Pegfp lc3b

BEAS‐2B cells were transfected with siRNA for nonspecific oligonucleotide control (NC) and Keap1 (Life Technologies) using HiPerfect (Qiagen) for 48 hours or 72 hours. HEK293 cells were transfected with different plasmids using Lipofectamine (Invitrogen). The following plasmids were characterized in a previous work by Lamark et al27: pDest‐EGFP‐p62 WT, pDest‐EGFP‐p62 ∆123‐170, pDest‐EGFP‐p62 ∆170‐256, pDest‐EGFP‐p62 ∆256‐370, pDest‐EGFP‐p62 ∆346‐385, pDest‐EGFP‐p62 (1‐385) that is ∆UBA (∆385‐440), pDest‐mCherry‐EGFP‐p62, pDest‐mCherry‐EGFP‐LC3B, and pDest‐EGFP‐p62 E352A (KIR point mutant). pBABE‐puro mCherry‐EGFP‐LC3B were a gift from Ana Maria Cuervo and pEGFP‐LC3B was purchased from Addgene.

AGS, SNU-216, NCI-N87 and SNU-620 cells (1 × 10⁵ cell/well) in 24-well plate were transfected with these double-stranded siRNAs (30 nmol/ml) such as siKLK6, siLC3B, and sip53 (Bioneer) for 24 hr by the Lipofectamine 2000 (Invitrogen) method according to the manufacturer's protocol and recovered in RPMI1640 medium (Welgene) containing 10% fetal bovine serum for 24 hrs. After recovering, viable cells were calculated by WST-1. pcDNA3.1-KLK6 and pEGFP-LC3B and –KLK6 made to study of target gene and pCMV-Neo-Bam p53 (addgene) purchased. KLK6 stable overexpressed cell line was constructed by using pcDNA 3.1 – KLK6 plasmid. In stable overexpressed cell lines, AGS cell (1x10⁵ cell/well) was seeded into 24-well plate. After 16 hr, KLK6-expressed vector was transfected into AGS cells using Lipofectamine 2000 (Invitrogen). The following day, the medium was changed, and G418 was added to the culture medium to a final concentration of 800 μg/mL and cultured in the presence of G418 for 4 weeks. Medium was exchanged every 3 day. The expression of KLK6 to identify establishment of KLK6 stable cell line was checked by RT-PCR and Western blot assay.