J774 cells

RAW-Blue (Invivogen), RAW cells and J774 cells (ATCC) were cultured as per vendor recommendations. Bone marrow derived macrophages (BMDM) were obtained as previously described by us and others 8 (link),13 (link).

J774 cells were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were grown under standard conditions in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Scientific, Massachusetts, USA), penicillin/streptomycin, and 10% fetal bovine serum (FBS) (Gibco) at 37 °C in a 5% CO₂ atmosphere. To obtain bone marrow-derived macrophages (BMDMs), C57BL/6 mice were used. Bone marrow cells were flushed from the femurs and cultured for 7 days in 30% L929-cell-conditioned medium to obtain BMDMs. When J774 or BMDM cells reached 80% confluence, they were separately treated with phosphate-buffered saline (PBS), lipopolysaccharides (LPS) (100 ng/mL), Och, IP6, PA, ADC, and ADC-PA for 24 h and then fixed in 4% paraformaldehyde for later immunofluorescence analyses. To determine the inflammatory responses, both J774 and BMDM cells were separately treated with PBS, LPS, ADC, ADC+LPS, ADC-PA, and ADC+LPS for 6 h. Cells were then preserved in RNAzol solution (Molecular Research Center, Taipei, Taiwan) and stored at −80 °C before use.

Macrophages (J774 cells from American Type Culture Collection) were seeded into 384-well black clear bottom plates. M. tuberculosis CDC1551 expressing mCherry was grown to mid-log phase in Middlebrook 7H9 OADC washed, and syringed 6-times with 25G⅝ tuberculin syringe. The de-clumped bacteria were diluted into pre-warmed infection media (DMEM, 10% fetal calf serum, 2.0 mM L-glutamine, and 1.0 mM sodium pyruvate) and were used to infect cells at an MOI of 4:1. Bacteria were added to the screening plates with a Janus Ministation (Perkin Elmer). Compounds were added to the screening plates 1 hour after infection to a final concentration of 10 μM. Following a six day incubation period at 37°C and 6% CO₂ Mtb mCherry fluorescence was quantified using an Envision Multilabel plate reader (Perkin Elmer). All screening plates contained negative (DMSO) and positive (10 μM rifampicin) controls. Percent inhibition for the experimental compounds was calculated using the formula, percent inhibition = 100x(DMSO signal—sample signal)/(DMSO signal—rifampicin signal). The Z′ factor, a measure of variability and reproducibility [21 (link)], was determined for each plate using the following formula: Z′ = 1-[3×(SD_rifampicin+SD_DMSO)/|M_rifampicin-M_DMSO|], where SD denotes the standard deviation and M denotes the mean for the samples and controls, respectively.

The S. pneumoniae strain D39 (ATCC49619) used in this study was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). and cultured according to previous research [8 (link)]. Briefly, S. pneumoniae was cultured in THY (Todd Hewitt Broth medium containing 2% yeast extract) at 37 °C statistically. Human-derived A549 alveolar adenocarcinoma basal epithelial cells and murine peritoneal macrophage J774 cells were all purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). A549 and J774 cells were cultured based on a previous method [19 (link)]. Briefly, both A549 and J774 cells were cultured in Dulbecco’s modified Eagle’s medium/high glucose (DMEM; HyClone, Logan, UT, USA) containing 10% fetal bovine serum and 1% penicillin–streptomycin (MRC, Madrid, Spain) at 37 °C and 5% CO₂. Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Shionone (purity > 98%) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China) an dissolved in DMSO.