Streptomycin sulfate

All mice used in this study are C57BL/6 mice, housed under the specific pathogen-free conditions in the Institute of Animal Experiments of the School of Pharmacy, Kitasato University, Japan. These infection experiments were performed as described previously [37 (link)]. Briefly, 6- to 13-week-old mice were deprived of food and water for 4 h, and then inoculated with 25 mg streptomycin sulfate (Wako Pure Chemical Industries) by gavage to attenuate the colonization resistance. Twenty-four hours later, mice were deprived of food and water, and then inoculated with bacteria (1 × 10⁸ CFU) by gavage. To determine the bacterial loads in the colonic content, fresh fecal pellets were freshly collected, and homogenized in sterile phosphate-buffered saline (PBS) for the plating on MacConkey agar plates (Nissui Pharmaceutical) supplemented with the appropriate antibiotic(s). To monitor bacterial loads in luminal content and organs, cecal content, mLN and spleen were recovered from mice sacrificed by cervical dislocation or carbon dioxide inhalation at the indicated time points, and then homogenized in sterile PBS containing 0.5% tergitol and plated on MacConkey agar plates supplemented with the appropriate antibiotic(s).

All reagents and solvents purchased were the highest commercial quality and used without further purification otherwise noted. Dimethyl sulfoxide (DMSO) was purchased from KANTO chemicals. Propidium iodide (PI), benzylpenicillin potassium, and streptomycin sulfate were purchased from Fujifilm Wako Chemicals. 1,3-Diphenylisobenzofuran (DPBF) was purchased from Sigma-Aldrich. Minimum essential medium (MEM) and phosphate-buffered saline (PBS) were purchased from Nacalai Tesque. Fetal bovine serum (FBS) was purchased from Capricorn Products Inc. The Ir complex 7 was synthesized according to our previous paper [26 (link)], a stock solution of which in DMSO was prepared and stored at 0 °C prior to use. UV-Vis absorption spectra of DPBF were measured on a JASCO V-550 UV-vis spectrophotometer. The microscopic images of HeLa S3 cells were observed on fluorescent microscopy (Biorevo, BZ-x800, Keyence, Amagasaki, Japan).

Cells from the breast cancer cell line MCF-7, obtained from the American type culture collection (ATCC), were cultured in DMEM and supplemented with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA), 100 U/mL penicillin G, and 0.1 mg/mL streptomycin sulfate (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan). The cells were incubated in a humidified atmosphere (5% CO₂ at 37 °C). Before start of the experiments, the cells were checked for mycoplasma contamination.

Reaction conditions were based on R-iSAT with ribosomal protein synthesis, but two-step reactions were performed for the mutational study. Translation of uS12 and subsequent assembly into 30S subunits were performed at 37 °C for 2 h in the first step, and then DNA templates for sfGFP-comp were added with or without 10 μg/mL streptomycin sulfate (FUJIFILM Wako Chemicals) for the second-step reactions. For negative controls, an equal volume of water was added to reactions. Second-step reactions were carried out at 37 °C for 4 h in Mx3005P (Agilent Technologies), and sfGFP fluorescence was monitored during incubation.

Mouse embryonic fibroblast26 (link) (MEF, gift from Dr. Akira Kitamura, Hokkaido University) and HEK293 cells (Riken BRC, Ibaraki, Japan) were maintained in a 5% CO₂ humidified atmosphere at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Hyclone Lab., Logan, UT, USA), 1 × 10⁵ U l⁻¹ penicillin G and 100 mg l⁻¹ streptomycin sulfate (Wako, Osaka, Japan). The day before the experiment, cells (early passage) were plated on a 35-mm glass-base dish (AGC Techno Glass, Ltd., Shizuoka, Japan) to 60–70% confluence for live-cell imaging or a 6-well plate (Thermo Fisher Scientific, Waltham, MA, USA) for expression assay. The medium was replaced by phenol red-free medium (Opti-MEM, Life Technologies, Gaithersburg, MD, USA) before confocal imaging. Microinjection of the DNA probe was conducted at the confocal microscope stage by combining Femtojet (Eppendorf, Hamburg, Germany) and Injectman NI2 (Eppendorf) with Femtotip (Eppendorf) as an injection needle. Injection pressure was adjusted to deliver the desired amount of DNA.