Phorbol myristate acetate (pma)

Briefly, mouse spleen and draining lymph node (dLN) and kidney grinded to cell suspension resuspended in a red blood cell lysing buffer at 37°C for 2 min, followed by passing through a 70 mm cell strainer and centrifuged.
For intracellular IL-17A staining, cells were treated with 5 ng/ml PMA (Invitrogen) and 1 ng/ml ionomycin (Enzo Life Sciences, Inc.) for 5 h and then added at 10 ng/ml brefeldin A (Enzo Life Sciences, Inc.) 30 min before staining with CD4- FITC (eBiosciences, San Diego, United States). Next cells were stained with IL-17A-PE (eBiosciences) followed by permeabilization of the cells with Cytofix/Cytoperm (BD Biosciences, San Diego, United States).
For intracellular Foxp3-PE staining (Pan et al., 2014 (link)), cells were permeabilized with Cytofix/Cytoperm, and stained with Foxp3-PE (eBiosciences), followed by stain with CD4-FITC and CD25-APC (eBiosciences). Cells were detected by FACS Calibur Flow Cytometer (Becton Dickinson, San Diego, United States) and analyzed with the Flow Jo software.

Human THP-1 monocytes were plated at 5×10⁵ cells/well in six-well culture dishes (Becton Dickinson) containing 10% fetal calf serum (FCS)-RPMI-1640 supplemented with PEST. The cells were incubated with PMA (50 ng/mL) (Sigma-Aldrich) for 72 hours to induce differentiation into macrophages and with Ac-LDL (50 µg/mL) for 48 hours to generate foam cells. Thereafter, for each master regulator, cells were transfected with siRNA (one at a time) (Ambion, Life Technologies, Table S17) using Lipofectamine 2000 as recommended by the manufacturer (Invitrogen), in medium without FCS, PEST, or PMA. Forty-eight hours after siRNA transfection, cells were examined for effects on the expression of network genes (see section Gene Expression Measurements and Hypergeometric Testing for Network Specificity) and CE accumulation (see section Lipid and Protein Measurements).
Ac-LDL was prepared as described [58] (link). The samples were then dialyzed against PBS at 4°C. Ac-LDL protein concentration was determined by the Bradford method. LDL was isolated from plasma of healthy donors by sequential ultracentrifugation [59] (link).

A variety of activating conditions were assessed for their capacity to induce citrullination. Cells were activated with a range of doses of either ionomycin (Invitrogen) for different times at 37°C, 100 nM PMA (Invitrogen) for 1 h or 4 h at 37°C, or 0.005% Triton-X for up to 1 h at 37°C. We chose 100 nM of PMA as this concentration is typically used as a standard concentration for inducing neutrophil extracellular trap formation (NETosis). The sublytic concentration of perforin (Enzo Life Sciences) was determined to be 500 ng/mL for human neutrophils in a previous study [10 (link)]. We used 150 ng/mL, 250 ng/mL, and 500 ng/mL to treat neutrophils at 37°C for various times (15 min, 30 min, 1 h, and 4 h). All reactions were stopped by adding lithium dodecyl sulfate (LDS) sample buffer (Invitrogen) and boiling. For controls, neutrophils were either immediately boiled in LDS sample buffer after isolation (time 0 control) or incubated for different times at 37°C before boiling in LDS sample buffer.

The mouse cell lines RAW 264.7 (ATCC® TIB-71™), L929 (ATCC® CCL-1™), GC-1(ATCC® CRL-2053™), GC-2(ATCC® CRL-2196™), NIH3T3 (ATCC® CRL-6442), 3T3-L1(ATCC® CL-173™), the human cell line THP-1 (ATCC® TIB-202), HeLa (ATCC® CCL-2) and HEK293T (ATCC® CRL-11268) and the insect cell line Sf 9 (ATCC^® CRL-1711^™) were purchased from ATCC. THP-1 cells were differentiated using PMA (Sigma-Aldrich, Dorest, UK) at a final concentration of 100 ng/mL and incubated for 48 h. THP-1 cell differentiation was enhanced by removing the PMA-containing media and adding fresh media for a further 24 h. Mycoplasma contamination was assessed using a MycoFluor™ Mycoplasma Detection Kit (M7006, Invitrogen™, USA), and cells used for the experiments were free of mycoplasma contamination. All experiments were three independent replicates.

Human liver cell line HL7702 and human liver carcinoma cell line HepG2 were cultured in RPMI 1640 medium (Hyclone, Logan, UT, USA) with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 mg/ml streptomycin medium (Hyclone), and incubated in a humidified incubator containing 5% CO2 at 37°C. Human monocyte cell line THP1 was stimulate with phorbol myristate acetate (PMA) (0.1μM, Invitrogen, Carlsbad, CA) for 3 hours, and then washed twice to remove PMA and seeded on 12-well plates (1×10⁶ cells/well) for 24 hours to generate polarized macrophages (Supplementary Figure 1). Sorafenib (Nexavar, BAY 43-9006, Germany) or DMSO (Sigma-Aldrich, St. Louis, MO, USA) carrier (mock) was added to cell culture for 3 hours. Treatment was followed by a medium exchange and stimulate with LPS (1ng/mlSigma-Aldrich, St. Louis, MO) for 24 hours. The supernatant of polarized macrophage was collected and centrifugated at 15,294×g for 15 min to remove cell debris.

Antibodies for detecting citrullinated histone H3 (R2 citrullination, Ab176843 and R2-8-17 citrullination, Ab5103), total histone H3 (Ab24834), NCF1, NCF2, and NCF4 were from Abcam (Cambridge, MA). Antibodies for detecting PAD4 (MABE254) and citrullination (F95) were from EMD Millipore (Billerica, MA). Antibodies for PAD IP were developed as previously described^{40 (link)}. Recombinant human PAD2 and recombinant human PAD4 were generated in-house. Recombinant human histone H3, pan PAD inhibitor BB-Cl-amidine, and PAD4 inhibitor GSK484 were from Cayman Chemicals (Ann Arbor, MI). The cell fraction isolation kit was from Cell Signaling (Danvers, MA). Protein G beads, DPI, ionomycin, PMA, Hoechst 33342, HRP-labeled anti-mouse immunoglobulin G (IgG), and anti-rabbit IgG secondary antibodies were from Invitrogen (Carlsbad, CA).