normal human pasmcs  (Lonza)

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: British journal of pharmacology

Article Title: Halofuginone, a promising drug for treatment of pulmonary hypertension

doi: 10.1111/bph.15442

Figure Lengend Snippet: Halofuginone (HF) inhibits receptor-operated and store-operated Ca2+ entry. (a) Representative traces showing changes in [Ca2+]cyt before, during and after superfusion of extracellular application of 1.8-mM Ca2+ (calcium) in HEK-293 cells transfected with the calcium-sensing receptor (CaSR) gene (CASR) with (HF) or without (vehicle [Veh]) treatment of 0.03-, 0.1- and 1-μM halofuginone. (b) Summarized data showing the concentration–response curve of 1.8-mM Ca2+-induced increases in [Ca2+]cyt in CASR-transfected HEK-293 cells treated with different concentrations of halofuginone (0.0001 to 1 μM). Data shown are means ± SEM, n = 6 independent experiments for each data points. *P < 0.05, significantly different from 0.0001-μM halofuginone. (c) Summarized data (means ± SEM) showing the basal [Ca2+]cyt in CASR-transfected HEK-293 cells treated with Veh (n = 5) and all concentrations of halofuginone (0.03–1 μM) (HF, n = 7). *P < 0.05, significantly different from Veh. (d) Representative traces showing changes in [Ca2+]cyt before and during extracellular application of cyclopiazonic acid (CPA, 10 μM) in the absence (0Ca) or presence (1.8Ca) of 1.8-mM Ca2+ in human PASMCs shortly (10 min) treated with Veh (left panel) or 0.1-μM halofuginone (right panel). (e) Summarized data (means ± SEM, n = 6 independent experiments) showing the CPA-induced increases in [Ca2+]cyt in the absence (first peak, 0Ca) and presence (second peak, 1.8Ca) of 1.8-mM extracellular Ca2+ in human PASMCs treated with Veh or halofuginone (0.1 μM). *P < 0.05, significantly different from Veh. (f) Summarized data (means ± SEM) showing the basal [Ca2+]cyt in human PASMCs treated with Veh (n = 6 independent experiments) and 0.1-μM halofuginone (n = 6 independent experiments)

Article Snippet: Normal human PASMCs (Lonza, RRID:SCR_000377) were cultured in smooth muscle growth medium (SMGM, Lonza), whereas HEK-293 cells were cultured in high-glucose DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen), 100-IU·ml −1 penicillin and 100-μg·ml −1 streptomycin (Sigma-Aldrich).

Techniques: Transfection, Concentration Assay

Journal: British journal of pharmacology

Article Title: Halofuginone, a promising drug for treatment of pulmonary hypertension

doi: 10.1111/bph.15442

Figure Lengend Snippet: Halofuginone (HF) inhibits Ca2+-sensitive Akt/mTOR phosphorylation in human PASMCs. Representative record (a) and summarized data (b, means ± SEM, n = 5 independent experiments) showing cyclopiazonic acid (CPA)-induced changes in [Ca2+]cyt in PASMCs superfused with 1.8-mM Ca2+ (1.8Ca)-containing solution in the absence (1.8Ca) or presence of BAPTA (BAPTA), BAPTA-AM (1.8Ca + BAPTA-AM) or BAPTA + BAPTA-AM. *P < 0.05, significantly different from 1.8Ca. (c) Western blot analysis on phosphorylated Akt (pAKT), total Akt (AKT), phosphorylated mTOR (pmTOR) and total mTOR (mTOR) in control PASMCs and PASMCs treated with PDGF (PDGF-BB, 10 ng·ml−1) in the absence (−) and presence (+) of BAPTA or BAPTA-AM. (d) Summarized data (means ± SEM, n = 8 independent experiments) showing the ratio of p-Akt/Akt and p-mTOR/mTOR in control PASMCs (control) and PDGF-treated PASMCs in the absence and presence of BAPTA or BAPTA-AM: PDGF alone, PDGF + BAPTA, PDGF + BAPTA-AM or PDGF + BAPTA + BAPTA-AM. *P < 0.05, significantly different from PDGF alone; #P < 0.05, significantly different as indicated. (e) Western blot analysis on p-Akt, Akt, p-mTOR and mTOR in control PASMCs and PASMCs treated with PDGF in the absence (vehicle control, Veh) and presence (HF) of 1-μM halofuginone (for 24 h). Summarized data (means ± SEM, n = 5–6 in each group) showing the ratio of p-Akt/Akt and p-mTOR/mTOR (f) as well as the relative p-Akt, Akt, p-mTOR and mTOR protein level (g, normalized to actin) in control PASMCs (control) and PDGF-treated PASMCs in the absence (PDGF) and presence (PDGF + HF) of 1-μM halofuginone. *P < 0.05, significantly different from control; *P < 0.05, significantly different from PDGF; #P < 0.05, significantly different as indicated.. (g) Summarized data (means ± SEM, n = 5–6 in each group) showing the ratio of p-Akt/Akt and p-mTOR/mTOR in control PASMCs (control) and PDGF-treated PASMCs in the absence (PDGF) and presence (PDGF + HF) of 1-μM halofuginone. *P < 0.05, significantly different from control; #P < 0.05, significantly different as indicated

Article Snippet: Normal human PASMCs (Lonza, RRID:SCR_000377) were cultured in smooth muscle growth medium (SMGM, Lonza), whereas HEK-293 cells were cultured in high-glucose DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen), 100-IU·ml −1 penicillin and 100-μg·ml −1 streptomycin (Sigma-Aldrich).

Techniques: Phospho-proteomics, Western Blot, Control