Kanamycin

Media used in this study include isolation agar plate, which was composed of carbon-free (cf) medium-peptone 20 g/L, yeast extracts 5 g/L, NaCl 5 g/L, K₂HPO₄ 0.05 g/L, KH₂PO₄ 0.05 g/L, L-cysteine HCl 1 g/L, hemin 0.01 g/L, vitamin K1 2 mg/L, kanamycin 0.1 g/L, and vancomycin 0.75 mg/L, and supplemented with 5% selected carbohydrate (xylan, rhamnose, or mannitol), 0.012 g/L bromocresol purple, and 15–20g/L agar; isolation control agar plate (CTRL), composed of brain heart infusion supplemented with 0.01 g/L hemin, 2 mg/L vitamin K1, 0.1 g/L kanamycin, 0.75 mg/L vancomycin, and 15–20g/L agar; and BHIS medium, composed of brain heart infusion supplemented with 0.01 g/L hemin and 2 mg/L vitamin K1.
Peptone and yeast extracts were purchased from Oxoid, United Kingdom; kanamycin, vancomycin, hemin, vitamin K1, rhamnose, and mannitol were purchased from Sangon Biotech, China; xylan was purchased from Sigma-Aldrich, United States; brain heart infusion was a product from Hopebio, China; and the rest reagents were obtained from Shanghai Hushi Laboratorial Equipment Co., Ltd., China.

For the transformation, 1 µl of DNA was mixed gently into 100 µl of JM109 competent cells (L200, Promega, Madison, Wisconsin, USA). The competent cell/DNA mixture was incubated on ice for 20–30 min, followed by a heat shock at 42 °C for 45 s. The tubes were then immediately put on ice for 2 min. 900 µl of SOC medium (15544034, Thermo Fisher Scientific, Inc.) was added, and the mixture was incubated at 37 °C for 45 min on a shaking incubator. Thereafter, 100 µl of the transformation mix were plated onto a 10 cm Luria Broth (LB) agar plate containing 25 µg/ml kanamycin (11815024, Thermo Fisher Scientific, Inc.) and incubated at 37 °C overnight. The next day, single bacterial clones were picked from the agar plates, added to liquid LB (10855001, Thermo Fisher Scientific, Inc.), mixed with 25 µg/ml kanamycin, and incubated at 37 °C on a shaker overnight. Vector DNA was isolated on the following day using the Plasmid MiniPrep kit (D4209, Zymo Research, Irvine, CA, USA) according to manufacturer’s instructions.

An authenticated human colon adenocarcinoma cell line (DLD1), MMR-deficient, was purchased from ATCC (Manassas, Virginia, USA). DLD1 cells were propagated in adhesion and cultured in RPMI 1640, supplemented with 10% inactivated fetal bovine serum, 1% GlutaMAX-I, and 1% kanamycin (all purchased by Thermo Fisher Scientific, Lucerne, Switzerland), at 37°C and 5% CO₂. All cultures were tested by PCR and proven to be mycoplasma free prior to experimental investigations.
PBMCs from healthy donors (Blood donor center of the University Hospital of Basel) were obtained by gradient centrifugation. All donors provided written informed consent. CD14+ monocytes were magnetically isolated from PBMCs by using antibody-coated beads (Miltenyi Biotech, Bergisch Gladbach, Germany) and cultured in RPMI 1640 supplemented with 1% GlutaMAX-I, 1% non-essential amino acids (NEAA), 1% sodium pyruvate, HEPES, 1% kanamycin sulfate, and 10% fetal bovine serum (all purchased by Thermo Fisher Scientific) for CD14+ monocytes. DCs were generated in presence of GM-CSF and IL4, as previously described [25 (link)].

Bacterial strains and plasmids are listed in Table S6 in the supplemental material. M. tuberculosis strains were grown with Middlebrook 7H9 broth or 7H10 agar (Difco) supplemented with 1× albumin dextrose saline (ADS), 0.5% glycerol, and 0.05% Tween 80 (7AGT) (68 (link)). As needed, growth medium was supplemented with 20 µg/ml kanamycin (Acros), 50 µg/ml hygromycin (Roche), or 50 µg/ml carbenicillin (Sigma). Escherichia coli strains were grown on Luria-Bertani medium (Fisher) supplemented as necessary with 40 μg/ml kanamycin, 150 μg/ml hygromycin, and 100 μg/ml carbenicillin.

An overnight culture of the cells (5 × 10⁵ cells/well) in a 6-well plate was infected with bacteria at a multiplicity of infection (MOI) of 2 for 1 h. The cells were washed twice with 1 mL of PBS to remove extracellular bacteria, and residual bacteria were killed by incubating the cells in DMEM containing 250 μg/mL kanamycin (Gibco) for 2 h. Thereafter, the infection was allowed to continue in the medium containing 20 μg/mL of kanamycin until the experiment was terminated (6 (link)).

Human mesenchymal stem cells (hMSCs, LONZA, PT-2501), normal human dermal fibroblasts (NHDFs, LONZA, CC-2511), HEK293T, NTERA2, HeLa cells, and induced pluripotent stem cells (iPSCs) were used in this research. hMSCs and NHDFs were maintained in Minimum Essential Medium Eagle (αMEM, MilliporeSigma, M4526) supplemented with 10% fetal bovine serum (Hyclone, SH30910.03), 1 × GlutaMAX (Gibco, Thermo Fisher Scientific, 35050–061), 1 ng/mL human basic FGF2 (Miltenyi Biotech, 130–093-840) and kanamycin (Gibco, 15160–054). Culture medium was exchanged every 2 days. FGF2 was kept at 4 °C for no longer than 1 week and was freshly added while preparing the growth medium. HEK293T, NTERA2, and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco, 11965–092) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate (Gibco, 11360–070), and kanamycin. iPSCs were induced from NHDFs as previously described [29 (link)] and maintained in StemFit AK02N (AJINOMOTO, RCAK02N). The inhibitors used in this study were LY294002 (Selleck, S1105), PD0325901 (Wako, 162–25291), and MK2206 (Selleck, S1078). All the cells were cultured in a humidified incubator with 5% CO₂ at 37 °C.