Pgc 1α

Western blotting was performed using 50–100 μg of nuclear fraction, microsomal fraction, cytosolic fraction or whole extract of the liver, as described previously^{14 (link)}. In some experiments, AML12 cells exposed to choline-deficient medium were lysed in 50 mM HEPES, pH 7.5, 150 mM NaCl, 1% CHAPS, 5 mM EDTA, and protease inhibitors (Nacalai Tesque, Kyoto, Japan), and resultant cell lysates (15 μg) were used. The expression of PGC-1α, lipin1, HIF-1α, lamin, and tubulin was detected using specific antibodies against the respective antigens as follows: PGC-1α (2178 S, Novus Biologicals; Littleton, CO, USA), lipin1 (AF3885, R&D Systems; Minneapolis, MN, USA), HIF-1α (36169 and 14179, Cell Signaling Technology, Danvers, MA, USA), lamin A/C (sc-20681, Santa Cruz Biotechnology; Santa Cruz, CA, USA), and tubulin (T9026, Sigma-Aldrich, St. Louis, MO, USA). Images of Western blots are shown in Supplementary Fig. S6.

The mice were euthanized by cervical dislocation, and the left lungs were harvested for histological and IHC analyses. The pulmonary artery and trachea were perfused with 4% (wt/vol) PFA at constant pressures (100 cm H₂O for the pulmonary artery and 25 cm H₂O for the trachea) to fully distend the pulmonary blood vessels and airway, respectively. The excised lungs were fixed in 4% PFA overnight at 4°C, embedded in paraffin, and cut into 4 μm–thick sections. The Abs used for IHC staining included α-SMA (catalog C6198; Merck KGaA), CD31 (catalog DIA-310; Dianova), PGC-1α (catalog NBP1-04676; Novus Biologicals), and γH2.AX (catalog 9178; Cell Signaling Technology). Images were captured using an Olympus BX51 fluorescence microscope. Morphometry was performed on lung sections obtained from randomly chosen animals. Pulmonary remodeling was assessed using the percent wall thickness of parenchymal pulmonary arteries classified into small arteries (terminal bronchioles) and arterioles (acini or alveolar ducts). The percent SMC thickness was calculated as [(SMC thickness × 2)/vessel diameter] × 100. The pulmonary artery diameter was determined using the ImageJ software (NIH).

AT samples were homogenized with a FastPrep Ribolyser (MP Biomedicals, Elsene, Belgium) in 10 mM Na phosphate, pH 7.2, 150 mM NaCl, 1% Triton, 0.1% sodium dodecyl sulphate (SDS), 0.5% Na deoxycholate, 0.2% NaN₃, containing a protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL, USA). Protein concentrations were determined with the BCA protein assay (Thermo Fisher Scientific, Rockford, IL, USA). An equal amount of protein was loaded in each well of a 10% SDS-PAGE (Bio-Rad, Hercules, CA, USA). Gels were transferred onto a 0.45 μm nitrocellulose membrane and blocked in 5% nonfat dry milk (Bio-Rad) in 10 mM Tris–HCl buffer containing 150 mM NaCl and 0.05% Tween 20 at pH 7.6 (TBST) for 3 h. Subsequently, membranes were probed with the following primary antibodies: β-actin (Cell Signaling Technology; CST, Leiden, The Netherlands), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, CST), peroxisome proliferator-activated receptor gamma coactivator 1 α (PGC1α, Novus Biologicals, Abingdon, UK), and UCP-1 (Sigma-Aldrich, Darmstadt, Germany). Secondary antibodies were species-appropriate horseradish peroxidase-conjugated antibodies (Dako, Glostrup, Denmark, 1:2000) diluted in TBST containing 5% nonfat dry milk. Signals were detected with Enhanced Chemiluminescence (Thermo Fisher Scientific) (http://dx.doi.org/10.17504/protocols.io.kbfcsjn).

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO (Grand Island, NY). Lipofectamine 2000 (Lipo 2000) reagent was obtained from Invitrogen (Carlsbad, CA). CD151 small interfering RNA (siCD151) and scrambled siRNA controls were synthesized by Vigenebio (Shandong, China). Real-time PCR primers of mRNA were synthesized and purchased from Wuhan AuGCT DNA-SYN Biotechnology Co., Ltd. (Wuhan, China). Antibodies against CD151 (Cat No: 66567-1-Ig), CD9 (Cat No: 60232-1-Ig) and GAPDH (Cat No: 60004-1-Ig) were purchased from Proteintech (Chicago, IL). Anti-PPAR-α (Cat No: NB300-537), and peroxisome proliferator-activated receptor coactivator 1α (PGC-1α) (Cat No: NB300-537) were purchased from Novus (Brazil, SP). Anti-CD151 (Cat No: ab33315), Anti-CD63(Cat No: ab134045), Anti-PPAR-γ (Cat No: ab59256) were obtained from Abcam (Cambridge, MA). Recombinant CD151 adenovirus (Adeno-CD151;1.02 × 10¹¹ plaque-forming-unit/mL), empty adenovirus (Adeno-CMV-3*flag-tagged) were generated by Shanghai Obio Technology Co. Ltd. (Shanghai, China). FITC-dextran (Cat No: 46945) and other reagents were purchased from the Sigma-Aldrich Company, unless otherwise specified.

Proteins (30 μg) in the cell lysate of healthy-MSCs and CKD-MSCs were resolved by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were transferred to an 8 μm pore size nitrocellulose membrane. The membrane was blocked using 5% skim milk prepared in TBST (10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.05% (v/v) Tween 20) for 1 h at room temperature. The membrane was then treated with primary antibodies. The antibodies used were directed against p-PERK, anti-PERK, p-JNK, JNK, MFN1, PrP^C, and β-actin (all 1:300 dilution, all from Santa Cruz Biotechnology, Dallas, TX, USA), p-eIF2α, eIF2α, ATF4, p-DRP1 (both 1:1000 dilution, both from Abcam, Cambridge, UK), p-IRE1α, IRE1α, CHOP, OPA1, and PGC-1α (all 1:1000 dilution, all from Novus, Centennial, CO, USA). Next, each membrane was washed twice, and the primary antibodies were detected using goat anti-rabbit IgG or goat anti-mouse IgG antibodies (Santa Cruz Biotechnology). The protein bands were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).