Ets 1

Immunoblot analysis was performed as described24 (link). SuperSep Ace gels (5–20%) were from Wako. Antibodies were against ETS1 (Abcam, Cambridge, UK), HSC70 (B-6, Santa Cruz Biotechnology), CXCR4 (ab124824, ab2074; Abcam), and ANGPTL2 (R&D Systems). Immunodetection was performed using an enhanced chemiluminescence (ECL) kit (GE Healthcare, Amersham, UK).

Dulbecco's modified Eagle's medium (DMEM/F12), foetal bovine serum (FBS) and collagenase II were obtained from HyClone. Trypsin was provided by Beyotime. All other cell culture reagents were purchased from Sigma Chemicals unless otherwise specified. Primary antibodies against KDM3A, ETS‐1, Bax, Bcl‐2, IL‐6, TNF‐α, GAPDH, ANP and BNP were purchased from Abcam. The secondary antibodies, either HRP‐linked goat anti‐mouse IgG or goat anti‐rabbit IgG, were also obtained from Abcam. The miR‐22‐3p mimic and its scrambled oligonucleotides (miRNA‐NC) were synthesized by GenePharma Co., Ltd.

Total RNA was extracted from tissue samples and cultured cells using the Trizol reagent (Takara), and qPCR was performed using the PrimeScript RT reagent kit (Takara) and SYBR Premix Ex Taq (Takara, USA) according to the manufacturer’s instructions. GAPDH was used as an internal control to detect the mRNA expression. For the detection of miRNA, Mir-X^TM miRNA First-Strand Synthesis Kit (Takara, USA) was used for synthesizing cDNA from different types of miRNAs and U6 small nuclear RNA was used as an internal control. Data analysis was performed using the 2^–ΔΔCt method. Primers are listed in Supplementary Table 1.
Standard western blot procedure was performed and the protein bands were transferred onto a nitrocellulose membrane (Bio-Rad, USA). Non-fat milk (5%) was used for blocking and the membranes were incubated with primary antibodies against GAPDH (Proteintech, 1:1000), β-catenin (CST, 1:1000); TGM2 (CST, 1:1000); ETS1 (Abcam, 1:1000) overnight. After incubation with an appropriate secondary antibody, the protein bands were visualized using Immobilon ECL (Millipore).

Cells were treated with or without 25, 50, or 100 μg/mL of O-PMs and harvested with RIPA buffer (H.M. Biological, Taoyuan, Taiwan) supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific, MA, USA). In addition, cytoplasmic and nuclear proteins were extracted using a Nuclear Extraction Kit (Cayman Chemical, MI, USA). Thirty micrograms of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The membranes were incubated overnight at 4 °C with primary antibodies against fibronectin, ETS-1 (1: 2000 dilution, Abcam, Cambridge, UK), E-cadherin, phosphorylated-NF-κB p65 (1:2000 dilution, Cell Signaling Technology, MA, USA), Lamin A + C, α-tubulin, β-actin (1:2000 dilution, GeneTex, CA, USA), or vimentin (1:2000 dilution, Santa Cruz Biotechnology, TX, USA). The anti-GAPDH antibody (1:10000 dilution, Tools, New Taipei City, Taiwan) was used as the loading control. Images were visualized by UVP BioSpectrum 815 imaging system (UVP, CA, USA), and the intensity of each band was quantified using ImageJ software.