Penicillin streptomycin

Mouse N9-microglial cells were cultured in Dulbecco modified eagle medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin and 2 mM glutamine (all cell cultures’ reagents were from Aurogene). At confluence, after a short wash with sterile phosphate-buffered saline (PBS), microglia were trypsinized for 5 min at 37 °C and trypsin was inactivated with complete DMEM medium. Detached cells were than collected, centrifuged for 5 min at 300× g and resuspended to be counted. For the immunomodulation assay, N9-microglial cells were plated at the density of 2.5 × 10⁵ cells in 35 mm dish and exposed to 100 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich), in presence or absence of the compound to be tested at 10 μM for 24 h. After treatment, microglial cells were lysed in ice-cold lysis buffer (50 mM Tris-HCl pH 7.4, 1% SDS, 0.05% protease inhibitor cocktail; all from Sigma-Aldrich), protein content was determined by using the Lowry method and samples were loaded for western blot analysis of iNOS (M1 microglia marker), TREM2 (M2 microglia marker), IL-1β (M1 microglia marker), BDNF (neurotrophic factor) and GAPDH (loading control) expression.

MEG-01 (ATCC^® CRL-2021) cells were grown in a humidified incubator at 37 °C, and 5% CO₂ in RPMI-1640 (Gibco^®, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) fetal serum bovine (FBS, Gibco^®, Thermo Fisher Scientific, Waltham, MA, USA), 1% (v/v) penicillin-streptomycin (Aurogene, Rome, Italy), 1% (v/v) MEM non-essential amino acids (MEM NEAA, Gibco^®, Thermo Fisher Scientific, Waltham, MA, USA), and 1% (v/v) sodium pyruvate (Aurogene, Rome, Italy). Differentiated MEG-01 cells were obtained upon 3 days of pretreatment with phorbol 12-myristate 13-acetate 5nM and thrompoietin 100 ng/mL. Both naive and differentiated MEG-01 cells were exposed to heat-inactivated SARS-CoV-2 lysate (5 μg/mL) for up to 24 h. All the results were compared to the effects of cells exposed to control lysate (5 μg/mL), here indicated as CTR, obtained from non-infected MEG-01 in the same conditions. At different time points, supernatants and/or cells were collected for subsequent assays.

VK2 E6/E7 cell line (ATCC CRL-2616) was established from the normal vaginal mucosal tissue taken from a premenopausal woman undergoing anterior–posterior vaginal repair surgery. We performed our experiments on a single type of cell line, since, to the best of our knowledge, VK2 E6/E7 is the only healthy vaginal model available on the market. VK2 E6/E7 cells were grown in a Keratinocyte-Serum Free Medium (K-SFM; GIBCO, Carlsbad, CA, USA) with 0.1 ng/mL human recombinant epidermal growth factor (EGF), 0.05 mg/mL bovine pituitary extract, additional calcium chloride 44.1 mg/L (final concentration 0.4 mM), and 1% penicillin/streptomycin (Aurogene, Rome, Italy). Cells were maintained at 37 °C in a saturated humidifying 5% CO₂ incubator. Cells were treated with vehicle (Veh; 0.0025% Tween20) and 25 and 250 µg/mL PE N/MPLs for 48, 72, and 120 h (acute exposure) for 21 days (chronic exposure), and with vehicle and 25 µg/mL PE N/MPLs for 1.5 months (low concentration, extended time chronic exposure). Cells were always sub-cultured at 80% confluence since keratinocytes spontaneously start differentiating at the higher confluence. Optical microscopy pictures of N/MPLs-treated cells were taken at 40× magnification by using EVOS XL Core Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).

PBMC proliferation at each time point was analyzed by evaluating the decrease in carboxyfluorescein succinimidyl ester (CFSE) fluorescence intensity (BD, Cat No. 51-9010817). Cells were thawed and resuspended in 5 mL of PBS with CFSE (2 µM) for 5 min at 37 °C. Reaction was blocked using a medium with 50% Fetal Bovine Serum (FBS, Gibco, Life Technologies, (Carlsbad, CA, USA). Cells were resuspended in culture medium RPMI 1640 (Corning) supplemented with 2 mm L-glutamine (EuroClone, Cat ECB3000D), 1% human serum AB (EuroClone, Cat ECS0219D), 100 U/mL penicillin–streptomycin (Aurogene, Cat AU-L0022-100), 0.1% β-Mercaptoethanol (Gibco, Cat 31350-010), 1% sodium pyruvate (Aurogene, Cat AU-L0642-500), 1% non-essential amino acids (Aurogene, AU-X0557-100). Cells were activated by anti-CD3 (Invitrogen, Cat 16-0039-85) and anti-CD28 (Invitrogen, Cat 16-0289-85) antibodies, and incubated for three days in 96-well plates at 37 °C and 5% CO₂. At the end of the incubation period, PBMCs were collected and stained with anti-CD4 and anti-CD8 antibodies as described in the previous section. CFSE dilution in CD4 and CD8 gates was measured through the Cytoflex S flow cytometer and data were analyzed with the Cytobank software version 2.4.

Human B cell lines derived from KSHV-positive PEL cell lines, BC3, and BCBL-1 (kindly supplied by Prof. P. Monini, National AIDS center, Istituto Superiore di Sanità, Rome, Italy) were grown in RPMI 1640 medium (Sigma-Aldrich, Burlington, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, Burlington, MA, USA), L-glutamine (2 mM) (Aurogene, Rome, Italy), and streptomycin/penicillin (100 μg/mL) (Aurogene, Rome, Italy) (complete medium) at 37 °C in a 5% CO₂ humified setting. Cells were seeded into 6-well plates at a density of 4 × 10⁵ per well in a final volume of 2 mL in complete medium and were treated for 24 h (h) singly or in combinations with the STAT3 inhibitor tyrphostin AG490 (50–100–200 µM) (Calbiochem, San Diego, CA, USA; 658411) and the NFE2L2 inhibitor brusatol (10–20–40 nM) (Sigma Aldrich, St. Louis, MO, USA; 1868). In some experiments, to evaluate the role of HSP90A and HSPB1 in the mechanism/s to which NFE2L2 could sustain STAT3 activity and vice versa, PEL cells were treated for 24 h with inhibitors of HSPB1 and HSP90A, respectively, J2 (10 µM) (MedChemExpress, Monmouth Junction, NJ, USA; HY-124653), 17-AAG (0.1 µM) (Selleckem, Planegg, Germany; S1141). All the drugs were dissolved in DMSO, and the control cells were supplemented with DMSO in the same amount used for the other samples.

MM cell lines SKO-007 (J3) (SKO) and RPMI-8226 (RPMI) and PEL cell lines BC3 and BCBL1 [23 (link)] were maintained in RPMI 1640 medium (Sigma-Aldrich, Burlington, MA, USA), supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, Burlington, MA, USA), L-glutamine (2 mM) (Aurogene, Rome, Italy), streptomycin/penicillin (100 μg/mL) (Aurogene, Rome, Italy) at 37 °C and 5% CO₂ humidified atmosphere. The cells were seeded into 6-well plates at a density of 6 × 10⁵ cells per well in a final volume of 2 mL. Subsequently, the cells were treated for 24 h (h) with c-Myc Inhibitor (I c-Myc) (50 μM) (Sigma-Aldrich, Burlington, MA, USA, 475956) or IRE1 RNAse inhibitor (4µ8c) (20 μM) (Sigma-Aldrich, Burlington, MA, USA, SML0949). Untreated cells were used as a control group (CTRL).