Raw264.7 mouse macrophage

RAW264.7 mouse macrophages were purchased from the American Type Culture Collection (Rockville, MD, USA). The cells were cultured in DMEM (Cellgro, Manassas, VA, USA) supplemented with 10% FBS, 1% penicillin/streptomycin (Invitrogen Co., Grand Island, NY, USA), and 4 mM l-glutamine in an atmosphere of 5% CO₂ at 37 °C. Cell viability was determined with an Ez-Cytox cell viability detection kit. When the cells were approximately 80% confluent, they were seeded in 96-well culture plates at 1 × 10⁵ cells per well and incubated for 24 h for adhesion. Then cells were treated with the control (0.5% DMSO) or with indicated concentrations of the EtOH extract of P. baumii, its fractions, and isolated compounds 1–8. After cells had been incubated with these treatments for 24 h, 10 μL of Ez-Cytox reagent was added to each well and incubated for 2 h. Cell viability was determined from the absorbance at 450 nm measured with a microplate reader (PowerWave XS; Bio-Tek Instruments, Winooski, VT, USA).

RAW 264.7 mouse macrophages were obtained from the American Type Culture Collection (Bethesda, MD, USA) and cultured in DMEM (containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin) in a 37 °C incubator with 5% CO₂. Cells in 48-well plates were treated with IPA (5, 10, 20, and 50 μM) for 24 h. Next, an MTT solution was added, followed by incubation for 30 min, and formazan crystals were solubilized by adding DMSO. The absorbance at 550 nm was measured using a BioTek Synergy HT microplate reader (BioTek Instruments, Winooski, VT, USA). Medium was collected for lactate dehydrogenase assay, and the absorbance at 490 nm was measured using a BioTek Synergy HT microplate reader (BioTek Instruments). Cell viability (%) and cytotoxicity (fold-change) were quantified based on the absorbance of treated cells relative to the control cells (exposed to DMSO alone).

The green fluorescent protein (GFP)-tagged H2B [28 (link)] was transduced into RAW264.7 mouse macrophages (American Type Culture Collection, Manassas, VA, USA) using pLenti-EF1a-Puro bearing a GFP-tagged H2B complementary DNA [27 (link)]. Preparation of lentiviral particles and transduction of target cells were performed as previously described [27 (link)]. To maintain GFP-H2B-positive cells, growth medium was supplemented with 0.5 μg/ml puromycin. To observe RAW264.7/GFP-H2B cell migration, cells were plated at 2 × 10⁴ cells/well of an eight-well Lab-Tek II chambered coverglass (Thermo Fisher Scientific, Waltham, MA, USA) in growth medium for 2 days before stimulating cell migration with CM. Fluorescence was visualized with an LSM710 confocal microscope equipped with a temperature- and CO₂-controlled chamber [29 (link), 30 (link)]. Before cell migration was analyzed, cells were rinsed twice and maintained in 400 μl of serum-free DMEM for 2 h. The action of RAW264.7/GFP-H2B cells was monitored at 5-minute intervals for more than 8 h. Cell migration was evaluated using time-lapse images with Imaris software (Bitplane, South Windsor, CT, USA).

Murine hematopoietic stem cells were isolated from the tibias and femurs of 6- to 15-week-old C57BL/6 mice. For differentiation into BMDMs, DMEM (Dulbecco’s modified Eagle’s medium [Gibco]) was supplemented with 10% fetal bovine serum (FBS) and 20% L929-conditioned medium for 7 days. The concentration of L929-conditioned medium was reduced to 10% before infections. HeLa cells obtained from Michael S. Diamond (Washington University School of Medicine) were grown in a mixture of DMEM, 2 mM l-glutamine, and 10% heat-inactivated fetal bovine serum (hiFBS [Invitrogen]). Penicillin/streptomycin (Gibco) was added for passaging of cells and excluded during infection of BMDMs. U2OS human osteosarcoma cells and RAW264.7 mouse macrophages originally from the American Type Culture Collection (ATCC [Manassas, VA]) were grown in DMEM supplemented with 10% FBS. Plasmids were transfected into HeLa cells with Effectene (Qiagen). Cells were grown at 37°C in a 5% CO₂ atmosphere.

RAW 264.7 mouse macrophages (American Type Culture Collection, ATCC, Rockville, MD, USA) were grown in modified Dulbecco Eagle medium (DMEM) that was high in glucose (Biowest, Riverside, MO, USA) and supplemented with 10% fetal bovine serum (FBS) (v/v) and 1% penicillin/streptomycin/amphotericin (v/v) (Biowest). Cells were grown at 37 °C under constant conditions of humidity, 5% CO₂, and 95% air.

To determine cathepsin activity in cell lysates, Raw 264.7 mouse macrophages were obtained from the American Type Culture Collection (ATCC TIB-71). Passages 25–29 were used in the experiments. Raw 264.7 mouse macrophages were stably transfected with stefin B (RMA cells), as reported before [11 (link)]. Cells were seeded in 24-well plates (2 × 10⁵ cells/well) and stimulated with LPS, as indicated. Negative controls were pretreated with 15 µm broad cathepsin inhibitor E-64d for 1 h. The supernatants were collected after treatment, and cells were washed with PBS. Cathepsin activity was measured as previously described [11 (link)] using fluorogenic substrates Z-ArgArg-AMC (Z-RR-AMC) and Z-PheArg-AMC (Z-FR-AMC) at a final concentration of 30 µM. The liberation of AMC was measured using a fluorimeter and normalised to the concentration of proteins in cell lysates determined using the Bradford assay. Results were normalised to the mean value of samples in the control group.