2.1 Cell culture and experimental agents:
Recombinant HIV-1 Tat protein (full-length HIV-1 Tat clade B sequence, isolate BH10, UniProtKB - P69697, TAT_HV1B1) was produced and purified as described in Silveira et al [7]. BV-2 microglial cells were purchase from Banco de Células do Rio de Janeiro (Brazil), and maintained in Dulbecco's Modified Eagle's Medium (Sigma-Aldrich, USA) supplemented with 10% of heat-inactivated fetal bovine serum (Cultilab, Brazil), 1mM sodium pyruvate, 2 mM L-glutamine, at 37°C in 5% CO2. Cells were seeded (5x106) in 12-well plates and cultured until they reached 80-100% of confluency for experiments, when media was replaced by serum-free medium. The IRE1 inhibitor 4μ8C was purchased from Tocris Bioscience (Minneapolis, USA, #4479/50). For experiments targeting IRE1 inhibition, the small-molecule 4μ8C was solubilized according to supplier’s instructions and assayed using a range of different concentrations with or without recombinant Tat, at different time-points. Heat-inactivated recombinant Tat (boiled at 90° C for 1 h) was used as an experimental control of non-stimulation.
2.2 Cell viability and apoptosis assessment:
BV-2 cells were assayed as described above and 100 µL of MTT [3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, Merck KGaA, Germany] solution (5 mg/mL in DMEM without serum) was added to each well. After 4 h incubation, supernatants were removed and 1 mL of dimethyl sulphoxide (Sigma-Aldrich, USA) was added to each well. Levels of MTT reduction were spectrophotometrically measured at 540 nm (SpectraMax Paradigm Multi-mode Microplate Reader, Molecular Devices, LLC, USA). In addition, the apoptotic profile of BV-2 cells was assessed by FITC Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend, USA). Apoptotic cells were analyzed in FACSCanto II flow cytometer (Becton & Dickinson, USA) and FlowJo-VX software. Double-stained cells (late apoptotic events) and Annexin V-stained cells (early apoptotic events) were considered.
2.3 Measurement of CD16/32 expression.
BV-2 cells were assayed as described, detached from plates after 48h and labelled with FITC anti-CD16/32 (Biolegend, USA, #101305, 1 µg/106 cells, 40 min at 4° C). Analyses were performed in BD FACSCanto II flow cytometer (Becton & Dickinson, USA) and FlowJo-VX software.
2.4 Cytometric bead array and NO dosage.
BV-2 cells were assayed as described above and supernatants collected after 48 h to assess inflammatory-related molecules using the CBA Mouse Inflammation kit (BD Pharmingen, USA) according to the supplier´s protocol. For NO dosage, supernatants were processed as previously described [7]. In brief, supernatants were mixed (1:1) with 0.1% naphthyl ethylenediamine and 1% sulfanilamide in 5% phosphoric acid solution (Sigma-Aldrich, USA) for 5 min at room temperature in the dark. Absorbance was then measured at 540 nm using a spectrophotometer (SpectraMax Paradigm Multi-mode Microplate Reader, Molecular Devices, LLC, USA).
2.5 Western blotting analysis:
BV-2 cells were assayed as described above and lysed after 48 h for western blot. Total protein concentrations were quantified by Bradford assay (Bio-Rad, USA). Samples were loaded (50 µg) and separated in 15% SDS-PAGE gel, then transferred (3 h at 200 mA) onto a nitrocellulose filter membrane (Bio-Rad, USA). The membranes were incubated at room temperature with 5% nonfat milk for 1 h, followed by incubation with primary anti-XBP-1 (Abcam, USA, #ab37152, 1:500), anti-eIF2α (Santa Cruz Biotechnology, USA, #sc-133132, 1:500), anti-p-eIF2α (Thermo Fischer, USA, #44-728G, 1:1000) or anti-β-actin (Abcam, USA, #ab8227, 1:5000). Membranes were then incubated with goat anti-rabbit (Sigma-Aldrich, USA, #A0545, 1:5000) or goat anti-mouse (Sigma-Aldrich, USA, #A9309, 1:1000) secondary antibodies in skimmed milk for 1 h at room temperature. ECL Western blot substrates (Pierce, Thermo Fisher Scientific, USA) were used to identify immunoreactive bands and analyzed using ImageJ software (http://rsbweb.nih.gov/ij/).
2.6 Quantitative real‑time PCR:
Total RNA was isolated from cells using a RNeasy Mini Kit (Qiagen, USA) and qRT-PCR was performed on a StepOne Real-Time PCR System (Applied Biosystems, Life Technologies, USA) using a FastSYBR® Green PCR Master Mix (Applied Biosystems, Life Technologies, USA). Reactions were performed in triplicate and the relative expression ratios calculated. Primer sequences were used as previously published [7].
2.7 Statistical analysis:
Statistical significance was evaluated using Student’s t test when considering differences between two experimental groups, or by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test in case of multiple groups. Statistical significance level was determined at p < 0.05.