Bafilomycin a1

E64-d (Sigma, E8640) and PepstatinA (Sigma, P5318) treatments were given at a concentration of 25 μg/ml and 10 μg/ml, respectively for 2 hours prior to fixation. Autophagy was inhibited by 3-Methyladenine (Sigma, M9281) treatment, added 48 hours post infection, at a concentration of 5 mM. Autophagy flux was monitored using BafilomycinA1 (Invivogen, tlrl-baf1) at 100 nM for 3 hours. LysoTracker Red DND-99 (Molecular Probes, L-7528) was used to monitor acidified lysosomes at 200 nM concentration for one hour.

Dovitinib (TKI258) was kindly provided by Novartis Pharmaceuticals. Bafilomycin A1 was purchased from Invivogen (California, USA). Thiazolyl blue tetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphe- nyltetrazolium bromide, MTT) and acridine orange were purchased from Sigma-Aldrich (Missouri, USA). SHP-1 inhibitor, the STAT3-specific inhibitor, was purchased from Merck Millipore (Massachusetts, USA). G418, being used for selecting transformed with STAT3 plasmid cell line, was purchased from Amresco (Ohio, USA). Antibody for immunoblotting, such as PARP, was purchased from Santa Cruz (Dallas, USA). Other antibodies, such as beclin 1, cyclin D1, Mcl-1, survivin, p-STAT3^Tyr705, STAT3, SQSTM1/p62, and SHP-1, were from Cell Signaling (Massachusetts, USA).

The primary antibodies used in this study were as follows: anti-Tubulin mAb (BioLegend, San Diego, CA, USA), anti-p62 mAb, anti-LC3A/B mAb (Cell Signaling Technology, Danvers, MA, USA), anti-Cytochrome C mAb, anti-COX IV pAb, anti-Lamin B1 mAb, anti-AIF mAb, anti-CTSB pAb (Beyotime, shanghai, China). HRP-conjugated goat anti-mouse IgG and goat anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA, USA) and Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (Molecular Probes, Waltham, MA, USA) were used for detection.
Ginsenoside 20 (S)-Rh2 was purchased from Chengdu Must Biotech (Chengdu, China). The Annexin V-FITC/PI apoptosis detection kit was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Rhodamine 123, Ac-DEVD-CHO, DAPI, the nuclear and cytoplasmic protein extraction kit and cell mitochondria isolation kit were purchased from Beyotime. CCK-8 was purchased from BOSTER Biotech (Wuhan, China). Rapamycin and Bafilomycin A1 (BA1) were purchased from InvivoGen (San Diego, CA, USA).

SB202190 was obtained from Sigma-Aldrich (Taufkirchen, Germany). SB202474 and SB203580 were bought from Invitrogen (Darmstadt, Germany) and Tocris Bioscience (Wiesbaden, Germany), respectively. SnPPIX was from Enzo Life Sciences GmbH (Lörrach, Germany). SiRNA targeting HO-1 was purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany; sc-35554). Negative control siRNA was from Qiagen (Hilden, Germany; cat. no. 1022076). Lipofectamine™ RNAiMAX and OptiMEM were from Thermo Fisher Scientific Inc. (Schwerte, Germany). Bafilomycin A₁ was bought from InvivoGen (Toulouse, France).

A549, A549-Cas9, and 293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Thermofisher) supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 2 mM L-Glutamine (Gibco) and 1% penicillin. A549 and 293T cells were obtained from ATCC. A549-Cas9 cell line was generated by transducing A549 cells with a lentiviral construct (pXPR101) expressing Cas9 and Blasticidin deaminase. Cas9 activity was confirmed by transducing A549-Cas9 cells with a lentiviral construct (pXPR_011-sgEGFP) expressing eGFP and an sgRNA specific for eGFP. Polyclonal population of the A549-Cas9 cell line was used for the CRISPR screen to maintain heterogeneity of the cells. Primary NHLF cells were cultured in Mesenchymal Stem Cell Growth Medium (MSCGM, Lonza). PR8/A/34, A/Udorn/72 and A/Aichi/68 (X:31) Influenza A viruses were grown in MDCK cells in serum-free DMEM supplemented with 1% BSA and 1 μg/ml TPCK trypsin. GFP-Vesicular stomatits virus (VSV) was kindly.pngted by Dr. Sean Whelan’s lab. Influenza A/New Caledonia/20/1999, A/California/04/2009 and A/Vietnam/1203/2004-PR8-IBCDC-RG/GLP viruses were kindly.pngted by Dr. Daniel Lingwood’s lab. Bafilomycin A1 was obtained from invivogen (88899-55-2). Chloroquine diphosphate was obtained from Sigma (C6628). Baloxavir was obtained from MedChemExpress (HY-109025A).

To allow LC3-II accumulation, PBMCs were treated with lysosome-targeting Bafilomycin A1 (InvivoGen) at 50 nM for 4 h or left untreated^{36 (link)}. To determine autophagy flux upon TCR-mediated activation, PBMCs were first activated for 16 h with soluble 2 μg/mL anti-CD3 and 2 μg/mL anti-CD28 (both Sanquin) followed by incubation with 50 nM Bafilomycin A1 for 4 h. Anti-LC3 monoclonal antibody was conjugated to AF488 using the Lightning-Link® Rapid Antibody Labeling kit (Expedeon) according to the manufacturer’s instructions. Viable cells were suspended in 0.05% saponin (Sigma-Aldrich) for 15 min at room temperature to flush out LC3-I^{36 (link)}. Cells were washed twice with PBS, then stained with the conjugated LC3-AF488 in combination with antibodies against surface receptors in 0.05% saponine buffer for 30 min, shielded from light, shaking at 600 strokes/min at 4 °C. After incubation, cells were washed twice with PBS, then fixated using 4% PFA, washed, stored in PBS and acquired on a Fortessa Cell Analyzer.