Phenol red

For testosterone treatment, we used RPMI 1640 Medium, without L-glutamine, without phenol red (Capricorn) supplemented with 1% GlutaMAX™ Supplement (Gibco), 1% Pen Strep (Gibco) and 5% charcoal stripped FBS Standard (PAN Biotech). For charcoal stripping of FBS, 2 g dextran-coated charcoal (DCC, Merck) were added to 100 ml FBS and incubated on a shaker overnight at 4 °C followed by centrifugation and filtration. Cryopreserved PBMCs from male healthy individuals were thawed and seeded at a density of 2 × 10⁵ cells per well in an anti-CD3 (10 µg/ml, clone OKT3, BioLegend) coated 96-well plate and supplemented with soluble anti-CD28 (2.5 µg/ml, clone CD28.2, BioLegend) or left unstimulated. Subsequently, testosterone (3, 30 and 300 ng/ml, Sigma Aldrich) or 5α-dihydrotestosterone (0.3, 3 and 30 ng/ml, SigmaAldrich) was added. Samples were incubated for 48 h at 37 °C and 5% CO₂, stained with antibodies listed in Table S4 and analyzed by flow cytometry after 2, 24 and 48 h.

Hanks’ Balanced Salt Solution (HBSS), no calcium, no magnesium, no phenol red, was obtained from Capricorn Scientific GmbH (Germany). All materials related to flow cytometry, such as focusing fluid, shutdown and wash solutions, Attune™ performance tracking beads, and PE-Annexin V, were obtained from Thermo Fisher Scientific.

For both in vitro and in vivo studies, the 4T1 murine mammary carcinoma cell line, provided by Judy Lieberman (Lieberman Laboratory, Harvard University, Boston, MA, USA), was used. The 4T1 cells were grown as adherent culture in Dulbecco’s Modified Eagle Medium (DMEM high glucose, 4.5 g/L without lglutamine and Phenol Red, Capricorn Scientific, Ebsdorfergrund, Germany, Cat-No. DMEM-HXRXA) supplemented with 10% Fetal Bovine Serum (FBS—South America Origen, EU approved, EuroClone S.p.A., Pero, Italy, CatNo. ECS0180L), l-glutamine 200 mM (Capricorn Scientific, Ebsdorfergrund, Germany, Cat-No. GLN-B), and penicillin/streptomycin 100x (Capricorn Scientific, Ebsdorfergrund, Germany, Cat-No. PS-B). Cells were submitted to passages every 2 or 3 days. Trypsin 10x (Lonza A. G., Basel, Switzerland, Cat-No. 17-160E) was used to release cells from sub-confluent monolayers. The detached cells were seeded back into cell culture flasks or prepared for experiments. KRIBB11 was purchased from MedChemExpress (Monmouth Junction, NJ, USA, Cat-No. HY-100872).