Atg7 sirna

For knocking down the expression of LIMP-2, the specific shRNA against LIMP-2 (shLIMP-2) and control shRNA (shCtrl) were constructed and packaged by Ubigene Biosciences (Guangzhou, China). The specific targeting sequence was as follows: shLIMP-2 (mouse): 5’-GGGTCTATGGATGAGGGAA-3’. The 4MOSC2 and SCC7 cells were infected with polybrene and lentiviral supernatant and then screened with 4 μg·mL⁻¹ puromycin (Sigma‒Aldrich, USA). The Protein levels were verified using Western blot.
Full-length mouse LIMP-2 complementary DNA was PCR amplified and cloned into the pcDNA3.1 plasmid vector (Tsingke Biotechnology Co. Ltd., China). β-catenin-siRNA (5ʹ-UACAUCAUUUGUAUUCUGCTT-3ʹ), ATG5-siRNA (5ʹ- GCGGUUGAGGCUCACUUUATT-3ʹ), and ATG7-siRNA (5ʹ-GCUAGAGACGUGACACAUATT-3ʹ) were from GenePharma (Suzhou, China). The mRFP-GFP-LC3 plasmid were kindly donated by Dr. T Yoshimori and distributed by Addgene. All the plasmids were transfected into HNSCC cells using Lipofectamine™ 3000 following protocol (Invitrogen, USA).

p53 siRNA, Parkin siRNA, ATG7 siRNA and the plasmid encoding Parkin were obtained from Genepharma (Shanghai, China). Transfection was performed using Lipofectamine 2000 reagent (Invitrogen) according to the instructions provided by the manufacturer.

The miR-106a mimics, miR-106a inhibitor, ATG7 siRNA, ATG16L1 siRNA, and ULK1 siRNA were purchased from GenePharma biotechnology company. To assay luciferase activity, HEK 293T cells were cotransfected with the pmirGLO luciferase constructs (WT or Mut) and miR-106a mimics or miR-106a inhibitor using Lipofectamine 2000 according to the manufacturer’s instruction. For autophagy analysis, THP-1 macrophages were transfected with 50 nM mimic negative control (mimic nc) or miR-106a mimics; inhibitor negative control (inhibitor nc) or miR-106a inhibitor; 50 pmol ATG7 siRNA, ATG16L1 siRNA, or ULK1 siRNA according to the manufacturer’s instructions. Several chemical reagents were also used to treat THP-1 macrophages: a lysosome inhibitor, bafilomycin A1 (100 nM; Baf A1, Selleck); An autophagy inducer, rapamycin (50 μg/ml; Rapa, Solarbio Science & Technology Co.).