Journal: bioRxiv

Article Title: Impaired Lung BCAA Metabolism Promotes Ferroptosis and Resultant Pulmonary Arterial Hypertension-Associated Hepatopathy

doi: 10.1101/2025.09.03.672819

Figure Lengend Snippet: Excess BCAAs promoted a pro-ferroptotic phenotype in human PASMC . (A) Representative confocal micrographs of PASMC stained with MitoTracker Orange and quantification of mitochondrial organization in control and BCAA-treated PASMC (Control PASMC [Con]: 1.1±0.6, BCAA-Treated PASMC [BCAA]: 0.7±0.3); p -values determined by Mann-Whitney U-test. (B) Confocal micrographs of TRME-stained PASMC in control and BCAA-treated media, with corresponding quantification of mitochondrial membrane hyperpolarization, indicated by fluorescence intensity (Con: 8.3±17.2, BCAA: 42.7±9.7). p -values determined by Mann-Whitney U-test. (C) Excess BCAAs increase mitochondrial ROS, demonstrated by confocal micrographs of control and BCAA-treated PASMCs stained with MitoSox Red (Con: 6.4±2.1, BCAA: 14.5±6.9 MFI). p -values determined by Mann-Whitney U-test. (D) Representative confocal micrographs of control, BCAA-treated, and BCAA and ferrostatin-1-treated PASMCs incubated with 50 μM oleate and 50 μM palmitate and stained for lipid peroxidation using BODIPY (Con: 1.8±0.3, BCAA: 1.4±0.1, BCAA-treated with 5 μM ferrostatin-1 [BCAA-FER1]: 1.7±0.3). p -values determined by Kruskal-Wallis test and Dunn’s multiple comparisons test. (E) Incubation with BCAAs induces ferroptotic cell death, demonstrated by viability staining of control, BCAA-treated, and BCAA-treated with ferrostatin-1 PASMCs via Trypan Blue (Con: 9.4±7.5, BCAA: 31.7±11.7, BCAA-FER1: 13.0±11.1). White arrows indicate trypan blue-positive cells. p -values determined by ordinary one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: Human PASMC (ATCC PCS-100-023) were grown with Sigma Basic Eagle Medium (Sigma, B1522-500) with supplements (Lonza, CC-3182) and passaged with subculture reagents (Lonza CC-5034).

Techniques: Staining, Control, MANN-WHITNEY, Membrane, Fluorescence, Incubation, Comparison

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: Orai1 and stromal-interacting molecule 1 (STIM1) expression in mouse pulmonary arterial smooth muscle cells (PASMCs). A: RT-PCR products from cultured mouse PASMCs amplified using primers for mouse Orai1 (269 bp), STIM1 (473 bp), and β-actin (498 bp). Three separate RT-PCR reactions were performed in the presence (+) and absence (−) of reverse transcriptase (RT). B: Orai1, STIM1, and GAPDH proteins were detected in cultured mouse PASMCs using Western blot analysis. Experiments were performed in 5 separate Western blot analyses.

Article Snippet: The cultured mouse PASMCs were fixed in 4% paraformaldehyde and stained with a rabbit polyclonal Orai1 antibody (1:100, ProSci) and mouse monoclonal STIM1 antibody (1:100, EXBIO) using Vector M.O.M. immunodetection kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instruction.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Amplification, Reverse Transcription, Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: Orai1 mediates capacitative Ca2+ entry (CCE) in mouse PASMCs. A and B: Orai1 protein and GAPDH were detected in nontransfected mouse PASMCs and in PASMCs transfected with 200 nM scrambled small interfering RNA (siRNA; negative control). The expression of Orai1 but not GAPDH was reduced significantly in cells transfected with 200 nM Orai1 siRNA. Experiments were performed in 3 separate Western blot analyses (**P < 0.01, ANOVA). C: siRNA knockdown of Orai1 reduced the cyclopiazonic acid (CPA)-induced transient but not the sustained increase in fura-2 fluorescence ratio in the presence of 10 μM nifedipine. 0Ca, Ca2+-free solution. D: bar graph showing mean changes in transient and sustained increase in intracellular Ca2+ concentration ([Ca2+]i) caused by 10 μM CPA after readdition of 2 mM Ca2+ in the presence of 10 μM nifedipine, in negative control cells (filled bars, n = 89), and in Orai1 siRNA-transfected cells (open bars, n = 86). **P < 0.01 (unpaired t-test). E: siRNA knockdown of Orai1 reduced the increase in Mn2+ quench of fura-2 fluorescence caused by 10 μM CPA in the presence of 10 μM nifedipine. AU, arbitrary units. F: bar graph showing percentage change in fura-2 quench rate after store-depletion in the presence of 10 μM nifedipine, in negative control cells (filled bar, n = 79), and in Orai1 siRNA-transfected cells (open bar, n = 86). **P < 0.01 (unpaired t-test).

Article Snippet: The cultured mouse PASMCs were fixed in 4% paraformaldehyde and stained with a rabbit polyclonal Orai1 antibody (1:100, ProSci) and mouse monoclonal STIM1 antibody (1:100, EXBIO) using Vector M.O.M. immunodetection kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instruction.

Techniques: Transfection, Small Interfering RNA, Negative Control, Expressing, Western Blot, Knockdown, Fluorescence, Concentration Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: STIM1 is associated with Orai1 to mediate CCE in mouse PASMCs. A and B: STIM1, Orai1, and GAPDH proteins were detected in nontransfected mouse PASMCs and in PASMCs transfected with 200 nM scrambled siRNA (negative control). The expression of STIM1 but not Orai1 or GAPDH was reduced significantly in cells transfected with 200 nM STIM1 siRNA. The expressions of STIM1 and Orai1 but not GAPDH were reduced significantly in cells transfected with both 200 nM STIM1 siRNA and 200 nM Orai1 siRNA. Experiments were performed in 3 separate Western blot analyses (**P < 0.01, ANOVA). C: siRNA knockdown of STIM1 reduced the CPA-induced transient and sustained increase in fura-2 fluorescence ratio in the presence of 10 μM nifedipine. siRNA knockdown of STIM1 and Orai1 further reduced the CPA-induced transient but not sustained increase in fura-2 fluorescence ratio in the presence of nifedipine. D: bar graph showing mean changes in transient and sustained increase in [Ca2+]i caused by 10 μM CPA after readdition of 2 mM Ca2+ in the presence of 10 μM nifedipine, in negative control cells (filled bars, n = 103), in STIM1 siRNA-transfected cells (shaded bars, n = 148), and in STIM1 siRNA and Orai1 siRNA-transfected cells (open bars, n = 70). **P < 0.01, compared with negative control cells (ANOVA); ++P < 0.01, compared with negative control cells and STIM1 siRNA-transfected cells (ANOVA). E: siRNA knockdown of STIM1 reduced the increase in Mn2+ quench of fura-2 fluorescence caused by 10 μM CPA in the presence of 10 μM nifedipine. siRNA knockdown of STIM1 and Orai1 further reduced the increase in Mn2+ quench of fura-2 fluorescence caused by CPA in the presence of nifedipine. F: bar graph showing percentage change in fura-2 quench rate after store-depletion in the presence of 10 μM nifedipine, in negative control cells (filled bar, n = 125), in STIM1 siRNA-transfected cells (shaded bar, n = 151), and in STIM1 siRNA and Orai1 siRNA-transfected cells (open bar, n = 137). **P < 0.01, compared with negative control cells (ANOVA); ++P < 0.01, compared with the negative control cells and STIM1 siRNA-transfected cells (ANOVA).

Article Snippet: The cultured mouse PASMCs were fixed in 4% paraformaldehyde and stained with a rabbit polyclonal Orai1 antibody (1:100, ProSci) and mouse monoclonal STIM1 antibody (1:100, EXBIO) using Vector M.O.M. immunodetection kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instruction.

Techniques: Transfection, Negative Control, Expressing, Western Blot, Knockdown, Fluorescence

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: knockdown of Orai1 reduced CCE in STIM1-overexpressing mouse PASMCs. A and B: STIM1, Orai1, and GAPDH proteins were detected in cells infected with adenovirus containing green fluorescent protein (Ad-GFP) transfected with 200 nM scrambled siRNA. The expression of STIM1 but not Orai1 or GAPDH increased markedly in cells infected with STIM1-GFP-adenovirus (Ad-GFP-STIM1) transfected with scrambled siRNA. The expression of Orai1 but not STIM1 or GAPDH was reduced significantly in STIM1-overexpressing cells transfected with 200 nM Orai1 siRNA. Experiments were performed in 3 separate Western blot analyses (**P < 0.01, ANOVA). C: overexpression of STIM1 in scrambled siRNA-transfected cells caused an increase in CPA-induced transient and sustained rise in fura-2 fluorescence ratio in the presence of 10 μM nifedipine. The increases in fluorescence ratio were reduced in Orai1 siRNA-transfected cells overexpressed with STIM1. D: bar graph showing mean changes in transient and sustained increase in [Ca2+]i caused by 10 μM CPA after readdition of 2 mM Ca2+ in the presence of 10 μM nifedipine, in GFP-infected cells transfected with scrambled siRNA (filled bars, n = 33), in STIM1-overexpressing cells transfected with scrambled siRNA (shaded bars, n = 65), and in STIM1-overexpressing cells transfected with Orai1 siRNA (open bars, n = 50). *P < 0.05, compared with GFP-infected cells transfected with scrambled siRNA and STIM1-overexpressing cells transfected with Orai1 siRNA (ANOVA). E: overexpression of STIM1 in scrambled siRNA-transfected cells caused a CPA-induced increase in Mn2+ quench of fura-2 fluorescence in the presence of 10 μM nifedipine. The increases in Mn2+ quench of fura-2 fluorescence was reduced in Orai1 siRNA-transfected cells overexpressed with STIM1. F: bar graph showing percentage change in fura-2 quench rate after store-depletion in the presence of 10 μM nifedipine, in GFP-infected cells transfected with scrambled siRNA (filled bars, n = 40), in STIM1-overexpressing cells transfected with scrambled siRNA (shaded bars, n = 95), and in STIM1-overexpressing cells transfected with Orai1 siRNA (open bars, n = 57). **P < 0.01, compared with GFP-infected cells transfected with scrambled siRNA and STIM1-overexpressing cells transfected with Orai1 siRNA (ANOVA).

Article Snippet: The cultured mouse PASMCs were fixed in 4% paraformaldehyde and stained with a rabbit polyclonal Orai1 antibody (1:100, ProSci) and mouse monoclonal STIM1 antibody (1:100, EXBIO) using Vector M.O.M. immunodetection kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instruction.

Techniques: Knockdown, Infection, Transfection, Expressing, Western Blot, Over Expression, Fluorescence

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: Orai1 coimmunoprecipitates with STIM1 in mouse PASMCs. A, left: Orai1 was detected in cultured mouse PASMCs in the absence and presence of store-depletion. Right: bar graph showing expression levels of Orai1 measured relative to GAPDH in control cells (denoted as 1, filled bar) and in cells subjected to store-depletion (open bar). Data are means ± SE of 7 separate Western blot analyses. B, left: STIM1 was detected in cultured mouse PASMCs in the absence and presence of store-depletion. Right: bar graph showing expression levels of STIM1 measured relative to GAPDH in control cells (denoted as 1, filled bar) and in cells subjected to store-depletion (open bar). Data are means ± SE of 7 separate Western blot analyses. C: STIM1 coimmunoprecipitated Orai1 in cultured mouse PASMCs in the absence and presence of store-depletion. STIM1 was first immunoprecipitated (IP) with EXBIO STIM1 antibody (10 μg), and the blot was subsequently probed with BD Biosciences STIM1 antibody (WB, 1:100). The blot was then probed for coimmunoprecipitation (co-IP) of Orai1 expression using Orai1 antibody (WB, 1:100, ProSci). Experiments were performed in 3 separate co-IP procedures and Western blot analyses.

Article Snippet: The cultured mouse PASMCs were fixed in 4% paraformaldehyde and stained with a rabbit polyclonal Orai1 antibody (1:100, ProSci) and mouse monoclonal STIM1 antibody (1:100, EXBIO) using Vector M.O.M. immunodetection kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instruction.

Techniques: Cell Culture, Expressing, Control, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: Colocalization of Orai1 and STIM1 in mouse PASMCs. A–D: staining of mouse cultured PASMCs after exposure of live cells with normal bath physiological salt solution (PSS). A: omission of Orai1 and STIM1 antibody resulted in no Orai1 or STIM1 staining. B–D: three representative cells dual-labeled with anti-Orai1 antibody (green) and STIM1 antibody (red). Orai1 and STIM1 colocalization (yellow/orange) is shown in the merged images. E–H: staining of mouse cultured PASMCs after exposure of live cells with Ca2+-free PSS containing 10 μM CPA. E: omission of Orai1 and STIM1 antibody resulted in no Orai1 or STIM1 staining. F–H: three representative cells dual-labeled with anti-Orai1 antibody (green) and STIM1 antibody (red). Colocalization of Orai1 and STIM1 is more apparent (yellow/orange) after store-depletion as shown in the merged images. Nuclei were stained with DAPI (blue). Experiments were performed in 3 separate immunostaining procedure, each with duplicate coverslips. Scale bars, 20 μm.

Article Snippet: The cultured mouse PASMCs were fixed in 4% paraformaldehyde and stained with a rabbit polyclonal Orai1 antibody (1:100, ProSci) and mouse monoclonal STIM1 antibody (1:100, EXBIO) using Vector M.O.M. immunodetection kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instruction.

Techniques: Staining, Cell Culture, Labeling, Immunostaining

Journal: The American journal of pathology

Article Title: BDNF/TrkB signaling augments smooth muscle cell proliferation in pulmonary hypertension.

doi: 10.1016/j.ajpath.2012.08.028

Figure Lengend Snippet: Figure 1. Ntrk2 is up-regulated in mouse lungs after 24 hours of hypoxia exposure (pO2 0.01). Total RNA from mice lung homogenates was isolated (n 18 per group), direct labeled, and hybridized to 44K 60mer oligonucleotide microarrays (MWG). A: Volcano plot: log2-fold regulation compared to probability of regulation. Genes with log odds values 5 were considered to be regulated; black spots, exemplary genes. B: Functional annotation of regulated genes was performed according to Gene Ontology (GO). Ntrk2 (TrkB) belongs to MAPK signaling pathway. C: A time course of TrkB expression was performed using real-time PCR of lung homogenates harvested 1, 7, or 21 days after hypoxia exposure (n 7 to 9, each). D: Immunohistochemical staining of TrkB and its ligand, BDNF in mouse lung sections. The arrow indicates positive staining. E and F: Real-time PCR analysis of TrkB and BDNF expression in (E) isolated major pulmonary arteries (n 6), and (F) laser-microdissected intrapulmonary vessels (40 vessels per animal, n 4), from mice exposed to 21 days of hypoxia or normoxia. G: Expression levels of TrkB and BDNF in mouse PASMC as indicated by real-time PCR; lower CT values represent a more abundant transcript level. H: Immunofluorescence staining of TrkB and BDNF in mouse PASMC. The arrows indicate TrkB localization. Inset, negative control staining. *P 0.05.

Article Snippet: To investigate proliferation of unstimulated or BDNF-stimulated human PASMC, cells were permeabilized for 5 minutes with 0.25% Triton X-100 in Tris-buffered saline, incubated with a mouse anti-human Ki-67 antibody (Santa Cruz Biotechnology), followed by incubation with fluorescein isothiocyanate–labeled secondary antibody (Dianova, Hamburg, Germany).

Techniques: Isolation, Labeling, Functional Assay, Expressing, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Staining, Immunofluorescence, Negative Control

Journal: The American journal of pathology

Article Title: BDNF/TrkB signaling augments smooth muscle cell proliferation in pulmonary hypertension.

doi: 10.1016/j.ajpath.2012.08.028

Figure Lengend Snippet: Figure 3. Up-regulation of TrkB and BDNF in human lung tissue from IPAH. A: Colocalization of BDNF and TrkB with SMA as indicated by immunofluorescence staining in donor and IPAH patients. B: Real-time PCR analysis of TrkB and BDNF in laser-microdissected intrapulmonary vessels from donor and IPAH patients. Expression is given in comparison to samples from donor lungs (n 5 per group). C: Real-time PCR analysis of expression levels of TrkB and BDNF in human PASMC; lower CT values represent a more abundant transcript level. D: Immunofluorescence staining of TrkB and BDNF in human PASMC derived from donor and IPAH patients. E: Colocalization of BDNF and TrkB with SMA in PASMC as indicated by immunofluorescence staining, IgG, isotype control staining. *P 0.05.

Article Snippet: To investigate proliferation of unstimulated or BDNF-stimulated human PASMC, cells were permeabilized for 5 minutes with 0.25% Triton X-100 in Tris-buffered saline, incubated with a mouse anti-human Ki-67 antibody (Santa Cruz Biotechnology), followed by incubation with fluorescein isothiocyanate–labeled secondary antibody (Dianova, Hamburg, Germany).

Techniques: Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Expressing, Comparison, Derivative Assay, Control

Journal: The American journal of pathology

Article Title: BDNF/TrkB signaling augments smooth muscle cell proliferation in pulmonary hypertension.

doi: 10.1016/j.ajpath.2012.08.028

Figure Lengend Snippet: Figure 6. BDNF regulates and activates Egr-1. A: Induction of early growth-response factor 1 (Egr-1) protein in human PASMC as analyzed by Western blotting. B: BDNF-induced Egr-1 protein localization in PASMC as assessed by immunofluorescence analysis. C: Electrophoretic mobility shift assay for Egr-1 induction and DNA–protein interactions from nuclear extracts prepared from BDNF (10 ng/mL)-treated PASMC, incubated with radiolabeled oligonucleotide containing Egr-1 consensus sequence. Incubation with an excess (10-fold) of an unlabeled probe served as a control for specific binding. PDGF (10 ng/mL) treated cells served as a positive control. D: Egr-1 expression in lung parenchyma of donor and IPAH patients. An Egr-1/ mouse (KO-Egr-1) served as negative control. E: Real-time PCR analysis for Egr-1 expression in IPAH patient and donor laser-microdissected intrapulmonary vessels (n 5). n.s., not significant. *P 0.05.

Article Snippet: To investigate proliferation of unstimulated or BDNF-stimulated human PASMC, cells were permeabilized for 5 minutes with 0.25% Triton X-100 in Tris-buffered saline, incubated with a mouse anti-human Ki-67 antibody (Santa Cruz Biotechnology), followed by incubation with fluorescein isothiocyanate–labeled secondary antibody (Dianova, Hamburg, Germany).

Techniques: Western Blot, Immunofluorescence, Electrophoretic Mobility Shift Assay, Incubation, Sequencing, Control, Binding Assay, Positive Control, Expressing, Negative Control, Real-time Polymerase Chain Reaction