Nahco3

Primary splenocyte cultures were prepared as previously described (Avitsur et al., 2001 (link); Kinsey et al., 2007 (link)) and were used to assess the ability of prolonged HIV-1 Tat and morphine to alter glucocorticoid-induced inhibition of peripheral immune cell viability. Briefly, mice were sacrificed 24 h after administration of the final dose of morphine (or vehicle) and spleens were harvested. Tissues were passed through a 70 μm pore nylon mesh (Greiner Bio-One) with 10 mL of RPMI media (#22400089, Life Technologies) to prepare a cell suspension. Cells were lysed with a hypotonic salt solution (0.16 M NH₄Cl, 10 mM KHCO₃, 0.13 mM EDTA) and suspended in RPMI media (#22400089, Life Technologies), supplemented with 10% heat-inactivated fetal bovine serum (FBS; #SH30071.03, Thermo Scientific), 7.5% NaHCO₃ (#25080094, Life Technologies), and antibiotic/antimycotic mixture (Life Technologies), centrifuged, and filtered through a 70 μm pore nylon mesh (Greiner Bio-One). Duplicate splenocyte samples were seeded onto 96-well plates with corticosterone and stimulated with LPS for 48 h.

The cell lines used in this study were human breast cancer cell lines MDA-MB-231, MDA-MB-361, HDQ-P1, MCF-7 and pancreatic ductal adenocarcinoma MIA-PaCa-2 (details in Supplementary Data 2). All breast cancer cell lines were purchased from ATCC and MIA-PaCa-2 was a generous gift from Professor Channing Der (University of North Carolina at Chapel Hill, NC, USA). All cells were maintained in DMEM with 2.2 g/L NaHCO₃ (Life Technologies) and 10% FBS at 37 °C with 5% CO₂ in a humidified incubator, according to provider’s instruction. The identity of all the cell lines were authenticated using the GenePrint 10 System (Promega) and were tested negative to be mycoplasma free. Mycoplasma test was based on the method described by Vojdani A et al.^{36 (link)} and was performed as a service by the sample management laboratory of THL Biobank, Helsinki, Finland.

3D7 line of P. falciparum was used for all experiments. Parasites were cultured at 2.5% hematocrit in Malaria Culture Medium (MCM): RPMI-HEPES supplemented with hypoxanthine (50 μg mL⁻¹, Life technologies), NaHCO₃ (25 mM), gentamicin (2.5 μg mL⁻¹, Life technologies), and Albumax II (0.5% wt/vol, Life technologies). To obtain synchronous parasites, schizont stage parasites (46–48 hpi) were collected through magnetic-activated cell sorting (MACS, Miltenyi Biotec, Singapore) and incubated with fresh RBCs for 3 hours, followed by treatment with 5% sorbitol (Sigma-Aldrich) to select ring-stage infections.

Chemicals and oligonucleotide primers were purchased from Sigma-Aldrich, unless stated otherwise. All reagents/buffers were prepared in RNase-free water and filter sterilised at 0.2 µm. Plasmid pRPS6-GFP was a kind gift of Prof F. Loreni^{40 (link)}, pGL3 was purchased from Invitrogen (#E1741). HeLaS3 and C8-D1A cells were purchased from ATCC, and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 5% fetal bovine serum (FBS). Simian VK219 cells (African green monkey, Chlorocebus aethiops, kidney epithelial cells latently infected with KSHV and expressing GFP from the EF-1α promoter, as a marker of latent infection^{38 (link)}) were cultured in Minimum Essential Medium Eagle (Sigma #M2279) supplemented with 10% FBS (Gibco), 2.2 g/L NaHCO₃, 5 μg/ml puromycin, and 10,000 U/ml penicillin-streptomycin (Life Technologies). The cells were grown in dishes in a humidified incubator at 37 °C in 5% CO₂. Saccharomyces cerevisiae strain BY4741 (MATahis3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was grown in yeast-peptone-dextrose (YPD) media at 30 °C.