Lipopolysaccharides from escherichia coli o26 b6

Synthetic honaucin A was used in all assays and measurements. For bioassays, lipopolysacchar-ides from Escherichia coli (O26:B6) as well as phorbol 12-myristate 13-acetate and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were purchased from Sigma-Aldrich (St. Louis, MO). NRF2 luciferase reporter MCF7 stable cells were purchased from Signosis (Santa Clara, CA). RNA sequencing was carried out using an Illumina Hiseq2000 (San Diego, CA) following RNA quality assessment via Bioanalyzer 2100 (Agilent, Santa Clara, CA). For in vitro alkylation experiments, reaction products were separated via liquid chromatography using a Phenomenex Kinetex 5 μm EVO C-18 column. Highresolution mass analysis was accomplished with an Agilent 6230 ESI-TOF-MS operating in positive mode.

Mice ears were co-injected intradermally with 10⁵ metacyclics of L. donovani and 10³ live Solibacillus bacteria or 1µg of LPS in a total volume of 10µl. After identification of Solibacillus from a Leishmania-positive egestion bite site, the agar bacterial colony was transferred to 30uL of sterile molecular biology grade water. An aliquot was transferred to LB and grown overnight at 28°C and an aliquot was stored as a glycerol stock. A sample of the bacteria was sequenced to confirm the identity of Solibacillus and to ensure that it was the only species present in the preparation. Further, a standard curve was established for the number of bacteria by counting colonies plated at various dilutions and optical densities (ODs). For co-injection, Solibacillus was grown in Luria broth overnight at 28°C. On the day of injection, 100µl of bacteria was inoculated into 5ml of fresh Luria broth and growth was monitored hourly until it reached an OD_600nm of 0.15. The culture was then diluted with LB medium according to the predetermined standard curve to give 10³ cfu per 5µl. As for LPS (Lipopolysaccharides from Escherichia coli O26:B6, Sigma), the stock solution (2mg/ml) was diluted to 200µg/ml immediately before use. For co-injection, 5µl containing 10⁵L. donovani metacyclics was mixed with 5µl of either 10³ live Solibacillus bacteria or 1µg of LPS.