4 6 diamidino 2 phenylindole c1005

The main primary antibodies used in this study were specific for MAP1LC3 (Cell Signaling Technology, Beverly, MA, USA; Cat 2775), ATG5 (Novus Biologicals, Littleton, CO, USA; Cat NB110-53818), SQSTM1/p62 (Santa Cruz Biotechnology Inc., Heidelberg, Germany; Cat sc-25575), DAPDH (Cell Signaling Technology; Cat 2118), IFNGR1 (Bioss Biotechnology Co., LTD., Beijing, China; Cat bs-1463R), IFNGR2 (Bioss Biotechnology Co., LTD.; Cat bs-2710R), LC3B (Bioss Biotechnology Co., LTD; Cat bs-4843R) and SQSTM1 (Bioss Biotechnology Co., LTD; Cat bs-2951R). 3-Methyladenine and rapamycin were purchased from Santa Cruz Biotechnology Inc. 4′,6-diamidino-2-phenylindole (C1005) and pifithrin-α were purchased from Beyotime (Beyotime Institute of Biotechnology, Shanghai, China). HRP-conjugated goat anti-rabbit secondary antibodies were purchased from Proteintech (Proteintech Group, Inc., Chicago, IL, USA). Bovine interferon was purchased from the Kingfisher Group (Kingfisher Biotech, Inc., Saint Paul, MN, USA).

For immunofluorescence, the cells were fixed with 4% aqueous paraformaldehyde and treated with 0.5% Triton X-100 in PBS. Cells were washed with PBS after each treatment. After blocking with 5% bovine serum albumin in PBS for 1 h at room temperature, the cells were incubated with primary antibodies against LCN2 (A2092; ABclonal) overnight at 4 °C. The cells were then incubated with fluorescent secondary anti-rabbit antibodies (A11034; Invitrogen) for 1 h at room temperature in the dark. The cells were washed with PBS, stained with 4′,6-diamidino-2-phenylindole (C1005; Beyotime) for 10 min at room temperature and then photographed using the LSM 880 confocal microscope (Carl Zeiss).

The cells were treated with 4% paraformaldehyde (E672002, Sangon) and 0.5% Triton X-100 (Sangon). The cells were treated with TWIST1 (1 : 500, #GTX60776, GeneTex) and EP300 (1 : 100, ab275378, Abcam) and stained with FITC-conjugated goat anti-rabbit antibody (ab6717, Abcam). The cells were then denatured with 2 M hydrochloric acid at 37°C for 10 min and then stained with Alexa Fluor 647-conjugated goat anti-mouse antibody (1 : 500, ab150083, Abcam). The cells were mounted in medium containing an antifluorescent quenching agent, and the nucleus was stained with 0.25 μg/mL 4′,6-diamidino-2-phenylindole (C1005, Beyotime Biotech). Then, the fluorescent images were captured with a laser scanning confocal microscope (Leica, Frankfurt, Germany) equipped with the appropriate FITC and Texas Red filters.