Journal: Scientific Reports

Article Title: The sonic hedgehog signaling inhibitor cyclopamine improves pulmonary arterial hypertension via regulating the bone morphogenetic protein receptor 2 pathway

doi: 10.1038/s41598-025-97627-7

Figure Lengend Snippet: Expression of SHH and BMPR2 protein in the pulmonary arteries of rats with MCT-induced PAH. Sprague–Dawley rats were treated with MCT (50 mg/kg) for 21 days and hPASMCs were treated with SHH-N for 48 h. ( a ) The location of SHH protein was detected using immunohistochemical staining ( n = 6 for each treatment group). ( b ) The SHH protein expression level was measured by western blot analysis. ( c ) Pulmonary hemodynamics were evaluated by RVSP and ( d ) right ventricular remodeling was measured by RVHI. Apoptosis was detected by TUNEL staining analysis ( e ) and proliferation was detected by BrdU staining in hPASMCs ( f ). ( g , h ) Western blot analysis showing the BMPR2 and OPN protein expression levels in the saline group and the MCT-treated group. The protein expression levels were normalized to GAPDH ( n = 3 for each treatment group). * P < 0.05 vs. the saline group; ** P < 0.001 vs. the saline group. α-SMA alpha-smooth muscle actin, BMPR2 bone morphogenetic protein receptor 2, HE hematoxylin-eosin, MCT monocrotaline, OPN osteopontin, PAH pulmonary arterial hypertension, RVHI right ventricular hypertrophy index, RVSP right ventricular systolic pressure, SHH Sonic hedgehog. Red labels the Brdu-positive cells, green labels the TUNEL-positive cells and blue labels the DAPI-stained nuclei.

Article Snippet: The membranes were blocked with 5% skim milk or 1% bovine serum albumin and probed with one of the following monoclonal antibodies: monoclonal rabbit anti-Bax antibody (1:1000, 2774, Cell Signaling, Danvers, MA, USA), monoclonal rabbit anti-Bcl2 antibody (1:1000; 2872, Cell Signaling), anti-BMPR2 (1:1000, 6979, Cell Signaling), monoclonal rabbit anti-SMAD1 antibody (1:1000, A14386-1, Boster Bio, Wuhan, China), monoclonal rabbit anti-p-SMAD1/5/8 antibody (1:400, 9511, Cell Signaling), polyclonal rabbit anti-SHH antibody (1:1500, 20697-1-AP, Proteintech, Chicago, IL, USA), polyclonal rabbit anti-osteopontin antibody (1:2000, 22952-1-AP, Proteintech), or anti-GAPDH (1:2500, ab9485, Abcam, Cambridge, MA, USA), followed by the matched secondary antibodies (Proteintech).

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, TUNEL Assay, BrdU Staining, Saline

Journal: Scientific Reports

Article Title: The sonic hedgehog signaling inhibitor cyclopamine improves pulmonary arterial hypertension via regulating the bone morphogenetic protein receptor 2 pathway

doi: 10.1038/s41598-025-97627-7

Figure Lengend Snippet: Effect of cyclopamine on proliferation and expression of α-SMA and osteopontin protein in BMPR2 knockdown hPASMCs. The hPASMCs were treated with Lv-NC, Lv-siBMPR2, and SHH-N in the presence or absence of cyclopamine (2.5, 5, or 10 µM) for 48 h. Exogenous BMP4 protein (10 ng/ml) was added in culture medium to promote proliferation. The transfection efficiency was evaluated by ( a ) BMPR2 protein levels and ( b ) BMPR2 mRNA levels. ( c ) Proliferation was detected by BrdU staining analysis. ( d ) The numbers of BrdU-positive cells were measured. Western blot analysis and quantification showing ( e ) α-SMA and ( f ) OPN protein expression levels in the various groups. The protein expression levels were normalized to GAPDH ( n = 3 for each treatment group). # P < 0.05 vs. the control group; * P < 0.05 vs. the Lv-siBMPR2 group. α-SMA alpha-smooth muscle actin, BMPR bone morphogenetic protein receptor, Cy cyclopamine, hPASMCs human pulmonary arterial smooth muscle cells, Lv lentivirus, OPN osteopontin, SHH Sonic hedgehog. Red labels the Brdu-positive cells and blue labels the DAPI-stained nuclei.

Article Snippet: The membranes were blocked with 5% skim milk or 1% bovine serum albumin and probed with one of the following monoclonal antibodies: monoclonal rabbit anti-Bax antibody (1:1000, 2774, Cell Signaling, Danvers, MA, USA), monoclonal rabbit anti-Bcl2 antibody (1:1000; 2872, Cell Signaling), anti-BMPR2 (1:1000, 6979, Cell Signaling), monoclonal rabbit anti-SMAD1 antibody (1:1000, A14386-1, Boster Bio, Wuhan, China), monoclonal rabbit anti-p-SMAD1/5/8 antibody (1:400, 9511, Cell Signaling), polyclonal rabbit anti-SHH antibody (1:1500, 20697-1-AP, Proteintech, Chicago, IL, USA), polyclonal rabbit anti-osteopontin antibody (1:2000, 22952-1-AP, Proteintech), or anti-GAPDH (1:2500, ab9485, Abcam, Cambridge, MA, USA), followed by the matched secondary antibodies (Proteintech).

Techniques: Expressing, Knockdown, Transfection, BrdU Staining, Western Blot, Control, Staining