Journal: Nature Communications

Article Title: Reduced FOXF1 links unrepaired DNA damage to pulmonary arterial hypertension

doi: 10.1038/s41467-023-43039-y

Figure Lengend Snippet: a – c Commercially available healthy donor PAEC were cultured under hypoxia (0.5% O 2 ) for 48 h followed by room air for 24 h (reoxy), or in room air for 72 h (normo). a Representative immunoblot of DNA damage markers (γH2AX and phosphorylated RPA) in human PAEC transfected with scrambled control siRNA (siC) or siRNA targeting ATM (siA). n = 3 individual experiments. b Comet assay shows DNA damage as reflected in the comet tail lengths in ATM-depleted cells in normoxia or reoxygenation. Scale bar, 20 µm. Cells n = 186, 154, 139, 124 for siC normoxia, siA normoxia, siC Reoxy, siA reoxy respectively. c Immunohistochemistry of the DNA damage markers in human PAEC transfected with Control or ATM siRNA. Scale bars, 20 μm. Cells n = 130, 133, 117, 102 for siC normoxia, siA normoxia, siC Reoxy, siA reoxy respectively. d Animal measurements - experimental design: Mice with EC-specific deletion of Atm (EC- Atm -/- ) were created using the strategy described for Fig. , and in the “Methods”. EC- Atm -/- or control mice were exposed to hypoxia (10% oxygen) for 3 weeks followed by 4 weeks of room air (reoxy), or maintained in room air for 7 weeks (normo). Schema created with BioRender.com. e γH2AX immunofluorescence foci in PAEC in control and EC- Atm -/- mice (arrowheads) were quantified in n = 6 mice. Scale bars, 20 μm. The bottom panels show a magnified merged image of the area delineated by the dotted line. Scale bars, 5 μm. In ( a and d ), bars represent mean ± S.E.M. P values determined by 2-way ANOVA with Holm-Sidak posthoc test. ns, not significant. In ( b and c ) The Bounds of boxes show the 25th and 75th percentiles, the whisker showed to the 10th to 90th percentiles and the centre in the box shows the median. Three independent experiments was performed. P values determined by Kruskal-Wallis ANOVA test with Dunn’s test Source data are provided as a Source Data file.

Article Snippet: Primary human PAEC were purchased from PromoCell (#C-12241) and grown in complete EC media (EC basal medium #1001, ECGS #1052, 5% FBS #0025, and Penicillin/Streptmycin #0503) (ScienCell).

Techniques: Cell Culture, Western Blot, Transfection, Control, Single Cell Gel Electrophoresis, Immunohistochemistry, Immunofluorescence, Whisker Assay

Journal: Nature Communications

Article Title: Reduced FOXF1 links unrepaired DNA damage to pulmonary arterial hypertension

doi: 10.1038/s41467-023-43039-y

Figure Lengend Snippet: a FOXF1 immunohistochemistry of pulmonary artery (PA) in EC- Bmpr2 -/- mice under normoxia (Normo) or reoxygenation (Reoxy). FOXF1 intensity quantified in 5 vessels/mouse ( n = 6 mice/group), Scale bar, 20 µm. b Representative images of an occlusive PAH lesion and a normal donor PA, with quantification of FOXF1 intensity in PAEC (von Willebrand factor (vWF)-positive cells), in PAH BMPR2 MUT, PAH-non BMPR2 MUT and healthy donors ( n = 4/group). αSMA indicates smooth muscle cells. Scale bar, 50 µm. Bottom panels show magnified merged image of the area delineated by the dotted line. Scale bars, 10 μm. Five arteries analyzed in each patient with n = 96, 114, 109 cells for Donor, PAH-BMPR2 MUT and PAH-not MPR2 MUT , respectively. c – e Angiogenesis and DNA damage following reduced FOXF1 : Commercially available human PAEC treated with siRNA targeting FOXF1 (si FOXF1) vs. control siRNA (siCon). c mRNA levels. n = 3 replicates. d Representative images of tube formation assay, with quantitative analysis 3 h after seeding. n = 5 replicates. Scale bar, 200 µm. e Wound closure (scratch) assay by cell migration. Representative images at 0 and 12 h, and quantified ratio of the area of the scratch in si FOXF1 relative to siCon at 12 h. n = 5 replicates. Scale bar, 450 µm. f – i Angiogenesis and DNA damage after restoration of FOXF1 expression. PAH PAEC transfected with lentivirus vector carrying the FOXF1 gene or GFP as control. f mRNA of angiogenesis and DNA damage response genes ( n = 3 replicates) in cells of PAH patients PAH1, PAH2 and PAH3. g , h Tube formation and scratch assays, in PAEC of PAH patients ( n = 5 replicates/patient). Scale bars, 200 μm in (g) 450 μm in ( h ). i Comet assay: DNA damage reflected by the comet tail length. Scale bar, 20 μm. PAH1, n = 173, 131; PAH2, n = 125, 129; PAH3; n = 116, 123 cells for GFP-vector, FOXF1- vector, respectively. Supplementary Fig. shows the images for tube formation, scratch and comet assays for patients PAH2, PAH3. a , c – h Bars represent mean ± S.E.M; ( b , i ), Box bounds = 25th and 75th percentiles, whiskers 10th to 90th percentiles, and box centre = the median. P values were determined by one-way ANOVA with Holm-Sidak posthoc test ( a ), 2-sided Mann–Whitney U test ( b , i ), or unpaired 2-sided t-test ( c – h ). ns, not significant. Source data are provided as a Source Data file.

Article Snippet: Primary human PAEC were purchased from PromoCell (#C-12241) and grown in complete EC media (EC basal medium #1001, ECGS #1052, 5% FBS #0025, and Penicillin/Streptmycin #0503) (ScienCell).

Techniques: Immunohistochemistry, Control, Tube Formation Assay, Wound Healing Assay, Migration, Expressing, Transfection, Plasmid Preparation, Single Cell Gel Electrophoresis, MANN-WHITNEY