Imidazole

Isopropyl β-d-1-thiogalactopyranoside (IPTG) was bought from INDOFINE Chemical Company Inc. (Hillsborough, NJ, USA). Sodium phosphate monobasic, sodium chloride, imidazole, Tris, HEPES and DMSO were all provided by VWR (Radnor, PA, USA). NTCB was purchased from TCI America (Portland, OR, USA). TCEP was obtained from Alfa Aesar (Tewksbury, MA, USA). LC/MS-grade water and acetonitrile for liquid chromatography were both purchased from Fisher Scientific (Waltham, MA, USA) and supplied with 0.1% Optima™ LC/MS-grade formic acid (Fisher Scientific, Waltham, MA, USA). All oligonucleotide primers and DNA sequences used for DNA mutagenesis were synthesized by Integrated DNA Technologies (Coralville, IA, USA). Sanger sequencing was performed by Eton Bioscience Inc. (San Diego, CA, USA).

pET30M-2 plasmids encoding His_tag-GST-TEV_site-Ddx4n1 or His_tag-GST-TEV_site-Ddx4n1-YFP (see sequence details below) were kind gifts from Dr. Tim Nott (Oxford University). ampicillin resistant TEV plasmid encoding MBP–TEV_site–His-tag–S-TEV was a kind gift from Charlotte O’Shea (University of Copenhagen). All constructs where sequenced by GATC (Eurofins, Germany) in order to ensure no point mutations had occurred. Tris, TCEP, NaCl, CaCl₂, PEG3000, PEG6000, Imidazole, LB broth, benzonase, ampicillin, kanamycin and reduced glutathione were all purchased from VWR (Denmark). The ssDNA as previously described to be partitioned by Ddx4n1 droplets by Nott et al.^{35 (link)} with the sequence 5'-TTT TTC CTA GAG AGT AGA GCC TGC TTC GTG G-3' as well as the 5'Alexa-488 labeled version was synthesized, HPLC purified and lyophilized by TAG Copenhagen (Denmark). The RP₃ peptide was synthesized and HPLC purified by Bachem (Switzerland) and delivered as TFA salt after MALDI-MS quality control. Fluoresbrite YG Microspheres of calibration grade were purchased from Polysciences Europe GmbH (Germany). All other relevant salts, buffer components and materials were purchased from Sigma (USA) & VWR (Denmark) and dissolved in RNAse and nuclease free milliQ water unless otherwise specified.

Mice were infected with ~100 CFU of Mtb H37Rv in an aerosol exposure chamber as outlined above. Next day, the infected mice were intranasally treated with ornithine (L-ornithine monohydrochloride; KYOWA HAKKO BIO Co., Ltd.) and imidazole (Sigma-Aldrich) at varying concentrations. In brief, mice were deeply anesthetized with ketamine plus xylazine (75 and 10 mg/kg i.p. injections) followed by intranasal administration of L-ornithine monohydrochloride (1000 mg/kg, 2500 mg/kg, or 5000 mg/kg) and imidazole (100 mg/kg, 300 mg/kg, or 600 mg/kg) twice a week for 30 days, while the control mice were injected with PBS (pharmaceutical grade; VWR life science). The highest dose was selected based on the LD50 provided in the manufacturer’s safety data sheet. After 30 days of treatment, the lungs were harvested to examine the CFU counts. In brief, the lungs were placed into 30-mm dishes containing 2 ml of 7H9 media and minced with scissors into pieces no larger than 2–3 mm. The fluid was discharged through a 70-μm filter (BD Biosciences, San Jose, CA) suspended over a 50-ml conical tube. The syringe plunger was then used to gently disrupt the lung tissue and the supernatant containing the cells was incubated for 2 h at room temperature. Finally, CFU assay was performed as outlined above. All the experiments were performed thrice with five mice in each group.