Cell culture

MIN6N8 cells, kindly gifted by Professor Josephine Forbes, were cultured as previously described⁵, in phenol-red–free Dulbecco’s modified eagle’s medium (DMEM; Life Technologies) containing 25 mM glucose (3.4 g L⁻¹ sodium bicarbonate, 50 U mL⁻¹ penicillin and streptomycin, 71.5 μM β-mercaptoethanol and 10% heat-inactivated fetal bovine serum (FBS).

HEPG2 cells were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). Cells were maintained in DMEM (Life Technologies) supplemented with 10% heat inactivated FBS, 100 U/ml penicillin and 100 mg/ml streptomycin, in a humidified atmosphere of 5% CO2 in air at 37 °C. HEPG2 cells were treated with 0-250 μM of palmitic acid to induce steatosis (Sigma-Aldrich). Solutions of palmitate-BSA complex used were prepared as previously described⁵. To assess the effects of IL-22-ScFv in steatotic hepatocytes, palmitate treated HEPG2 cells were further treated with hIL-22-ScFv for up to 72 h (with IL-22-ScFv being replenished every 24 h).

Following treatment, cells were stained with 10 μM of Bodipy_493/503 (D3922, Invitrogen) for 20 min in the dark before washing extensively with PBS. A subset of cells was incubated with primary antibodies against ACC1 (1:200; 3676; Cell Signaling Technology) for 1 h at room temperature, washed extensively with PBS, and further incubated with Alexa Fluor 647 Goat anti-rabbit IgG (1:500; A32733, Invitrogen) for 1 h at room temperature. Following extensive washing with PBS, ACC1 and Bodipy staining were determined using a PHERAstar plate reader. Representative images of Bodipy staining were captured using the Olympus confocal microscope FV3000.

For mass spec analyses: Treated and control cell pellets were lysed by resuspension in 100 mM Tris pH 8.5, 1% Sodium Deoxycholate, 40 mM 2-chloroacetamide, 10 mM Tris (2-carboxyethyl) phosphine. Lysates were disrupted by sonication on a Bioruptor and heat inactivation (90 °C for 5 min). 20 µg of each lysate sample was digested with trypsin according to the manufacturer’s instructions (Promega catalog V5111). Digests were acidified and peptides desalted using C18 STAGE tips according to the manufacturer’s instructions (Agilent catalog A57003100). Tryptic peptides resolved by C-18 separation on an UltiMate 3000 HPLC nano-flow HPLC and analyzed on Q Exactive Plus orbitrap mass spectrometer running in positive ion data-dependent analysis (DDA) mode, with settings typical of peptide analyses. Raw data was searched against the human proteome using the Proteome Discoverer 3.0 platform and the Chimerys search engine. Peptides were quantified by precursor abundance, and group comparisons were made using pairwise peptide ratios and a background-based t-test.

LX-2 cells, kindly gifted by Professor Grant Ramm, were maintained in DMEM (Life Technologies) supplemented with 2% heat-inactivated FBS, 100 U/ml penicillin and 100 mg/ml streptomycin in a humidified atmosphere of 5% CO2 in air at 37 °C. Prior to experiments, cells were serum starved in media supplemented with 0.1% FBS overnight.