Nb600 502

Cells were washed with ice cold PBS and lysed in RIPA buffer (50 mM Tris/150 mM NaCl/2.5 mM Na₂EDTA/1% Triton X-100/0.5% sodium deoxycholate/0.1% SDS in dH₂0) with 1% phenylmethanesulphonyl fluoride (PMSF) for 30–45 min on ice. Protein concentration was determined using the Pierce™ BCA Protein Assay Kit (#23227; Thermo Scientific). Lanes were loaded with 40 μg protein and electrophoresis and blotting were carried out according to standard protocols. Staining was performed with primary antibodies for BCL-2 (Abcam, Cambridge, UK), BCL-XL, BCL-W, MCL-1 (Cell Signaling Technology, Danvers, MA, USA), ERβ (PPZ0506, Invitrogen, Waltham, MA, USA), ERα (Abcam), and GAPDH (1:10.000; NB600-502, Novus Biologicals) overnight at 4 °C in 5% milk.

Lung tissue samples, pulmonary artery samples and cells were homogenized in RIPA lysis buffer (Thermo scientific), quantified and lysates were separated on 10% polyacrylamide gels and transferred to nitrocellulose membranes. After blocking, the membranes were probed with one of the following antibodies: anti-HIF1A (1:1000, BD Biosciences), anti-HIF2A (1:1000, Novus Biologicals), anti-ACTB as a loading control (1:5000, NB600–502, Novus Biologicals) and anti-RASSF1A (1:500, ab23950), anti-HK2 (1:2000, ab9464), anti-PDK1 (1:500, ab9461) and anti-LDHA (1:1000) all from Abcam Ltd. This was followed by 1 h incubation with secondary antibodies conjugated with horseradish peroxidase (HRP). Bound antibodies were detected by chemiluminescence with the ECL detection system (Thermo Scientific,) using Image reader (GE Healthcare) and densitometric analysis of the blots was obtained using multi gauge software (Fujifilm, Tokyo, Japan). In some experiments, Image reader (Fujifilm) was used for visualizing and quantifying western blot bands. Expression was quantified using band intensity values (in arbitrary units), which were normalized to ACTB. Uncropped images of all western blots are provided in the Supplementary Figs. 12–27. Detailed information of antibodies is provided in Supplementary Table 3.

The following primary antibodies were used: mouse anti-HA-tag (6E2) (immunocytochemistry [ICC], 1:100; western blot [WB], 1:1,000; #2367, Cell Signaling Technology), mouse anti-V5-tag (ICC, 1:300; WB, 1:1,000; R960-25, Invitrogen), rabbit anti-TOM20 (1:100; sc-11415, Santa Cruz Biotechnology), mouse anti-GAPDH (1:1,000; NB600-502, Novus Biologicals), mouse anti-SSEA-4 (1:100; MAB4304, Millipore), goat anti-NANOG (1:20; AF1997, R&D Systems), rabbit anti-OCT4 (1:200; ab19857, Abcam), mouse anti-αSMA (1:200; M0851, Dako), goat anti-Brachyury (1:20; AF2085, R&D Systems), goat anti-SOX17 (1:200; AF1924, R&D Systems), mouse anti-NESTIN (1:200; MAB5326, Millipore), rabbit anti-TUBB3 (1:1,500; PRB-435P, BioLegend), and mouse anti-MyHC (1:200; MAB4470, R&D Systems). Alexa Fluor 488 (A11055 or A11034, Molecular Probes)-, Alexa Fluor 594 (A11005, Molecular Probes; ab150132, Abcam)-, and Alexa Fluor 647 (ab150075, Abcam)-conjugated secondary antibodies were used for immunofluorescence study.

Western blot analysis were performed as described previously (Park et al., 2001 (link); Park et al., 2002 (link)). Antibodies were anti‐4‐hydroxynonenal (4‐HNE; ab48506, Abcam, Cambridge, MA, USA), anti‐manganese superoxide dismutase (MnSOD; 574,516, Calbiochem, San Diego, CA, USA), anti‐isocitrate dehydrogenase 2 (IDH2; sc‐134,923, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti‐fission 1 (Fis1; SAB1405076, Sigma‐Aldrich), anti‐optic atrophy 1 (Opa1; 612,607, BD Bioscience, San Diego, CA, USA), cytochrome c oxidase subunit IV (COX IV; ab33985, Abcam), and anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; NB600‐502, NOVUS, Littleton, CO, USA).