α actinin

The primary antibodies used were RhoA (#2117, for WB), Parkin (#2132), COX-IV (#11967), VDAC (#4661), lamin A/C (#2032), Rho-GDI (#2564), HA (#3724), PKD (#90039), P-PKD S916 (#2051), GAPDH (#2118), α-actinin (#3134) and LC3B (#3868) from Cell Signaling Technology; PINK1 from Novus Biologicals (#NB600-973); RhoA (SC-418, for IP) and ubiquitin (SC-8017) from Santa Cruz Biotechnology; miniSOG from Kerafast (#EFH004). Horseradish peroxidase (HRP)-conjugated secondary antibodies for WB, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), cycloheximide (CHX), MG-132, CID755673, Bafilomycin A1, Evans blue and triphenyltetrazolium chloride (TTC) were purchased from MilliporeSigma. Y-27632 was purchased from Cell Signaling Technology. DharmaFECT-1 and LysoTracker Blue were purchased from Thermo Fisher Scientific. C3 exoenzyme was purchased from Cytoskeletton, Inc.

Global mRNA expression profiling by microarray was performed as previously described [15 (link)].
To quantify mRNA and miRNA expression levels by quantitative real-time PCR (qRT-PCR), total RNA was isolated with the Direct-zol RNA MiniPrep Kit (Zymo Research, Irvine, CA, USA). RNA was transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). Relative mRNA and miRNA expression was measured in triplicate using TaqMan Gene Expression and MicroRNA Assays (Life Technologies, Carlsbad, CA, USA). For normalization, TBP and RNU6B were used as reference genes for mRNA and miRNA expression analyses, respectively.
For protein analysis, whole cell lysates were prepared with RIPA buffer using equal cell numbers per sample. Protein levels were analyzed using a standard western blot protocol with antibodies against HDGF (AF1606; R&D Systems, Minneapolis, MN, USA), SOX4 (C15310129; Diagenode, Seraing, Belgium), ERK 1/2 (4695; Cell Signaling Technology), phospho-ERK 1/2 (4370; Cell Signaling Technology), cofilin (5175; Cell Signaling Technology), β-actin (3700; Cell Signaling Technology), and α-actinin (4233; Cell Signaling Technology).

Cells were homogenized in ice cold lysis buffer containing proteinase and phosphatase inhibitor cocktail. The primary antibodies utilized in this study were as follows: OCT4 (sc-5279; Santa Cruz Biotechnology), Nanog (A3233 ABclonal), NKX2.5 (ab91196; Abcam), α-actinin (6487; Cell Signaling Technology), CX43 (3512 Cell Signaling Technology), GATA4 (5851; Cell Signaling Technology), MEF2C (5030; Cell Signaling Technology), cTnI (ab47003; Abcam), p-ERK (4370; Cell Signaling Technology), ERK (5013 Cell Signaling Technology), β-Catenin (8480; Cell Signaling Technology), p-cadherin (2189; Cell Signaling Technology), p-STAT3 (RT1490; HuaBio), STAT3 (ET1605; HuaBio), and GAPDH (5174; Cell Signaling Technology). Blots were developed using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA, USA).

Ang II was purchased from ENZO (ALX-151-039-M025); collagenase and trypsin were purchased from Gibco (Grand Island, NY, United States); the BCA protein assay kit was bought from Pierce (Rockford, United States); and 2,7-dichlorofluorescindiacetate (DCFH-DA) was obtained from the Bioengineering Institute (Nanjing, China). The following primary antibodies were obtained from Cell Signaling Technology (CST, United States): glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#2118), p-mTORC1 (#2971), T-mTORC1 (#2983), α-actinin (#69758S), P-p70 S6 kinase (Thr389) (#9234P), T-p70 S6 kinase (#2708), P-JNK (T183/Y185) (#4668P),T-JNK (#9258), P-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#4370P), T-ERK (#4695), P-p38 (#4511P), p38 MAPK (#9212P), T-TAK1 (#5206), P-TAK1 (#4508), T-AKT (#4691), P-AKT (#4060), acetyl-CoA carboxylase antibody (#3676), and P-acetyl-CoA carboxylase antibody (#3661S). ABCAM provided the following primary antibodies: Anti-AMPKα2 (ab3760), p-AMPKα2 (S491, ab109402), anti-SOD1 (ab16831), anti-SOD2 (ab38155), Nrf2 (ab15323), 4-hydroxynonenal (ab46545), sarcomeric α-actinin (ab68167), heme oxygenase1 (ab-13243), and NOX2/gp91phox (ab129068). The Sesn2 antibody was acquired from Proteintech (no. 10795-1-AP). Antibodies were used at 1:1,000 dilutions for Western blotting. The secondary antibodies were obtained from LI-COR Biosciences (Lincoln, United States).

The details of the experiments protocol were performed as described previously [15 (link)]. Primary antibodies against hERG (1:50, Santa Cruz) and α-actinin (1:200, Cell Signaling, Beverly, MA, USA) were applied following by incubation with the appropriate fluorescence-labeled secondary antibodies (Invitrogen) for 1 h at room temperature. After several washes, the samples were air-dried, mounted with a drop of ProLong Diamond Antifade Mountant with DAPI (Molecular Probes) and subjected to microscopy. Images were acquired using a 100× oil objective (Plan-Apochromat 100× [numerical aperture, 1.46] oil immersion objective for differential interference contrast [DIC]; Carl Zeiss). All sections were analyzed using a confocal laser microscopy system and software (LSM710; Carl Zeiss) that was built around an inverted microscope (Axio Observer Z1; Carl Zeiss, Germany). Images were saved in TIFF format and analyzed by ImageJ software (Wayne Rasband, National Institutes of Health, USA).

Tissues and cultured myocytes were lysed by RIPA buffer and then were centrifugated for 20 min at 4°C, 12000g to remove cell membranes. The supernatant was quantified by reacting with BCA working buffer (Thermo Fisher Scientific, Bremen, Germany). 12% or 8% SDS-PAGE gels were used to separate the target proteins, which was transferred to a PVDF membrane after wards. The membrane was blocked with 5% BSA buffer for 60 min at room temperature, followed by incubation with corresponding primary antibodies overnight at 4°C. After three washes with TBST buffer and incubated with horseradish peroxidase-conjugated secondary antibody, the bands were detected in LAS-3000 imaging system (FUJIFILM Inc. Tokyo, Japan). The antibodies used in this study were listed below: BACH2 (Affinity, DF2461; Cell Signaling, 80,775), β-actin (Cell Signaling, 3,700), α-actinin (Cell Signaling, 6,487), Myh7 (AB clonal, A7564). The original gel images were provided in the Supplementary Material.