Lentiviral particles

Stable CCRF-CEM-CRISPR-sgRNA-dCK⁻ cell line was established by lentiviral transduction using purified lentiviral particles (Horizon Discovery) and blasticidin selection (10 µg/ml). Doxycycline-inducible Cas9 integration was confirmed after doxycycline exposition (1 µg/ml) by western blot with CRISPR-Cas9 antibody (Santa Cruz, SC-517386, 1:1000). Cas9-expressing cell line was further transduced with human DCK sgRNA purified lentiviral particles (Horizon Discovery, sequences are provided in Supplementary Table 6) and puromycin selection (1 µg/ml). Finally, two successive gemcitabine selections (2 µM for 3 days and 10 µM for 2 days) were performed to obtain the cell line deficient for dCK. Experiments were performed as in the dCK cellular assay.

Stable NQO1 knockdown was generated as previously published^{19 (link)}. Briefly, lentiviral particles expressing shRNA against non-targeted control (NTC) and NQO1 (shNQ) were purchased from Dharmacon (Lafayette, CO). LNCaP and PC3 cells were transduced with either NTC or shNQ in the presence of 8 μg/ml polybrene (Sigma-Aldrich) in 96-well plate. Transduction was continued for 48 h. Cells were transferred into a 24-well plate and selected with 1 μg/ml puromycin (Sigma-Aldrich). Serially diluted cells were grown and efficiency of transduction was confirmed in isolated clones by detecting GFP fluorescence. For transient knockdown, cells were transfected with either scramble or NQO1 SMARTpool siRNA (Dharmacon) using Lipofectamine (Invitrogen). Knockdown was verified by qPCR and immunoblot analysis. For stable overexpression, cells were transfected with pcDNA3.1 vector control or pcDNA-NQO1 (pcNQ). After selection with G418, NQO1 overexpression was verified by western blotting.

Islets from human cadaver donor pancreas were obtained via the NIDDK-sponsored Integrated Islet Distribution Program, CA. Details regarding the human islet donors used in this study are provided (Supplementary Table 1). After overnight recovery at 37 °C, islets were handpicked and used for western blot analyses and the TGF-β1 secretion assay. Similar sized 15 islets per well were treated with 0.5 mM palmitate (PA, Sigma) in 2% charcoal-treated FBS containing CMRL 1066 media (Gibco, ThermoFisher Scientific, Waltham, MA, USA) for 24 h and the TGF-β1 level measured in the media was normalized to protein extracted from 15 islets. For the apoptosis detection, handpicked islets were dissociated with trypsin at 37 °C and dispersed islets were used. Lentiviral particles containing nontargeting short hairpin RNA (CON) or shRNA against to FoxO1 (shFoxO1) were purchased from Dharmacon (Lafayette, CO, USA). The sequence of FoxO1 shRNA was 5′ -ACATATGGCCAATCCAGCA-3′. Dissociated T2D human islet cells were infected with control or shFoxO1 Lentiviral particles with polybrene (8 µg/ml) for 3 days and subsequently the islet cells were treated with for FFA palmitate or SB431542 (Sigma) for additional 24 h.