Oxythiamine chloride hydrochloride

The ptfLC3 plasmid used here was a generous gift from Tamotsu Yoshimori (Addgene plasmid #21074; http://n2t.net/addgene:21074; accessed on: 30 January 2018; RRID: Addgene_21074, Watertown, MA, USA) [28 (link)]. Lipofectamine 2000 (ThermoFisher, Barcelona, Spain) was used to achieve transient transfection following the manufacturer’s instructions. The silencing of CHOP gene expression was performed by transient transfection of commercial siRNAs from Dharmacon (#J-004819-06, #J-004819-07, and #J-004819-08), which were introduced into the cells using RNAiMax (Life Technologies, Barcelona, Spain) during 24 h incubations. The DDIT3 sequence of CHOP was used to design the siRNA (accession number NM_004083 NCBI) and a negative siRNA control (siNeg) from Life Technologies was used as a control (unknown mixed sequences). The commercial inhibitors employed were: 3-methyladenine (3-MA, 5 mM; Merck KGaA, Darmstadt, Germany), Bafilomycin A1 (BafA1, 10 nM, Sigma), LY294002 (25 µM, Biogen Cientifica, Madrid, Spain), Oxythiamine chloride hydrochloride (OT, 1, 10 mM, or 100, 200, or 300 mg/kg mice; Santa Cruz Biotechnology, Heidelberg, Germany), and SP600125 (25 µM, Santa Cruz Biotechnology). The cells were seeded in culture plates and pre-treated with these commercial inhibitors for 1.5 h prior to their exposure to HCA for different times.

The cell cycle was analyzed as described previously [62 (link)]. Ros scavengers used were: α-tocopherol (100 µM, Vit. E) or Ferrostatin-1 (1 µM). The commercial inhibitors used were: Z-IETD-FMZ (50 µM: Selleckchem, Houston, TX, USA), Z-LEHD-FMK (50 µM: Selleckchem, Houston, TX, USA) and Oxythiamine chloride hydrochloride (OT, 1 and 10 mM: Santa Cruz Biotechnology, Heidelberg, Germany). The cells were seeded in culture plates and pre-treated with commercial inhibitors for 1.5 h before they were exposed to HCA for different times. Caspase-3/7 activity was measured using the Caspase-Glo^® 3/7 assay according to the manufacturer’s instructions (Promega, Madison, WI, USA).