Lc3 1 2

The cells in all groups were lysed with 200 μl of cell lysate and placed on ice for 1 hour. Subsequently, lysing cells were centrifuged at 12,500 rpm for 15 minutes at 4° C. Then transferred the centrifuged supernatant to a clean centrifuge tube. Bicinchoninic acid (BCA) protein quantification kit was used to determine protein concentration and stored the proteins at -80° C. In electrophoresis, the loading concentration was 50 mg per well. After electrophoresis, the membrane was transferred and blocked. SPRED2, caspase-3, BAX, BCL-2, P16, P21, P53, LC3 II/I, beclin1 and GAPDH (1:500, anti-human, Abcam, USA) were diluted and incubated at 4° C overnight. The secondary antibody was incubated in the dark at room temperature for 45 minutes (1:2000, Abcam, USA). Developers were used for development and photography.

Inflammatory and autophagy-related proteins were evaluated by Western blot. Proteins were extracted from the lung tissues using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China). Approximately 40 μg of protein was loaded and separated with the 12% SDS-polyacrylamide gel (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, MIT, USA). The membrane was incubated with 5% nonfat dry milk in TBST (Tris-buffered saline/0.1% Tween-20, pH 7.4) for 1 h at room temperature, followed by incubation overnight with primary rabbit anti-mouse antibodies to NF-κB (1 : 1000, Abcam, USA), p-NF-κB (1 : 1000, Abcam, USA), TLR1 (1 : 1000, Abcam, USA), TLR2 (1 : 1000, Abcam, USA), TLR3 (1 : 1000, Abcam, USA), TLR4 (1 : 1000, Abcam, USA), Myd88 (1 : 1000, Abcam, USA), ATG5 (1 : 1000, Abcam, USA), LC3I/II (1 : 1000, Abcam, USA), Beclin1 (1 : 1000, Abcam, USA), and GAPDH (1 : 1000, Abcam, USA). A horseradish peroxidase-conjugated antibody against rabbit IgG (1 : 5000, Abcam, USA) was used as a secondary antibody. Blots were incubated with the ECL reagents (Beyetime, Jiangsu Province, China) and exposed to Tanon 5200-multi to detect protein expression. Three independent assays were performed.

Quercetin (>98% pure), and Green tea were obtained from Sigma. The CYCLIN D, cyclin E, CYCLIN A, CDK4, CDK2, CDK6, P21, P27, BCL-XL, BAX, BCL-2, cytocrome c, MCL-1, actin, and GAPDH from Santa Cruz Biotechnology. BECLIN-1 and PI3K class III from Cell Signaling Technology. pJNK from Invitrogen. ATG5-ATG12, ATG7 and LC3I/II from Abcam Inc. Anti-rabbit, anti-mouse, and anti-goat peroxidase–conjugated antibodies from KPL, Inc. The HL-60 cell line, derived from a 36-year-old woman with acute promyelocytic leukemia, was obtained from ATCC, Philadelphia, PA. Cytogenetic analyses of the cell line, during the procedures of this study, identified the same alterations as previously described^{27 (link)} in 100% of metaphases analysed.

One hundred microliters RIPA lysis buffer (Beyotime, Zhejiang, China) containing 1 μL PMSF (Sigma, USA) was used to extract the cellular proteins. Concentrations of proteins were measured by BCA kit (Thermo Scientific, USA). Antibodies against Livin (Cell Signaling Technology, USA), H2A.X (Abcam, USA), H2A.XY142F (Abcam, USA), GAPDH (Santa Cruz, USA), ATG5 (Cell Signaling Technology, USA), ATG13 (Cell Signaling Technology, USA), ATG14 (Cell Signaling Technology, USA), LC3-I/II (Abcam, USA) and β-actin (Santa Cruz, USA) were used at 1:1000 dilution.