Nih 3t3 cell line

The NIH‐3T3 cell line was obtained from the American Type Culture Collection (ATCC, CRL‐1658) and cultured in DMEM (HyClone, GE) with 10% foetal bovine serum (Lonsera) and 1% penicillin/streptomycin (HyClone, GE) at 37°C in 5% CO₂ according to the protocol provided by the ATCC. The NIH‐3T3 cells were cultivated in 24‐well plates (1 × 10⁵ cells/well). The next day, the cells were stimulated with the following agents: 10 mmol/L metformin (D150959‐5G; Sigma), 250 ng/mL HMGB1 (50913‐M01H; Sino Biological), 10 ng/mL TGF‐β1 (100‐21‐10UG; PeproTech) and 1 ng/mL SRI‐011381 hydrochloride (Smad3 agonist, HY‐100347A; MCE), respectively. In the control group, the cells were treated with the equal volume of PBS (HyClone, GE). The cells were harvested after 48 hours for further analysis.

Primary human nasal epithelial cells (hNEPCs) were maintained in Dulbecco’s Modified Eagle Medium (DMEM)/Nutrient Mixture F-12 (Cat.No.11320033, Gibco), which contained human epithelial growth factor (Cat.No.100-15-500, Peprotech), insulin (Cat.No.I5500-50MG, Sigma), cholera toxin (Cat.No.C9903-0.5MG, Sigma), hydrocortisone (Cat.No.H0888-1G, Sigma), 3,3’,5-triiodo-l-thyronine (Cat.No.T6397-100MG, Sigma), and N-2 supplement (Cat.No.17502001,Gibco-Invitrogen). Further, a 1% Antibiotic-Antimycotic solution (Cat. No.MIR 5970, Mirusbio) was added. hNEPCs were cultured at 37°C in a 5% CO2 cell incubator.
The NIH/3T3 cell line purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) was cultured in DMEM (Cat.No.C11995500BT, Gibco) containing 10% fetal bovine serum (FBS) (Cat. No. 10099-141, Gibco-Invitrogen) and 1% penicillin-streptomycin (Cat. No.15140-122, Gibco). When the cell density reached about 80%, mitomycin C (Cat.No.M4287-2MG, Sigma) was added, treated in a 37°C cell incubator for 2 h, washed three times with PBS, and then co-cultured with the isolated primary cells.
Madin-Darby canine kidney (MDCK) cells were cultured in the following manner: 5% fetal bovine serum (FBS), 100 IU/mL penicillin, 100 mg/ml streptomycin, and 2 mM glutamine were added to the DMEM. Further, the cells were cultured at 5% CO2 and 37°C.

The NIH/3 T3 cell line (American Type Culture Collection, Rockville, MD) was used to detect the mitogenic activity of oil body bound oleosin-rhFGF9. Briefly, cells were grown in a culture flask containing DMEM, 10% fetal bovine serum (FBS), and ampicillin and streptomycin (both 100 U/ml). Cells at the appropriate growth phase were seeded into a 96-well plate (5 × 10³ cells per well), cultivated under normal or cell starvation conditions for 24 h, and then treated for 48 h with oil body bound oleosin-rhFGF9 or rhFGF9 (purified rhFGF9 from E.coli) at various concentrations from 0.82–212 ng/ml. The cells were then incubated with 20 μl MTT for 4 h, the culture medium was added to 100 μl dimethyl sulfoxide and mixed to homogeneity by shaking, and optical absorbance values at 570/630 nm were measured using a microplate reader.

The fibroblast NIH/3T3 cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in Dulbecco’s modified essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 4 mM L-glutamine, streptomycin, and penicillin, maintained in an incubator with a humidified atmosphere containing 5% CO ${}_2$ at 37 °C.
Caco-2 intestinal epithelial cells, obtained from ATCC, were grown in DMEM supplemented with 10% FBS, 4 mM L-glutamine, 1% non-essential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were maintained at 37 °C in a humidified atmosphere with 95% air and 5% CO ${}_2$ .

The National Institute of Health (NIH) 3T3 cell line was obtained from American Type Culture Collection (ATCC, USA). The cell line was maintained in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution. The cells were grown in a humidified incubator supplemented with 5% CO₂ at 37°C and cell morphological examinations of the culture flasks were performed daily under inverted light microscope (NIKON, Japan). After the cells were completely grown by covering the entire 75 cm² culture flask (90% confluence), old media was discarded and the cells were washed with sterile phosphate buffer saline (PBS, pH 7.4) thrice, detached from the culture flasks using trypLE^TM Express (animal origin free) solution and passaged as single cells in a DMEM culture medium. The detached cells were collected, added with 10 mL of fresh DMEM medium to inactivate the action of trypLE^TM and centrifuged at 3000 rpm for 5 minutes to obtain the cell pellet. After centrifugation, supernatant was discarded, and 10 mL of fresh DMEM medium was added and was vortexed to obtain homogenous solution. The viable cells in the solution were counted by Trypan blue using hemocytometer.