Charcoal stripped fbs

Tissues were obtained from eight women undergoing surgery to remove parts of their endometrium. These tissues were identified as Type I endometrial cancers. None of the subjects received any hormonal therapy for at least 6 months prior to surgery. Patients were categorized into two groups which are non-obese (BMI: 18.5–24.9 kg/m²) and obese (BMI > 30 kg/m²). About 1 g of tissues was subjected to stromal isolation using anti-fibroblast magnetic microbeads (Miltenyi Biotec, Cologne, Germany) as described previously [29 (link)]. The fibroblasts were categorized as CAFs isolated from non-obese (CN) and obese (CO) women and were labeled according to each individual patient. Apart from different category of BMI, both CN and CO have similar molecular expression of basal PR (S1 Table). Moreover, the expression of fibroblast (vimentin and alpha-smooth muscle actin) and epithelial (EpCAM and E-cadherin) markers in both group was also similar. All treatment with vehicle and MPA were performed in high glucose condition using phenol red-free DMEM/F12 media, which contained 18 mM glucose, supplemented with 10% charcoal-stripped FBS (Biowest, USA) and 1% penicillin/streptomycin (Life Technologies, USA). Experiment was performed with cultures of passage 12 and below to maintain the closest phenotype to its original excised tissue.

T47D and CAMA‐1 cell lines were purchased from DSMZ (Brunswick, Germany) and ATCC (Manassas, VA, USA), respectively. After reconstitution, all cells were passaged for a maximum of 2–3 months and regularly tested for mycoplasma contamination. T47D cells were grown in DMEM (Corning, NY, USA) and CAMA‐1 cells in MEM (Corning), both supplemented with 10% FBS (Biowest, Nuaillé, France), 100 U·mL⁻¹ penicillin and 100 μg·mL⁻¹ streptomycin (HyClone, Logan, UT, USA). For all treatments, the standard media were replaced with phenol red‐free DMEM (HyClone), serum‐free or, when appropriate, supplemented with 10% charcoal‐stripped FBS (Biowest). FGF7 (50 ng·mL⁻¹, PeproTech, Rocky Hill, NJ, USA) was always applied with heparin sodium salt (50 ng·mL⁻¹; Sigma‐Aldrich, St. Louis, MO, USA). 17β‐oestradiol (E2, oestrogen, 10 nm), progesterone (P4, 100 nm), 4‐hydroxytamoxifen (OHT, 1 μm), LY294002 (2 μm), SP600125 (10 μm), SU6656 (10 μm), UO126 (10 μm), and LiCl (20 mm) were purchased from Sigma‐Aldrich, AZD4547 (0.5 μm), SB216763 (10 μm), ABT‐199 (5 μm), SB202190 (10 μm) and Magnolol (10 μm) from Selleckchem (Houston, TX, USA) and BI‐D1870 (1 μm) from Axon Medchem (Groningen, the Netherlands).

HEK293T, LnCap, and PC3 were obtained from ATCC and were propagated according to ATCC datasheets. LnCap-CR were generated by culturing LnCap cells in RPMI medium supplemented with 10% charcoal-stripped FBS (#S181F; Biowest) for more than 2 months. PrEC cells were purchased from Lonza (CC-2555) and propagated according to the manufacturer’s data sheet.