*2.4. RT-qPCR*

Total RNA was isolated from the cells using Qiazol reagen<sup>t</sup> (QIAGEN, Germantown, AR, USA), and chloroform extraction was followed by purification on RNeasy Mini columns with DNAse digestion step (QIAGEN). cDNA was prepared with a Maxima First Strand cDNA Synthesis Kit for a quantitative real-time polymerase-chain reaction (RT-qPCR) (ThermoFisher Scientific, Waltham, MA, USA), in accordance with the protocol of the manufacturer, from 400–600 ng of total RNA per sample. Q-PCR was performed using the PerfeCTa qPCR FastMix (Quanta Biosciences, Gaithersburg, MD, USA) in replicates and cycled in a StepOnePlus ™ Real-Time PCR cycler (ThermoFisher Scientific, Waltham, MA, USA). The following TaqMan® Gene Expression Assays

(Applied Biosystems®, ThermoFisher Scientific, Waltham, MA, USA) were used: *IFNβ* (Hs01077958\_s1), *TNF* (Hs00174128\_m1), *TBP* (Hs00427620\_m1), *TIRAP* (Hs00364644\_m1), *IL-6* (Hs00985639\_m1), *IL-1β* (Hs01555410\_m1), *IL-12A* (Hs01073447\_m1), and *IL-12B* (Hs01011518\_m1). The level of *TBP* mRNA was used for normalization and the results presented as a relative expression compared to the control's untreated sample. Relative expression was calculated using Pfaffl's mathematical model [25]. Graphs and statistical analyses were made with GraphPad Prism v9.1.2 (Dotmatics, Bishops Stortford, UK), with additional details provided in the figure legends and statistics paragraph (Section 2.11).

### *2.5. ELISA and BioPlex Assays*

TNF level in supernatants of human macrophages was determined using human TNF-alpha DuoSet ELISA (DY210-05) (R&D Systems, Minneapolis, MN, USA), IFNβ level—using VeriKine-HSTM Human Interferon-Beta Serum ELISA Kit from PBL Assay Science (Piscataway, NJ, USA). Other cytokines (IL-12p70, IL-6, MCP-1, IL-8) were analyzed using BioPlex cytokine assays from Bio-Rad, in accordance with the instructions of the manufacturer, using the Bio-Plex Pro ™ Reagent Kit III and Bio-Plex ™ 200 System (Bio-Rad, Hercules, CA, USA).

### *2.6. Cell Fractionation*

Stimulated human primary monocytes were detached by accutase treatment and collected by centrifugation. Cell pellets were washed by once by PBS with 2% FCS. For preparation of total lysate, a cell pellet from one of the wells per condition was collected and lysed by RIPA lysis buffer (150 mM NaCl, 50 mM TrisHCl (pH7.5), 1% Triton X100, 5 mM EDTA, protease inhibitors, phosphatase inhibitors). The remaining cells were resuspended in Buffer A (50 mM NaCl, 10 mM HEPES pH = 8, 500 mM sucrose, 1 mM EDTA, 0.2% Triton-X100), and the samples were vortexed and centrifuged (5000 rpm, 5 min, 4 ◦C). Cytosolic fraction in the supernatant was transferred to clean tubes. Pellets with nuclei were washed with Buffer B (50 mM NaCl, 10 mM HEPES (pH = 8), 25% glycerol, 0.1 mM EDTA), and centrifuged, the supernatants were discarded, and the pellets were resuspended in Buffer C (350 mM NaCl, 10 mM HEPES (pH = 8), 25% glycerol, 0.1 mM EDTA) with added Benzonaze endonuclease (Merck) and incubated on ice for 30 min, followed by centrifugation (15,000 rpm, 15 min, 4 ◦C) to extract nuclear proteins.

### *2.7. Western Blotting*

Cell lysates for pSTAT1 analysis were prepared by simultaneous extraction of proteins and total RNA using Qiazol reagen<sup>t</sup> (QIAGEN, Germantown, AR, USA), as suggested by the manufacturer. Extracted total RNA was used for RT-qPCR, while protein samples were used for simultaneous analysis of protein expression/post-translational modifications. Protein pellets were dissolved by heating the samples for 10 min at 95 ◦C in a buffer containing 4 M urea, 1% SDS (Sigma, Merck, Darmstadt, Germany), and NuPAGE® LDS Sample Buffer (4X) (Thermo Fisher Scientific, Waltham, MA, USA), with a final 25 mM DTT in the samples. Otherwise, lysates were made using 1X RIPA lysis buffer. For traditional Western blot analysis, we used pre-cast protein gels NuPAGE ™ Novex ™. Proteins were transferred to iBlot Transfer Stacks by using the iBlot Gel Transfer Device (ThermoFisher Scientific, Waltham, MA, USA). The blots were developed with the SuperSignal West Femto (ThermoFisher Scientific) and visualized with the LI-COR ODYSSEY Fc Imaging System (LI-COR Biotechnology, Lincoln, NE, USA). For densitometry analysis of the bands, Odyssey Image Studio 5.2 software (LI-COR Biotechnology, Lincoln, NE, USA) was used, and the relative numbers of bands' intensity were normalized to the intensities of the respective loading-control protein (GAPDH, or PCNA, or β-tubulin). Loading-controlprotein expression was always performed on the same membrane as the protein of interest.
