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
![Excess BCAAs promoted a pro-ferroptotic phenotype in <t>human</t> <t>PASMC</t> . (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.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_19/10__1101_slash_2025__09__03__672819/10__1101_slash_2025__09__03__672819___F3.large.jpg)