Tubastatin a tuba

The immortalized human endometrial stromal cell line (ATCC, CRL-4003^TM) cells were cultured as previously described [66 (link)]. Human endometrial stromal cells were seeded in 12-well plates and cultured with DMEM/F12 (Merck) containing 10% cFBS (Biological Industries) until ~80% confluency at 37 °C and 5% CO₂. In vitro decidualization was performed as previously described [67 (link)]. Briefly, human endometrial stromal cells were induced by 100 or 500 μM dibutyryl cyclic adenosine monophosphate (db-cAMP, D0627, Merck) and 1 μM medroxyprogesterone acetate (MPA, M1629, Merck) for indicated times. Insulin-like growth factor binding protein-1 (IGFBP1) and prolactin (PRL) are reliable markers for evaluating human in vitro decidualization [67 (link)]. The reagents used in this study included Tubastatin A (TubA, S8049, Selleck Chemicals, Houston, TX, USA), ciliobrevin A (CBA, S8249, Selleck Chemicals), ammonium sulfate ((NH₄)₂SO₄, A610060, Sangon, China), BPV (HOpic) (S8651, Selleck Chemicals), LY294002 (S1105, Selleck Chemicals), AMG-900 (S2719, Selleck Chemicals), TC-S 7010 (S1451, Selleck Chemicals), and prostaglandin E₂ (PGE2, 14010, Cayman chemical).

The ovarian culture procedure was according to previous reports 20 , 26 (link), 58 (link). Briefly, 1.2-1.4 mL of Dulbecco's Modified Eagle's Medium (DMEM) medium was added to a 6-well plate with low adsorption, placed in a cell culture incubator, and equilibrated for 15-20 min. A 20-50 μL pipette was used to transfer 6-10 newborn mouse ovaries to each well of the pre-warmed 6-well plate which were cultured in suspension. The ovaries were cultured with 4 μM Tubastatin A (TubA) (S8049, Selleck, USA), 30 nM K252a (HY-N6732, MedChemExpress, USA), 20 μM ANA-12 (S7745, Selleck, USA) at 37 °C with 5% CO₂. The medium was changed every other day.