SE) showing protein expression levels of CALHM1 (n = 7) and CALHM2 (n = 17) in the PA and in PASMCs. Data are presented as arbitrary units (upper panels) or normalized to PA tissue (lower panels). The horizontal broken lines indicate the level of the CALHM1/2 proteins in the PA. *p < 0.05 vs. PA (Contractile PASMC).
Fig 3: Upregulated CALHM1/2 in proliferating PASMCs from patients with idiopathic pulmonary arterial hypertension (IPAH) and inhibitor or downregulation of CALHM1/2 significantly inhibits PASMC proliferation. (A,B): Representative images (A) and summarized data (mean ± SE, B) showing protein level of CALHM1 (n = 8) and CALHM2 (n = 6) in primary cultured PASMCs isolated from normal subjects (Normol) and patients with IPAH. **p < 0.01 vs. Normol PASMC. (C): Summarized data (mean ± SE, n = 18) showing number of viable PASMCs (determined by absorbance at 450 nm) before (Basal, 0 h) and after 48 h of incubation in 5% FBS media in the absence (5% FBS) and presence of CGP 37157 (CGP, 0.1 μM, 48 h) (5% FBS + CGP). *p < 0.01 between 5% FBS and 5% FBS+CGP. (D): Representative image (left) and summarized data (mean ± SE, n = 6, right) showing Western blot analyses on CALHM1 in normal PASMC treated with control siRNA (siNT) and siRNA for CALHM1 (siCALHM1). **p < 0.01 vs. siNT. (E): Summarized data (mean ± SE, n = 18) showing number of Normal PASMCs and IPAH PASMCs before (Basal, 0 h) and 48 h after incubation in 10% FBS media in the presence of control siRNA (siNT) and siRNA for CALHM1 (siCALHM1). ***p < 0.001 between Normal and IPAH; ###p < 0.001 vs. siNT. (F): 10% FBS (48 h)-mediated increases in viable PASMC numbers (or 10% FBS-induced PASMC proliferation, % of the basal cell number at 0 h) in normal and IPAH PASMCs treated with siNT and siCALHM1. Data are constructed from the number of viable cells shown in panel E. ***p < 0.001 vs. Normal PASMC; ###p < 0.001 vs. siNT. (G): Representative image (left) and summarized data (mean ± SE, n = 6, right) showing Western blot analyses on CALHM2 in normal PASMC treated with siNT and siRNA for CALHM2 (siCALHM2). ***p < 0.01 vs. siNT. (H): Summarized data (mean ± SE, n = 18) showing number of normal and IPAH PASMCs before (Basal, 0 h) and 48 h after incubation in 10% FBS media containing siNT and siCALHM2. ***p < 0.001 between Normal and IPAH PASMC; ###p < 0.001 vs. siNT. (I): 10% FBS (48 h)-mediated increases in viable PASMC numbers (or 10% FBS-induced PASMC proliferation, % of the basal cell number at 0 h) in normal and IPAH PASMCs treated with siNT and siCALHM2. Data are constructed from the number of viable cells shown in panel H. ***p < 0.001 vs. Normal PASMC; ###p < 0.001 vs. siNT.
Fig 4: Serum starvation inhibits PASMC proliferation and downregulates protein expression of CALHM1/2 in proliferating PASMCs. (A): Representative Western blot analyses on CALHM1, CALHM2 and PCNA in primary cultured PASMCs incubated in culture media containing 1%, 5%, and 10% fetal bovine serum (FBS, for 48 h). (B): Summarized data (mean ± SE, n = 6 experiments) showing levels of CALHM1, CALHM2, and PCNA in PASMCs cultured in 1%, 5%, and 10% FBS-containing media. *p < 0.05 and #p < 0.05 vs. PASMC cultured in 1% FBS medium.
Fig 5: Upregulated CALHM1 and CALHM2 in freshly isolated PA from rats with monocrotaline (MCT)-induced pulmonary hypertension (PH). (A–C): Representative images (A) and summarized data (mean ± SE, n = 5–13) showing protein levels of MHC11 (n = 5), PCNA (n = 13), PDGF receptor α (PDGFRα, n = 9) (B) as well as CALHM1 and CALHM2 (C) in freshly isolated PA (mainly containing contractile PASMC) from vehicle- (Cont) or MCT-injected rats. *p < 0.05 vs. Cont rats. (D): Representative records of right ventricular pressure (RVP, upper panels) and right ventricule (RV) contractility (RV- ± dP/dt, lower panels) in control rats (Cont) and MCT-injected rats (MCT). (E): Summarized data (mean ± SE, n = 5 for Cont rats and n = 6 for MCT rats) showing right ventricular systolic pressure (RVSP, a surrogate measure of pulmonary arterial systolic pressure), mean PAP (mPAP), RV-+dP/dtmax and heart rate in control rats and MCT-injected rats. *p < 0.05 vs. Cont rats. (F): Summarized data (mean ± SE, n = 9 for each group) showing the dry weight of the RV and the left ventricle (LV) and septum (S) (upper panel), as well as Fulton Index (lower panel), the ratio of the weight of RV to the weight of LV and S [RV/(LV+S)] in vehicle- (Cont) or MCT-injected rats. *p < 0.05 vs. RV in Control rats; #p < 0.05 vs. RV in MCT-injected rats (upper panel); *p < 0.05 vs. Cont rats (lower panel).
Supplier Page from Abcam for Anti-CALHM2 antibody