Lentiviral particles

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.

Lentiviral particles encoding shRNA sequences for specific target genes were obtained from Sigma. MEFs or H9c2 cells were seed at appropriate density in growth media containing 5 μg/ml hexadimethrine bromide (Sigma), and cells were then infected by adding shRNA Lentiviral particles to the culture. Stable clones expressing the shRNA were selected using 2–10 μg/ml puromycin dihydrochloride. shRNA-mediated knockdown was confirmed by western blotting.

Primary normal human lung fibroblasts (NHLFs) were cultured in FGMTM-2 Fibroblast Growth Medium-2 Bullet Kit (Lonza). Human lung fibroblast lines (MRC-5 and IMR-90) and the human lung epithelial cell line (A549) were cultured in DMEM containing 10% FBS, penicillin and streptomycin (Pen+Strep), plus supplements of L-glutamine, sodium pyruvate, and non-essential amino acid (NEAA). RAW264.7 cells were cultured in complete RPMI. Cells were washed in PBS and detached from culture flasks with 5 ml Accutase.
To generate cell lines with stable transgene expression, lentiviral particles were purchased commercially (Sigma) or generated by the AbbVie lentiviral core facility. To enhance transduction efficiency, polybrene was added to the filtered supernatants (5 µg/ml) prior to transduction of A549 and RAW264.7 cells via spin-fection at 1250 x g for 60 min at room temperature. For establishing the pSTAT3 luciferase reporter RAW264.7 cell line, cells expressing the reporter construct were selected by puromycin (10 µg/ml), single cell cloning was performed to isolated stable clones via the standard limiting dilution approach.