Tib 202

THP-1 cells (ATCC® TIB-202) were maintained as suspension cultures in RPMI 1640 media, supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, 1 mg/ml streptomycin, 0.25 μg/ml amphotericin B, 1% ml of 100× non-essential amino acids (Gibco Thermo Fisher), 10 mM HEPES buffer, 1 mM sodium pyruvate, and 2 mM glutamine. Differentiation of THP-1 cells into monolayers of macrophage-like cells was achieved by seeding the cells on plates and stimulating for 24 h with 100 nM phorbol-12-myristate-13-acetate (PMA; EMD Chemicals). THP-1 cells stably expressing an shRNA directed against ASC (47 (link)) or stably expressing a YFP-ASC fusion protein were maintained in RPMI 1640 media, supplemented as above. HEK293 cells (ATCC) and HEK293 stably expressing TLR4-MyD 88 signaling along with YFP-ASC (69 (link)) (Gavrilin et al., personal communication) were maintained in DMEM containing 10% FBS, 100 IU/ml penicillin, 1 mg/ml streptomycin, and 0.25 μg/ml amphotericin B.

Uninfected and A. phagocytophilum NCH-1 strain–infected human promyelocytic HL-60 cells (CCL-240; American Type Culture Collections [ATCC]) and RF/6A rhesus monkey choroidal endothelial cells (CRL-1780; ATCC) were cultured as previously described (Huang et al, 2010a (link)). HeLa human cervical epithelial cells (CCL-2; ATCC) were maintained as described (Justis et al, 2017 (link)). C. burnetii Nine Mile Phase II (NMII; clone 4, RSA439) and mCherry-C. burnetii NMII were purified from Vero cells (African green monkey kidney epithelial cells; CCL-81; ATCC) or acidified citrate cysteine medium-2 (ACCM-2) and stored as described (Cockrell et al, 2008 (link); Beare et al, 2009 (link)). Mouse alveolar macrophages (MH-S; CRL-2019; ATCC) and THP-1 human monocytic cells (TIB-202; ATCC) were maintained as described (Mulye et al, 2018 (link)). THP-1 cells were differentiated into macrophage-like cells by overnight treatment with 200 nM phorbol 12-myrisate 13-acetate (MilliporeSigma). C. trachomatis serovar L2 (LGV 434) was maintained in HeLa cells at 37°C as described (Rucks et al, 2017 (link)). C. pneumoniae AR39 was maintained in HeLa cells at 35°C as described (Ouellette et al, 2016 (link)). Mammalian cell cultures were low passage and confirmed to be mycoplasma free using the Universal Mycoplasma Detection kit (ATCC) or Mycoplasma PCR Detection kit (MilliporeSigma).

The human hepatocellular carcinoma cell line HepG2 (ATCC^® HB-8065™), the colorectal adenocarcinoma cell line HT-29 (ATCC^® HTB-38^TM), and the acute monocytic leukemia cell line THP-1 (ATCC^® TIB-202™) were cultured in Roswell Park Memorial Institute 1640 media (RPMI; ThermoFisher Scientific, Waltham, MA, USA) with 10% (v/v) fetal calf serum (FCS, Sigma-Aldrich, Steinheim, Germany), 1% (v/v) penicillin-streptomycin (ThermoFisher Scientific), and 1% GlutaMAX (ThermoFisher Scientific) at 37 °C in a 5% CO₂ atmosphere. For experiments, the cells were seeded for 24 h and accordingly were treated with or without 1 mM NAC (Sigma-Aldrich) dissolved in culture medium without FCS. In parallel, copper sulfate (CuSO₄) (Sigma-Aldrich/Merck) and zinc sulfate (ZnSO₄) (Sigma-Aldrich/Merck) were added in a final concentration of 50 µM for 6 h. The concentrations of Cu and Zn were chosen based on previous experiments [17 (link)]. Cells were harvest 30 h after seeding.

M. tuberculosis laboratory strains H37Ra (ATCC 25177) and H37Rv (ATCC 27294) were used in the HFS-TB experiments. Culture conditions for stock bacterial strains and the human-derived THP-1 cells (ATCC TIB-202) were the same as reported in our previous publications.^10–12 (link) Study drugs were purchased from Sigma−Aldrich (St Louis, MO, USA), Baylor Medical Center campus pharmacy and BOC Sciences (NY, USA). The mycobacterial growth indicator tube (MGIT) system and related supplies were purchased from Becton Dickinson (Franklin Lakes, NJ, USA).

Cell lines used in the study were human acute monocytic leukemia cell line THP-1 (ATCC^® TIB-202), normal human skin fibroblast CCD-1070Sk (ATCC^® CRL-2091, ATCC, Manassas, VA, USA.), and a spontaneously transformed aneuploid immortal keratinocyte cell line from adult human skin HaCaT (330493, CLS Cell Lines Service GmbH, Eppelheim, Germany). All cell lines were grown in DMEM medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal calf serum (FCS) (GIBCO, Grand Island, NY, USA) and antibiotic antimycotic solution (ATB) (Sigma-Aldrich, St. Louis, MO, USA).

To investigate the effect of transfection on osteoclast differentiation, a coculture of PDLF and human macrophages (THP‑1; TIB-202; ATCC; Manassas, VA, USA) was established. THP‑1 monocytes were cultured in RPMI (61870-010, Gibco™, Carlsbad, CA, USA) supplemented with 20% FBS (P30-3302; PAN-Biontech, Aidenbach, Germany) and 1% antibiotics/antimycotic (A5955, Sigma-Aldrich, St. Louis, MO, USA). First, suspension THP‑1 monocytes were differentiated to adherent macrophages by addition of 25 ng/ml phorbol 12-myristate 13-acetate (PMA; 19-144, Sigma-Aldrich, St. Louis, MO, USA) for 3 days. PDLFs were seeded in 24-well plates (662 160; Greiner Bio-One, Frickenhausen, Germany) according to the experimental setup for transfection and compressed with 6 g/cm² for 6 h (supplemental Fig. 1c). After the compression period, we checked, whether the suspension THP‑1 monocytes were differentiated into adherent macrophages under the microscope. The macrophages were scraped off using a cell scraper (83.3951, Sarstedt, Nümbrecht, Germany) and 50,000 macrophages per well of the 24-well plate were added to the previously compressed PDLFs and incubated at 37 °C for an additional 3 days. Finally, TRAP staining was performed.