Ishikawa cells

The Ishikawa cells were purchased from the American Type Culture Collection (Manassas, VA) and were passaged in our laboratory for less than 6 months. Cells were grown in Dulbecco’s Modified Eagle’s Medium supplemented with glutamine, pyruvate, antibiotics, and 10% fetal calf serum in a humidified atmosphere containing 5% CO2 at 37°C.
Cell lysate proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10% gels) and transferred to nitro- cellulose membranes. Protein amounts were quantified using the Bradford method, and equal protein amounts were loaded to the gel. Membranes were blocked in TBS with 0.05% Tween 20 (TBST) containing 5% nonfat dry milk powder for 1 hour. Western blots were probed with primary antibodies for 1 hour, washed three times with TBST, and then incubated with the appropriate secondary anti- bodies for 30 minutes. Membranes were then washed with TBST three times before developing with SuperSignal West Dura chemi-luminescent substrate (Pierce, Rockford, IL).

Ishikawa cells (a well-differentiated human endometrial adenocarcinoma cell line), obtained from American Type Cell Culture (Manassas, VA, USA), were maintained in Dulbecco’s Modified Eagle Medium/F12 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin (Gibco), 100 mg/mL streptomycin (Gibco) and 2 mM L-glutamine (Gibco). Recombinant human TGF-β (cat# L1718; Peprotech, Rocky Hill, NJ, USA) was used at concentrations of 1, 5, or 10 ng/mL. The anti-fibrotic effects of eupatilin (cat# S3846; Selleckchem, Houston, TX, USA) were examined at final concentrations of 25, 50, and 100 μM. Cells were grown on a Matrigel-coated cover glass (1:8 dilution, growth factor-reduced; Corning, Tewksbury, MA, USA) for further investigation of morphological changes and immunofluorescence (IF) analyses.

Ishikawa cells (a well-differentiated human endometrial adenocarcinoma line) obtained from American Type Cell Culture (Manassas, VA, USA) were maintained in a Dulbecco’s Modified Eagle Medium/F12 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin (Gibco), 100 mg/mL streptomycin (Gibco), and 2 mM L-glutamine (Gibco). Cells were grown on Matrigel-coated cover glass (1:8 dilution, growth factor-reduced; Corning, Tewksbury, MA, USA) for further attachment assays.