Hepes

Gallium(III) meso-tetraphenylporphyrin chloride (GaTP) was purchased from Frontier Scientific (Logan, UT). THP-1 cells were purchased from ATCC. Difco Middlebrook 7H9 broth and BBL Middlebrook oleic acid-albumin-dextrose-catalase (OADC) enrichment medium were purchased from BD (Sparks, MD, USA). 7H10 agar plates were purchased from Remel Inc. (Lenexa, KS). HyClone RPMI 1640, Dulbecco modified Eagle medium (DMEM), l-glutamine, HEPES, sodium pyruvate, and fetal bovine serum were purchased from GE Life Sciences (Logan, UT). Macrophage colony-stimulating factor (MCSF) was purchased from BioLegend (San Diego, CA). Fe-free 7H9 medium was prepared as described previously (11 (link)). Human monocytes that had been purified by countercurrent centrifugal elutriation from normal human donors were purchased from the UNMC Elutriation core facility using an institutional IRB-approved protocol. HIV-1_ADA was obtained from UNMC, Omaha, and its potency was tested by reverse transcriptase (RT) assay before infection. H37Rv was obtained from ATCC and grown in a biosafety level 3 (BSL3) lab, and its potency was tested by counting CFU before infection.

16HBE14o- (16HBE) cells were a gift from Dieter Gruenert (Mt Zion, Cancer Center, San Francisco, CA, USA). The cells were cultured in MEM medium (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS; Life Technologies), 2 mM L-glutamine (Invitrogen), 10 mM HEPES (GE Healthcare) and 1% (v/v) Minimal Essential Amino Acids (GE Healthcare) in fibronectin-coated flasks (Corning) at 37 °C and 5% CO₂ atmosphere.

Male and female neonatal rat pups from independent litters (n = 3 pups/sex/treatment) were used for transcriptome profiling and qRT-PCR analysis. Prefrontal cortex tissues were identified and dissected under a Nikon SMZ18 Stereo Microscope (Nikon, Tokyo, Japan) as previously described with slight modifications [177 ]. According to the protocol by Guo et al. [178 (link)], rat pups (postnatal days (PNDs) 1–2) were deeply anesthetized by intraperitoneal injection of 100 mg/kg·BW sodium pentobarbital and euthanized by decapitation. The brain was quickly and carefully removed from the skull and placed in a prechilled cell culture dish containing ice-cold, freshly prepared 1× HBSS (Invitrogen, Waltham, MA, USA) containing 30 mM glucose (Sigma-Aldrich, Saint Louis, MO, USA), 2 mM HEPES (GE Healthcare Bio-Sciences, Piscataway, NJ, USA), and 26 mM NaHCO₃ (Sigma-Aldrich, Saint Louis, MO, USA). The meninges were completely removed. The prefrontal cortex was dissected and immediately placed in a tube with RNA stabilization reagent (RNAlater) (Ambion, Austin, TX, USA) and stored at −80 °C according to the manufacturer’s protocol until use.

Epithelial cells were plated in 0.4 μm 24-well plate Matrigel-coated transwell inserts (Corning Life Sciences, Tewksbury, MA) and grown in defined media consisting of DMEM/F12 (Thermo Fisher) supplemented with NuSerum (Corning Life Sciences), Hyclone Defined FBS (GE Life Sciences, Logan, UT), Penicillin–Streptomycin (Thermo Fisher), l-Glutamine (Thermo Fisher) and HEPES (GE Life Sciences) as previously described by us^{30 (link),31} . Growth on transwell inserts allows for the formation of distinct apical and basolateral compartments^{30 (link),31} . Cells were grown to confluence and allowed to polarize as determined by transepithelial resistance (TER) of greater than 1000 ohms per insert^{30 (link),31} . TER was measured using an EVOM electrode and Voltohmmeter (World Precision Instruments, Sarasota, FL). Only polarized preparations of epithelial cells above 1000 ohms were used in this study. 24 h before progestin treatment, cells were transferred to stripped media.

Fourteen patients carrying STAT1 GOF mutations were enrolled (2014–2017) on approved NIH protocols and provided written informed consent. Healthy donor blood samples were obtained under approved protocols through the Department of Transfusion Medicine, Clinical Center, NIH.
Patient and healthy donor peripheral blood mononuclear cells (PBMC) were isolated by density-gradient centrifugation using lymphocyte separation media (Lonza). PBMC were re-suspended in RPMI culture media (Gibco), supplemented with pyruvate (100 mM, Sigma Aldrich), glutamate (200 mM, Life Technologies), penicillin/streptomycin (100 U/100 μg/ml, Life Technologies), 10% fetal bovine serum (Serum Source International), and 20 mM HEPES (GE).

The EO771 murine epithelial-like carcinoma cell line (ATCC #CRL-3461; American Type Culture Collection, Manassas, VA, USA) was grown in DMEM culture medium containing 4.5 g/L D-glucose, supplemented with 10% fetal bovine serum (FBS, Hyclone, Pittsburgh, PA, USA), 100 units/mL of penicillin and 100 µg/mL of streptomycin and 2% of HEPES (GE Healthcare, Pittsburgh, PA, USA). For experiments and sub-culturing, cells were rinsed with PBS and detached from T75 flasks by incubating with 0.25% (w/v) trypsin 0.53 mM EDTA for 2 min at 37 °C followed by resuspension in culture medium.