Bafilomycin a1 baf a1

APY0201, bafilomycin A1 (BafA1), and rapamycin (Rapa) were obtained from MedChemExpress (MCE, Shanghai, China) and were dissolved in DMSO (Sigma, USA) for in vitro experiments. APY0201 was dissolved in PEG300 (MCE, Shanghai, China) for animal research in vivo. Anti-LC3A/B (#12741S), CTSD (#2284S), CTSB (#311718T), CDK2 (#2546T), CDK4 (#12790T), cyclin E1 (#4129T), p21 (#2947T), p27 (#3686T), and ATG5 (#12994T) were obtained from Cell Signaling Technology (Danvers, USA). Anti-p62 (#PM045) was obtained from MBL (Tokyo, Japan). Anti-CDK6 (#ab124821) and Anti-Ki67 (#ab15580) were obtained from Abcam (Cambridge, UK). Anti-cyclin D1 (#ab143498) and GAPDH (#60004-1-Ig) were obtained from Proteintech (Wuhan, China). Anti-tubulin (#AF0001) was obtained from Beyotime (Shanghai, China).

HEK293T cells and shelduck embryo fibroblast (DEF) cells were maintained at 37°C in DMEM (Gibco), enriched with 10% FBS (Gibco). Anti-HA (26183), anti-MYC (MA1-980), anti-FLAG (MA1-91878) and anti-GAPDH (MA515738) antibodies were purchased from Invitrogen. Anti-HA agarose beads (26182) and anti-V5 agarose beads (A7345) were purchased from Invitrogen and Sigma, respectively. Low molecular weight (LMW)-poly(I:C) and anti-V5 (ab27671) antibody were obtained from Invivogen and Abcam, respectively. IRDye^® 800CW goat anti-rabbit IgG (C50331-03) and IRDye^® 800CW goat anti-mouse IgG (C41028-02) secondary antibodies were from LI-COR. DMSO, MG132, bortezomib, bafilomycin A1 (Baf A1), chloroquine (CQ) and 3-Methyladenine (3-MA) were purchased from MedChemExpress.

Rapamycin (Rapa), 3-methyladenine (3MA), chloroquine (CQ), and Bafilomycin A1 (Baf-A1) were purchased from MedChem Express (Monmouth Junction, NJ, USA). Cells were pretreated with 1 μM rapamycin, 3 μM 3 MA, 10 μM CQ or 10 nM Baf-A1 before infection with H. parasuis. Recombinant adenovirus tandom expressing GFP-RFP-LC3 was obtained from HANBIO (Shanghai, China). Primary antibodies used in this study were: rabbit anti-LC3A/B polyclonal IgG (12741), NF-κB p65 (6956), phospho-NF-κB p65 (Ser536) mAb (3031), p38 MAPK (8690), phospho-p38 MAPK (Thr180/Tyr182) rabbit mAb (4511), JNK2 rabbit mAb (9258), phospho-SAPK/JNK (Thr183/Tyr185) rabbit mAb (4668), AMPKα rabbit mAb (5831), phospho-AMPKα (Thr172) rabbit mAb (2535), mammalian target of rapamycin (mTOR) rabbit mAb (2983), and phospho-mTOR (Ser2448) rabbit mAb (5536) were purchased from Cell Signaling Technology (CST, Boston, MA, USA). Rabbit anti-ATG5 polyclonal IgG, rabbit anti-Beclin-1 polyclonal IgG, rabbit anti-ATG7 polyclonal IgG, rabbit anti-ATG16L1 polyclonal IgG, and rabbit monoclonal antibody against β-actin were purchased from ABclonal Biotech (Cambridge, MA, USA). The siRNA targeting ATG5, Beclin-1, ATG7 and ATG16L1 mRNA were synthesized by GenePharma (China) and the sequences are listed in Table 1. Pharmacological treatments and RNA interference do not affect cell viability (Figure S1).

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).

Splenocytes from wild type (WT) mice and P2RY12^-/- mice were isolated and cultured in 96-well plates at a density of 1×10⁶ cells per well, then stimulated with ConA (2 μg/ml) (Sigma-Aldrich) for 12 hr or 24 hr and co-cultured with the P2RY12 inhibitors clopidogrel (1, 3, 10 μM) or ticagrelor (1, 3, 10 μM), HK2 inhibitor 3-bromopyruvate (3-BrPA, MedKoo Biosciences Inc., Morrisville, NC, USA), proteasome inhibitor MG-132 (Med-Chem-Express LLC, Monmouth Junction, NJ, USA), lysosomal inhibitor bafilomycin A1 (BafA-1) (Med Chem Express LLC) or Akt activator SC79 (Med-Chem-Express LLC). The cells were analyzed by flow cytometry and supernatants were collected for measurement of cytokines.

Cadmium acetate (CdAc₂), Pue (for in vivo studies), and poly L-lysine hydrobromide (L-glutamine) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium Carboxymethylcellulose (CMC-Na) and penicillin–streptomycin were purchased from Solarbio (Beijing, China). Neurobasal^TM medium, B-27 supplement, Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 Nutrient Mixture (DMEM/F-12), and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, USA). Rapamycin (Rap), Lyso-Tracker Red (LTR), Hoechst33342 staining, and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Beyotime (Shanghai, China). DQ-BSA-Green was purchased from Invitrogen (Carlsbad, CA, USA). Pue (for in vitro studies) was purchased from Selleck Chemicals (Houston, TX, USA). Bafilomycin A1 (Baf A1) was purchased from MedChem Express (Monmouth Junction, NJ, USA). All other chemicals and reagents used were of analytical grade.

The ASTC‐a‐1 cells were obtained from the Department of Medicine, Jinan University in Guangzhou, China. All other cell lines were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (CAS) in Shanghai. All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Thermo Fisher Scientific) supplemented with 10% foetal bovine serum (FBS, Sigma cat# F2442), 50 units/mL penicillin and 50 mg/mL streptomycin. Prior to experimentation, all cell lines were tested for mycoplasma using the PlasmoTest™‐Mycoplasma Detection Kit (InvivoGen, China). The cells were incubated at 37°C with 5% CO₂. The PRMT5‐specific inhibitor GSK3326591 (cat# SML‐1751) and cycloheximide (cat# C4859) were purchased from Sigma. Lysosome inhibitor bafilomycin A1 (BAF‐A1, cat# HY‐100558), GSK3326595 (cat#, HY‐101563) and MG‐132 (cat#, HY‐13259) were purchased from MedChemExpress (MCE).