T akt

Proteins were extracted with cell lysis buffer containing 1% Triton X-100, sonicated using QSonica-Q800R2, quantified using bicinchoninic acid assay (Sigma) and separated on NuPAGE™ 4-12% Bis-Tris precast gels (Thermo Fisher). Immunoblot analyses were conducted according to standard protocol with the following antisera: caspase-3 (#9662), PARP (#9542), β-actin (#3770), IRE1α (#3294), p-eIF2α (#3597), t-eIF2α (#2103), p-P38 MAPK (#4511), t-P38 MAPK (#8690), p-JNK1/2 (#4668), t-JNK1/2 (#9252), p-ERK1/2 (#9101), t-ERK1/2 (#9102), CHOP (#5554), Bim (#2933), Bcl-2 (#4223), Bax (#5023), p-Akt (#4060), t-Akt (#9272), GAPDH (#5174), cyclin b1 (#12231), p-CDK1 substrates (#9477) and FLAG (#14793) from Cell Signaling Technology; p62 (#ab56416) and Mcl-1 (#ab31948) antibodies were purchased from Abcam, while LC3 antibody (#NB100-2220) was obtained from Novus Biologicals. Images were captured using a ChemiDoc™ Touch Imaging System and processed using ImageLab™ Software.

Pterostilbene with a purity >98% was purchased from Chengdu Pufeide Biotech Co., Ltd. (Chengdu, China). Sodium arsenite (NaAsO₂, 99.0%) and DCFH-DA were provided by Sigma Chemical (St. Louis, MO, United States). Antibodies against Nrf2, HO-1, NQO1, Bax, Bcl-2, Bcl-xL, Caspase-3, Bad, Cytochrome c, p-AMPK, T-AMPK, p-AKT, T-AKT, Lamin B and β-actin were supplied by Cell Signaling (Boston, MA, United States) or Abcam (Cambridge, MA, United States). The MDA and SOD test kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The Annexin V fluorescein isothiocyanate (FITC)/Propidium Iodide (PI) apoptosis kit was purchased from BD (San Jose, CA, United States). The control siRNA and Nrf2 siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Mitochondrial membrane potential assay kit with JC-1 were offered from Beyotime Institute of Biotechnology (Jiangsu, China).

ASMCs were washed once with cold DPBS (#14190, Thermo Fisher Scientific) and lysed by RIPA buffer (#R0278, Sigma-Aldrich). Cell lysates were centrifuged at 12,000 rpm (10 min) and the protein concentration of the supernatant was measured by using the BCA protein assay kit (#23227, Thermo Fisher Scientific). Equal amounts of denatured proteins (20 μg) were separated in 4%–12% SDS–PAGE (#M41212, GenScript, Piscataway, NJ, USA), and, subsequently, transferred onto a nitrocellulose membrane (#88018, Themo Fisher Scientific). Proteins of interest were detected by specific antibodies, including total- (t-)PI3K (#4292), and phosphorylated- (p-)PI3K (#GTX132597), t-AKT (#4691) and p-AKT (#4060), t-p70S6 kinase (#2708) and p-p70S6 kinase (#9205), t-STAT3 (#9139) and p-STAT3 (#9145), PTEN (#9188, which were all purchased from Cell Signaling Technology, Cambridge, UK), PGC-1α (#ab54481, Abcam), t-PPARγ (#sc-7273, Santa Cruz), and p-PPARγ (#sc-28001, Santa Cruz). GAPDH (#2118, Cell Signaling Technology) was used as an endogenous control for a semi-quantification measure. Protein bands were visualized by Luminata Forte Western HRP substrate (#WBLUF, Sigma-Aldrich) after binding specie-specific secondary HRP conjugated antibodies (#7076 and #7074, Cell Signaling Technology).

Proteins were extracted from cell lines, resolved by SDS-PAGE and transferred to PVDF membrane. After blocking for 1 h at room temperature, blots were incubated overnight at 4°C with antibodies to PTEN, P-AKT, T-AKT, β-actin (Cell Signaling Technology) diluted according to the manufacturer’s recommendations. After wash, secondary HRP-conjugated antibodies (dilution 1:10,000; Pierce) were added and blots were incubated at room temperature for 1 h. The protein bands were visualized using ECL chemiluminescence (Pierce).

Proteins were collected from 3D spheroids of UCB-MSCs using the radioimmunoprecipitation assay buffer (RIPA), which is a lysis buffer containing protease inhibitors (Thermo Fisher Scientific, Waltham, MA, USA); proteins were then sonicated. Protein samples were separated and transferred onto nitrocellulose membranes. After blocking, the membranes were stained with primary antibodies: PARP, p-ERK, t-ERK, p-AKT and t-AKT (cell signaling, Danvers, MA, USA), BAX, SOD2, E-cadherin, PI3K and p-Nrf2 (Abcam, Cambridge, UK), caspase-3 (Santa Cruz Biotech., Dallas, TX, USA), GAPDH (AbClon, Seoul, Korea), and β-actin (Sigma), respectively. After washing, the membranes were incubated with the HRP-conjugated secondary antibody. Protein bands were visualized using an Amersham™ ECL Plus system (GE Healthcare, Chicago, IL, USA) and imaged using a ChemiDoc XRS camera (Bio-Rad Laboratories, Hercules, CA, USA). Band densities were analyzed via Image Lab software 6.0.1 (BioRad) and calculated by normalization to GAPDH or β-actin.

Unfixed frozen hearts from WT mice treated with telmisartan for two weeks were homogenized in lysis buffer and centrifuged^{16 (link)}. Following protein quantification, samples were resolved using SDS-PAGE and transferred onto nitrocellulose membrane. The membrane was cut just below 100kDs and the top and bottom sections were blocked and probed overnight with primary antibodies for eNOS (1:1000, Cell Signaling, Cat# 32027) on the top membrane, and T-AKT (1:1000, Cell Signaling, Cat# 2920), p-AKT (1:1000, Cell Signaling, Cat# 4060), Cav-1 (1:1000, Santa Cruz Biotechnology, Cat# sc-894) and GAPDH (1:4000, Cell Signaling, Cat# 2118) on the bottom membrane, followed by incubation with goat anti-rabbit AlexaFluor700 (1:5000, Thermo Fisher Scientific, Cat# A-21038) and goat anti-mouse AlexaFluor800 (1:5000, Thermo Fisher Scientific, Cat# A-32730) secondary antibodies before imaging with a Li-COR scanner. The Li-COR software was used to designated the area of interest to be scanned for the bottom membrane.

Antibodies used were mouse monoclonals; γH2AX (Millipore (U.K.) Ltd, Watford, UK), Pimonidazole (Hydroxyprobe 1 clone 4.3.11.3, Natural Pharmacia International Inc, Burlington, USA), AKT-phospho (p) S473 (Cell Signaling Technology, CA, USA), and β-actin (Sigma-Aldrich, Gillingham, UK). Rabbit monoclonals used were: ATM-pS981, total (t)-ATM, DNA-PK-pS2056, tDNA-PK, Isotype control rabbit IgG (Epitomics, CA, USA). Rabbit polyclonals used were; ATR-pS428, tATR, tAKT (Cell Signaling Technology, CA, USA) and rabbit IgG (Vector Laboratories Ltd, Peterborough, UK).