Calf serum

HEK293T, PaTu-8988T, PANC-1, C2C12, and 3T3-L1 cells were purchased from the Cell Resource Center and authenticated by authentication testing (Chinese Academy of Sciences, Shanghai, China). All cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich) containing 12% calf serum (Sigma-Aldrich), 100 U/mL penicillin (Beyotime Biotechnology, Shanghai, China), 100 μg/mL streptomycin (Beyotime Biotechnology), and 0.25 μg/mL amphotericin B (Sangon Biotech, Shanghai, China) at 37 °C in a 5% CO₂ incubator (Thermo Fisher Scientific, Waltham, MA, USA). During metabolic studies, all cells were tested for mycoplasma and declared mycoplasma-free using a MycoAlert™ PLUS detection kit (Lonza Corporation, Basel, Switzerland).

Blood was collected from a male corn snake and a chicken; 0.5 mL of blood was mixed with 5 mL of 0.7 mM EDTA in HBSS (no. 14175095; Gibco) and stored at 4 °C. The cells were washed with 2% calf serum (no. 12238C; Sigma-Aldrich) in DPBS (D8537; Sigma-Aldrich), resuspended in 1 mL of propidium iodide solution (20 μg/mL PI; no. 81845; Sigma-Aldrich) with 100 μg/mL RNase A in DPBS–2% calf serum, and incubated at 37 °C for 30 min. The samples were stored at 4 °C and analyzed the same day on a BD Accuri C6 flow cytometer.

Mesenteric fat preadipocytes from non-IBD, CD, and UC patients were collected from a previous study and stored in liquid nitrogen [28 (link)]. The human preadipocytes were thawed and cultured in DMEM/F12 media containing 10% calf serum and 1% P/S (Invitrogen) until >60% confluence was achieved. The preadipocytes were dissociated by trypsin/EDTA solution (Invitrogen) and seeded to 6-well plates (400,000 cells per plate) in DMEM/F12 media containing 10% calf serum and 1% P/S. Two days later, the preadipocytes underwent differentiation process by incubating with induction media (DMEM with FBS, P/S/G, bovine insulin (Sigma I-5500; 1μg/mL), dexamethasone (Sigma D-4902; 1μM) and isobutylmethylxanthine (IBMX; Sigma I-5500; 115μg/mL) for two days, insulin media (DMEM with FBS, P/S/G and insulin (1 μg/mL)) for two days, and DMEM + FBS + P/S for two days [29 (link)]. The adipocytes were regarded as differentiated by the observation of lipid droplet deposition under microscope. The differentiated adipocytes were serum-starved for 8 hours, followed by incubation with human serum exosomes (100μg/mL) for 16 hours. The conditioned cells were then switched to serum-free DMEM media for 6 hours to let the cells secrete elafin. The conditioned media were collected for elafin ELISA.

HEK293 (ATCC), RAS-less mouse embryonic fibroblasts (MEF) isogenic cell lines [17 (link)] (from the RAS initiative at Frederick National Laboratory), and HKe-3 (Shirasawa et al. 1993) cells were maintained in T75 flasks at 37 °C in 95% humidified air containing 5% CO₂ in HEPA class 100 steri-cycle CO₂ incubator (Thermo Electron Corporation, Vantaa Finland) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% Foetal Bovine Serum and 2 mM L glutamine (all from Gibco, Waltham, MA, USA). NIH3T3 (ATCC) cells were maintained at similar conditions and cultured in filtered DMEM containing 10% Calf Serum (Sigma-Aldrich, Burlington MA, USA) and 2 mM L-glutamine. Cells were transfected with Lipofectamine 2000 (Invitrogen, Waltham, MA, USA), according to the manufacturer’s instructions. All cells were validated by genome sequencing in the last 12 months. For stimulation with EGF (Roche, Basel, Switzerland), following 24 h transfection of HEK293 cells, cells were starved for 16 h prior to stimulation with 3 µM EGF for 5 and 10 min, and cells were then lysed. Cells were treated with 5 µM Sotorasib (MedChemExpress, Monmouth Junction, NJ, USA) and 5 µM Ruxolitinib (SelleckChem, Berlin, Germany) for 24 h in normal growth conditions prior to protein extraction by cell lysis.