Nb100 2220ss

25HC was purchased from Sigma-Aldrich. Caspase-1 activity assay kit was purchased from Beyotime Institute of Biotechnology. Lipofectamine 3000 was purchased from Invitrogen. Annexin V-FITC and PI apoptosis detection kits were purchased from Keygen Biotech Company (Nanjing, China). Antibodies used in the present study were as follows: ALV-J envelope protein JE9-specific mouse monoclonal antibody (provided by Professor Qin Aijian of Yangzhou University), rabbit anti-LC3II antibody (NB100-2220SS, Novus Biologicals), rabbit anti-ATG5 antibody (12994, Cellsignal), mouse monoclonal anti-flag antibody (AP007M, Bioworld), rabbit anti- SQSTM1/p62 antibody (P0067, Sigma-Aldrich), and anti-myc antibody (A02060, Abbkine), rabbit anti-β-actin antibody (AF5003, Beyotime), goat anti-rabbit IgG (H+L) antibody (A21020, Abbkine), Goat anti-mouse IgG/HRP antibody (A25112, Abbkine).

The experiment was designed in 6-well plates with sterilized coverslips in each well. 50.000 cells/well were seeded and the next day cells were treated either with DMSO (0.1%), ABT-199 (10 µM), or Bcl-2 inhibitor BAU-243 (20 µM) for 72 h. Primary antibody against LC3B (#NB-1002220SS, Novus Biologicals, Littleton, CO, USA) and F(ab’)2-Goat anti-Mouse IgG (H + L) cross-adsorbed secondary antibody Alexa Fluor-488 (#A-11017, Invitrogen, Waltham, MA, USA) were used for the staining. Cell nuclei were stained with DAPI (10 mg/ml) (#A4099, AppliChem GmbH, Darmstadt, Germany). Cells were then observed with a Leica DM1000 fluorescent microscope (Leica Microsystems, Wetzlar, Germany).