Calpastatin

Lentiviral constructs expressing shRNAs for caspases 1, 3, 6, 7, 9, CAD, EndoG, calpastatin, calpain S1, PAD2, PAD4, Atg5, Vps34 and Beclin1 were purchased from Sigma-Aldrich. cDNAs of caspase 3, CAD, EndoG, calpain 1 and calpain 2 were purchased from OriGene (Rockville, MD, USA), calpastatin cDNA was from Shi-Yong Sun (Emory University School of Medicine), being subcloned into pCDH-EF2-MCS-T2A-Puro, a lentiviral vector for cDNA expression (System Biosciences, Mountain View, CA, USA). Mutation of calpastatin at cleavage sites of caspases was accomplished using a QuickChange site-directed mutagenesis kit (Stratagene, Santa Clara, CA, USA). All the lentiviral vectors were transfected to 293TN cells (System Biosciences) with Lipofectamine 2000 transfection reagent (Invitrogen, Seoul, Korea). Particles were collected 2 days after the transfection of lentiviral plasmids, and infected into the cells. Lentivirus-infected cells were puromycin-selected for 1 week.

MG132, PS-341, Lactacystin, Calpastatin, Brefeldin A, emetine, puromycin, penicillin, streptomycin, Guanabana and Thapsigargin were purchased from Sigma (St. Louis, MO, USA).
Normal human hepatocyte and human Macrophage were obtained from Lonza, Hepatocellular cell line C3A and ubiquitin-proteasome system reporter cell line GFPu-1 were from ATCC, and these hepatocytes were cultured in DMEM/F12 1:1 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The α1ATD disease specific Human induced pluripotent stem cells were provided by David A. Lomas. The mutant and wide type iPSC cells lines were differentiated in definitive endoderm over 5 days (T5), and then into early hepatocytes over 24 days following standard protocol. The successfully differentiated hepatocytes were then confirmed by α1AT’s gene expression using qPCR. Mature hiPSC hepatocytes cultured in Hepatocyte Culture Medium (GIBCO, Columbia, USA).
Polyclonal Rabbit anti-Human alpha-1-antitrypsin (A0012) was purchased from Biocompare. Antibodies for G3BP1 and GADD34 were purchased from Proteintech and antibodies for eIF2α, p-eIF2α, p-S6, S6, p-4EBP1 and 4EBP1 were purchased from Cell Signaling Technology (Danvers, MA USA).