Micrococcus luteus

Rat embryonic cardiomyo-blast-derived H9C2 cells were purchased from the Typical Culture Preservation Commission Cell Bank, Chinese Academy of Sciences (Shanghai, China). H9C2 cells mimic most of the characteristics of adult cardiomyocytes and this is an ideal cell line with which to explore the role of UCP2 in the septic myocardium in a cell culture system. The cells were cultured in DMEM (Gibco-BRL, Beijing, China) supplemented with 10% fetal calf serum and 5% CO₂ at 37°C. The H9C2 cells were passaged regularly and subcultured to 75% confluence prior to use in the experiments. In order to simulate sepsis, some cells were cultured in the presence of 2 μg/ml lipopolysaccharide (LPS, from Escherichia coli O111:B4; Sigma-Aldrich, St. Louis, MO, USA) plus 20 μg/ml peptidoglycan G (PepG, from Micrococcus luteus; Sigma-Aldrich). The experimental design consisted the following 4 groups: i) the control group, cells were treated with saline only; ii) the LPS/PepG group, cells treated with LPS and PepG as described above; iii) the LPS/PepG + siRNA group, cells transfected with siRNA2 and 24 h later treated with LPS plus PepG as described above; and iv) the LPS/PepG + ncRNA group, cells transfected with ncRNA and 24 h later treated with LPS plus PepG as described above. Further experiments were carried out 24 h following stimulation with LPS plus PepG.

Bombyx mori (Nistari), Helicoverpa armigera larvae, and Drosophila melanogaster adults (w¹¹¹⁸, PPO1^Δ, PPO2^Δ and PPO1^Δ, PPO2^Δ) were reared as described (15 (link)–17 (link)). The strains of fungal pathogens B. bassiana ARSEF 2860, M. robertsii ARSEF2575, and its mutant ΔMBZ1 were routinely maintained for the preparations of spore (conidia), hyphae, and blastospore as described (18 (link)). The yeast strains of Saccharomyces cerevisiae and Candida albicans were also routinely cultured for spore preparations (19 (link)). Spores (Chlamydospores) of C. albicans were collected (19 (link)). Escherichia coli (BL21) was cultured in lab. Micrococcus luteus (Sigma) was suspended in buffer for pull-down assay.

Heat-killed Micrococcus luteus (ATCC No. 4698; Sigma-Aldrich, Catalog # M3770-5G) was used as a substrate for recombinant His-Gmd at final concentration of 0.075% (750 μg/ml) in phosphate-buffered saline (PBS) as we previously described (Gedbjerg et al., 2013 (link)). Triton X-100 (Sigma, Catalog # T8787-250ML) was used as a substrate-solubilizing agent. Briefly, 50 μl of 200 μg/ml of Gmd was diluted twofold in 96-well plate and 50 μl of 0.15% M. luteus containing various concentrations of cell wall solubilizing agent Triton X-100 was added and incubated at 37°C, and OD₆₀₀ was measured after 5, 60, and 120 min of incubation. Percentage of lysis was calculated by subtracting OD₆₀₀ of M. luteus treated with various concentrations of Gmd from OD₆₀₀ of M. luteus treated with Triton X-100 and dividing the product by OD₆₀₀ of M. luteus treated with Triton X-100, expressing it as a percentage.

For preparation of robotic arrays, cell wall samples from S. pneumoniae R6, with choline- or ethanolamine-containing (lipo)teichoic acids, and Micrococcus luteus (Sigma) were printed on 16-pad nitrocellulose-coated glass slides (FAST-slides, Maine manufacturing) using a non-contact arrayer (Sprint, Arrayjet Ltd.). Pneumococcal cell walls were prepared, as described^{75 (link)}, from cultures grown without shaking at 37 °C to an OD₆₂₀ of 0.5 in C medium supplemented with yeast extract (0.8 mg/ml; Difco Laboratories) or in ethanolamine-containing Cden-EA medium^{76 (link),77 (link)}. Cell wall sample suspensions in PBS were diluted with two volumes of 70.5% glycerol, 0.09% Triton X-100 (final concentrations 47% and 0.06%, respectively) and printed in a four-level dose-response format by applying 100 pl/spot. Spots were printed as triplicates. 1 µl of Cy3 fluorophore (GE Healthcare) was added per millilitre of sample suspensions, to enable post-array monitoring of the spots^{78 (link)} by scanning fluorescence signals upon excitation at 532 nm (green laser) with a GenePix 200-AL scanner (Axon, Molecular Devices).

The mucoid bioluminescent P. aeruginosa strain Xen05 was purchased from Caliper life Sciences, Inc. The nonmucoid bioluminescent strain H1001 was the kind gift of Robert Hancock (University of British Columbia, Vancouver, BC, Canada). Both strains were stored as glycerol stocks at −80 °C. Recombinant hLYS and freeze‐dried Micrococcus luteus were purchased from Sigma Millipore (Burlington, MA, USA). Protegrin 1 AMP (PTG1) and HB71 peptides (> 95% purity) were obtained from Peptide 2.0. LL‐37, Melittin, and Tet009 peptides (> 95% purity) were purchased from GenScript (Piscataway, NJ, USA). Human beta defensin 3 AMP (hBD3) peptide was purchased from AnaSpec. Peptide stock solutions were stored at −20 °C. All other reagents and materials were purchased from Fisher Scientific (Waltham, MA, USA).

Serum respiratory burst activity was measured by reducing nitro-blue tetrazolium (NBT) by intracellular superoxide radicals, as mentioned by (29 (link)). The serum lysozyme activity was measured using lyophilized Micrococcus luteus (Sigma, United States) proposed by (30 ). The serum myeloperoxidase activity was calculated according to (31 (link)) using 3, 3’, 5, 5’-tetramethylbenzidine (TMB) and hydrogen peroxide.