Interleukin 6 (il 6)

Rat ADM (molecular formula: C₂₄₂H₃₈₁N₇₇O₇₅S₅) was obtained from Bachem (Bubendorff, Switzerland). PA and Compound C were purchased from Sigma Aldrich (St. Louis, MO, USA). ADM22-52 was purchased from Anaspec (Fremont, CA, USA). DMEM, 0.25% trypsin-EDTA, fetal bovine serum, trypsin and streptomycin/penicillin were from Thermo Fisher Scientific (Waltham, MA, USA). The primary antibodies against α-actin and Runx2 were purchased from Abcam (Burlingame, CA, USA). ADM, CRLR, RAMP2, RAMP3, TNF-α and IL6 were obtained from Affinity Biosciences (Pottstown, PA, USA). The antibodies of IL-1β, NOX2, NOX4 and GAPDH were from Proteintech (SANYING, Wuhan, China).

The heart apical tissue was lysed for protein extraction. Totally 100 μg of total protein was separated by SDS-PAGE and then electronically transferred onto the PVDF membrane. After blocking with 10% non-fat milk at room temperature for 2 h, the membrane was treated with the primary antibodies against GAPDH (1:10,000 dilution; Abcam, Cambridge, UK), NICD (1:1,000 dilution; Abcam), NF-κB P65 (1:1,000 dilution; Abcam), P-NF-κB P65 (1:1,000 dilution; Abcam), Hes1 (1:1,000 dilution; Abcam), TNF-α (1:1,000 dilution; Abcam), IL-6 (1:1,000 dilution; Affinity Biosciences, China), and caspase-3 (1:2,000 dilution; Abcam), respectively, at 4°C overnight. Then, the membrane was treated with goat anti-rabbit IgG H&L (HRP) (1:20,000, Abcam) at 37°C for 2 h. Protein bands were developed with the ECL kit (Boster, Wuhan, Hubei, China) and analyzed with the Image Lab system. GAPDH was used as an internal reference.

Bafilomycin A1 (Baf A1) was obtained from MedChem Express (catalog no. HY‐100558, Princeton, USA). Dorsomorphin dihydrochloride (Compound C dihydrochloride) was purchased from MedChem Express (catalog no. HY‐13418, Princeton, USA). Antibodies against p‐AMPK (1:1000; catalog no. AF3423), AMPK (1:1000; catalog no. DF6361), TFEB (1:1000; catalog no. AF7015), IL‐6 (1:1000; catalog no. DF6087) and H3 (1:3000; catalog no. BF9211) were purchased from Affinity Biosciences (OH, USA). LC3A/B (1:1000; catalog no. 4108), IL‐1β (1:1000; catalog no. 12242), GAPDH (1:1000; catalog no. 5174), p‐FOXO1/FoxO3a (1:1000; catalog no. 9464) and FOXO1 (1:1000; catalog no. 2880) were purchased from Cell Signaling Technology (Danvers, MA, USA). LAMP1 (1:1000; catalog no. A16894), ABCA1 (1:1000; catalog no. A16337), IL‐18 (1:1000; catalog no. A16737), Beclin 1 (1:1000; catalog no. A11761), CD36 (1:1000; catalog no. A5792), TNF‐α (1:1000; catalog no. A11534) and CTSD (1:1000; catalog no. A13292) were purchased from ABclonal Technology (Wuhan, China). SQSTM 1/p62 (1:1000; catalog no. ab240635) was purchased from Abcam (Cambridge, the United Kingdom). The secondary antibodies HRP‐conjugated anti‐mouse IgG and HRP‐conjugated anti‐rabbit IgG were purchased from Affinity Biosciences (OH, USA).

Ibrutinib was purchased from Macklin (Shanghai, China). Poly I:C was purchased from APExBIO (Houston, TX, USA). LPS was purchased from Sigma (St Louis, MO, USA). Mouse IL-1β, IL-6, and TNF-α ELISA kits were procured from Solaibao (Beijing, China). Anti-myeloperoxidase mouse mAb, anti-IL1 beta rabbit pAb, anti-TNF-α rabbit pAb, anti-CD8 mouse mAb, and anti-Ly6g rabbit pAb were obtained from Servicebio (Wuhan, China). APC Anti-Mouse Ly6G Antibody [1A8] and PerCP/Cyanine5.5 Anti-Mouse CD45 Antibody [30-F11] were purchased from Elabsciense (Shanghai, China). Antibodies against BTK/p-BTK (Tyr223), ERK/p-ERK, AKT/p-AKT, FLT3/p-FLT3, and EGFR/p-EGFR were bought from Cell Signaling Technology (Beverly, MA, USA). Antibodies against GAPDH, NFκB/p-NFκB, IL-1β, IL-6, IFN-γ, and TNF-α were purchased from Affinity Biosciences (Inc., Cincinnati, OH, USA). The RIPA lysis buffer and a Bradford protein assay kit were purchased from Biyuntian Biotechnology (Shanghai, China). PBS was purchased from Zhongshan Jinqiao Biotechnology (Co., Ltd., Beijing, China). A SuperLumia ECL HRP kit was procured from Abbkine (Redlands, CA, USA).

Western blot was performed as previously described^{20 (link)}. Antibodies used in this study were PDLIM1 (Affinity, DF3003), α-SMA (Affinity, AF1032), COL1A1 (Cell Signaling Technology, #91144), CTCF (Millipore, 07-729), TNF-α (Affinity, AF7014), IL-6 (Affinity, DF6087), p65(Affinity, AF5006), anti-p-Smad2/3 (Abcam, ab254407), anti-Smad2/3 (Abcam, ab202445), anti-p-ERK (Abcam, ab201015), anti-ERK (Abcam, ab184699), anti-p-P38 (Proteintech, 28796-1-AP), anti-P38 (Proteintech, 66234-1-Ig), anti-p-JNK (Proteintech, 80024-1-RR), anti-JNK (Proteintech, 66210-1-Ig), GAPDH (Cell Signaling Technology, #2118). Western blot analysis was performed on at least three independent biological replicates.