Mir 155 mimic

The miR-155 mimic, the miR-155 inhibitor, and the controls were purchased from Bioneer (Korea Bioneer Biotech Corp, Daejeon, South Korea). According to the manufacturer’s instructions, cells were seeded in 6-well plates at a density of 2 × 10⁵ per well overnight in media without antibiotics. Cells were then transfected with 40 pmol of the miR-155 mimic, the miR-155 inhibitor, and the controls with transfection reagent Lipofectamine 2000 (Invitrogen) in fresh medium without serum and antibiotics. Cells were cultured for 5 hours and subsequently were supplied with fresh medium with serum and cultured for additional 48 hours before they were harvested.

MDMs were plated in 12-well dishes at 1.5×10⁵ cells/well and were transfected the next day with 50 nM miR-155 mimic (Ambion, Austin, TX) or 100 nM miR-155 inhibitor (Dharmacon, Thermo Scientific) along with the negative control pre-miR or anti-miR using SiPORT-NeoFx transfection reagent (Ambion, Austin, TX) in complete medium. Macrophages were infected with bacteria, stimulated with 100 ng/ml E. coli O111:B4 LPS (Sigma-Aldrich), or subjected to other treatments 48 h after transfection.

RAW264.7 macrophages were seeded in 6-well plates and transfected using the siPORT NeoX transfection reagent (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. AGO2-specific siRNA and a scrambled negative control were purchased from Ambion (Austin, TX, USA). Additionally, miR-155 mimic, mimic control, miR-155 inhibitor, and inhibitor control were obtained from Ambion (Austin, TX, USA). After 48 h of transfection, cells were harvested for analysis.

MTB H37R strain (number: 9302025), purchased from Beijing Strain Preservation Center of China Institute of Drug Control, was incubated in 7H11 slant solid medium at 37°C for 21 days and then transferred to 7H9 liquid medium for 4-week culture. After that, bacterial fluid was collected, centrifuged at 3800 rpm for 15 min and diluted into suspension in RPMI-1640 medium with 10% FBS but without antibiotic, and its concentration was adjusted to 5 × 10⁶ CFU. Each mouse was caused renal injury by injecting with 5 × 10⁶ CFU of MTB H37Ra in tail vein. The mice were divided into six groups, with ten mice in each group: control group, MTB group, mimic group, inhibitor group, inhibitor + ESAT6 group, and inhibitor + ESAT6 + TAK242 group. The treatment regimens are presented in Table 1. Recombinant protein ESAT6 was purchased from Nanjing Sai Hongrui Biotech Company (number: PRO-563) as immunizing antigen (dose: 50 μg/kg). MiR-155 mimic (number: PM12601) and miR-155 inhibitor (number: AM12601) were both bought from Ambion company. Each mouse was infected by tail-vein injection with 0.5 ml mimics, inhibitor, and ESAT6 using EntransterTM-in vivo kit (number: 18668-11-1) only once during the experiment.