Streptomycin sulfate

PBL from patients and controls were isolated via centrifugation on a Ficoll-Hypaque gradient for 20 min, washed twice, and resuspended in Roswell Park Memorial Institute 1640 medium with HEPES, supplemented with 2 mM glutamine (Sigma-Aldrich Japan, Tokyo, Japan), 100 units/ml penicillin G (Sigma-Aldrich Japan, Tokyo, Japan), 100 μg/ml streptomycin sulfate, and 10% heat-inactivated human AB serum (Biowest, Nuaillé, France). Cells (1 × 10⁵/well) were incubated in a total volume of 100 ml with the peptides or concanavalin A (4 μg/ml, Fujifilm Wako Pure Chemical Co., Osaka, Japan) in triplicates in 96-well round-bottom culture plates (Nunc, Roskilde, Denmark) at 37°C in a humidified atmosphere with 5% CO₂. The cultures were incubated for 4 days and then pulsed with bromodeoxyuridine (BrdU) during the last 16 h of incubation. Cellular proliferation was assessed using a BrdU enzyme-linked immunosorbent assay kit (Cell Proliferation ELISA BrdU, Sigma), according to the manufacturer’s instructions. After co-culture with Brdu for 16 h, the BrdU labeling solution was removed and anti-BrdU antibody was added. The OD at 450 nm for BrdU was measured using a luminescence plate reader (ARVO X3, Perkin Elmer Japan, Yokohama, Japan). The results were expressed as stimulation index (SI: mean OD_450nm in stimulated cultures / mean OD_{450 nm} in unstimulated control cultures).

Human GB cell lines U251, LN229, T98G, and U87 (unknown origin) were purchased from ATCC. U251 and U87 cells were authenticated before by STR profiling. All cell lines were cultured in high glucose phenol red Dulbecco’s Modified Eagle Medium (DMEM, Biowest, FRA) supplemented with 10% Fetal Bovine Serum (FBS, Biowest, FRA), pyruvate 1 mM (InVitro SA, MEX), non-essential amino acids 0.1 mM (InVitro SA, MEX), and 10 mL/L of antibiotic-antimycotic (Catalog number: L0010; Amphotericin B, Penicillin G Sodium Salt, and Streptomycin Sulfate; Biowest, FRA). Cells were maintained in a 5% CO₂ humidified atmosphere at 37 °C. Before steroid treatments, the medium was replaced by DMEM supplemented with charcoal dextran filtered 10% FBS (without steroid hormones) for 24 h. Cells were treated with vehicle (V; 0.1% ethanol), progesterone (P4; 10 nM), and different concentrations of 3α-THP (10, 100 nM, and 1 µM), or as indicated in each section. P4 was purchased from Sigma Aldrich (St. Louis, MO, USA), and 3α-THP was purchased from MP Biomedicals (Santa Ana, CA, USA). To evaluate the involvement of c-Src in the 3α-THP effects on migration and invasion, c-Src was pharmacologically inhibited with pyrazolopyrimidine (PP2, 1 μM) (Sigma Aldrich, MO, USA).

Human blood products used in the in vitro assays (for cell stimulation and stability) were obtained from the center for transfusion medicine in Tübingen, Germany (Zentrum für Klinische Transfusionmedizin Tübingen GmbH, (ethical approval number ZKT-FoPro202106-2305-01). Test compounds (1 µM) in either culture medium (RPMI-1640 medium containing 10% fetal bovine serum, 60 mg/l Penicillin G sodium salt and 100 mg/l Streptomycin sulfate (all Biowest)), human blood (diluted 1:1 with culture medium) or a suspension of 5x10⁶ cells/ml U937 in culture medium were incubated at 37°C, 450 rpm on a shaking incubator. At the indicated time points 50 µL of blood, cell suspension or medium were collected and prepared for HPLC-MS/MS-Analysis as detailed below.