Betamethasone

2,4,6-dinitrobenzenesulfonic acid (DNBS), croton oil, fluorescein isothiocyanate (FITC)-conjugated dextran (molecular mass 3–5 kDa), betamethasone, atropine, tubocurarine and neutral red (NR) solution were purchased from Sigma (Milan, Italy). TNF-α was obtained from R&D Systems, Space Import-Export SRL (Milano, Italy). All reagents for cell cultures were obtained from Sigma, Bio-Rad Laboratories (Milan, Italy) and Microtech Srl (Naples, Italy). All chemicals and reagents employed in this study were of analytical grade.

C57BL/6J, MRL/MpJ-Fas^lpr/J, NOD mice (The Jackson Laboratory, Bar Harbor, ME, USA), and Foxp3^RFP reporter mice (30 (link)), kindly provided by S. Huber, were housed and maintained under specific pathogen-free conditions. Mice were mated and the presence of a vaginal plug was considered day 0.5 of pregnancy (E0.5). On day 18.5 (E18.5) mice were treated by i.p. injection of 0.1 mg betamethasone (Sigma-Aldrich, Germany) in PBS or vehicle control (PBS). When indicated, 4- to 5-week-old animals were given 0.1 mg betamethasone i.p. or vehicle control (PBS) 24 h prior to organ harvesting. All animal experiments were performed in accordance with national and institutional guidelines on animal care and ethics.

Experiments were approved by the Institutional Animal Care and Use Committee of the Ulsan University College of Medicine and conformed to the Revised Guide for the Care and Use of Laboratory Animals [NIH GUIDE, 25(28), 1996] (18 ). Timed-pregnant Sprague-Dawley rats were purchased from an approved source (Orient Bio Inc., Seoul, Korea). The rats were housed individually in the animal facility during the remainder of their pregnancy with free access to standard rat chow and water on a regular 12-h light-dark cycle with the lights on at 08:00. On gestational day 15, pregnant rats received two injections of 0.4 mg/kg betamethasone (Sigma-Aldrich, St. Louis, MO, USA) at 08:30 and 18:30. Delivery occurred consistently on gestational day 22, which was considered postnatal day (P) 0 for the offspring.
Spasms were triggered by intraperitoneal injection of NMDA on P12 (6 mg/kg), P13 (10 mg/kg), and P15 (15 mg/kg); control groups received the same volume of saline (Figure 1). Immediately after NMDA administration, the rats were observed for 90 min and only the animals confirmed to have had three bouts of spasms at the appropriate time points on each day (i.e., on P12, P13, and P15) were included in the analyses (4 (link)).

Stock solutions were prepared by dissolving DEX (25 mM, ≥98%) and mifepristone (RU486, 50 mM, ≥98%) in ethanol, cortisone (25 mM, ≥98%) in methanol, chloroquine (100 mM, ≥98.5%) in PBS, and bafilomycin A1 (100 μM, ≥90%), betamethasone (50 mM, ≥98%), budesonid (100 mM, ≥99%), and prednisone (100 mM, ≥98%) in DMSO (all reagents were from Sigma‒Aldrich, Munich, Germany). Rapamycin (20 mM, ≥98%, Axxora, Lörrach, Germany) was prepared in DMSO. Gemcitabine (126 mM, Lilly Deutschland, Bad Homburg, Germany) was freshly diluted in cell culture medium to a 100 μM stock solution. The final concentrations of the solvents in the media were 0.1% or less.

The osteoblast cell lines MC3T3 and hFOB1.19 were purchased from American Type Culture Collection (ATCC, CRL-2593, and CRL11372) and cultured as per the manufacturer’s protocol. Dexamethasone (Sigma-Aldrich, D1756, St. Louis, MO, USA) and Betamethasone (Sigma-Aldrich, B7005, St. Louis, MO, USA) treatments were performed as described in the experiments. Lentiviral vectors containing CMV-aurora A and CMV-glucocorticoid receptor were all purchased from GeneCopeia^TM (Rockville, MD, USA) and applied to the cells as per the manufacturer’s protocols.