Fig 2: Assay specificity. Sample (2.5 µL) was added to 10 µL reagent mix then incubated at 65°C for 60 minutes, 80°C for 2 minutes, and cooled to 4°C for 10 minutes using T100 Thermal Cycler (S/N 621BR14432; Bio-Rad Laboratories, Inc., CA, USA). Results were viewed using the EasyViewTM LED Transilluminator (Manila HealthTek, Inc., Marikina, Philippines) which has a blue LED light and yellow plastic gel filter, and through agarose gel electrophoresis (1.5% agarose, 135 V for 25 minutes). Salmonella enterica subsp. enterica positives appear bright yellow green. Positive control panel includes the following: SPA-Salmonella enterica sv. Paratyphi A (typhoidal), SPB-Salmonella enterica sv. Paratyphi B (typhoidal), STY-Salmonella enterica sv. Typhi (typhoidal), and SNT-Salmonella enterica sv. Typhimurium (nontyphoidal). Negative control panel includes the following: Pa–Pseudomonas aeruginosa, Lm–Listeria monocytogenes, Kp–Klebsiella pneumoniae, Ec–Escherichia coli, Ef–Enterococcus faecalis, Se–Staphylococcus epidermidis (Gram-positive bacteria), and Sa–Staphylococcus aureus. Samples were assayed in triplicate. All images were taken using a Canon EOS 750D camera. Interpretation of results are as follows: (+)–Positive using the LED transilluminator and positive in 1.5% AGE; (-)–Negative using the LED transilluminator and negative in 1.5% AGE.

Fig 3: Optimization of assay incubation time. Sample (2.5 µL) was added to 10 µL reagent mix then incubated at 65°C for 30–60 minutes, 80°C for 2 minutes, and cooled to 4°C for 10 minutes using a T100 Thermal Cycler (S/N 621BR14432; Bio-Rad Laboratories, Inc., CA, USA). Samples used were the No Template Control (NTC) and the Positive Control (PC; 106 copies/µL). Samples were run in triplicate. Results were viewed using the EasyView™ LED Transilluminator (Manila HealthTek, Inc., Marikina, Philippines) which has a blue LED light and a yellow plastic gel filter, and through agarose gel electrophoresis (1.5% agarose, 135 V for 25 minutes). Salmonella enterica subsp. enterica positives appear bright yellow-green. All images were taken using a Canon EOS 750D camera. Interpretation of results are as follows: (+)–Positive using the LED transilluminator and positive in 1.5% AGE. (-)–Negative using the LED transilluminator and negative in 1.5% AGE. (-/+)–Negative using the LED transilluminator but positive in 1.5% AGE.

Fig 4: TR-57 and IMT1 both inhibit mitochondrial transcription from the heavy and light strand. HEK293T cells were plated at 4 × 105 cells/well in 6 well plates, allowed to adhere overnight, and treated with TR-57 (150 nM) and IMT1 (10 µM) for indicated times. Cells were washed 3 times with cold DPBS, scraped, and pelleted. RNA was isolated using the RNeasy Plus Mini Kit (Qiagen) and residual DNA was removed using the RNase-Free DNase Set (Qiagen). cDNA was synthesized from 2 µg of extracted RNA in 20 µL total volume using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and the Bio-Rad T100 thermal cycler. Quantitative PCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad). The nuclear reference gene 18S, mitochondrial heavy strand gene, ND1, and mitochondrial light strand gene, ND6, were quantified using the CFX96 Touch Real-Time System. A one-way ANOVA analysis was performed to determine significance; p-value < 0.05 (*), 0.01 (**), 0.001 (***). N = 2 biological replicates.

Fig 5: Detection of CD45+ human cells in the BM of NSGSCF mice by immunohistochemistry staining and comparison with expression of tryptase mRNA and KIT DNA levels.Immunohistochemistry was performed on slides prepared from paraffin-embedded, formalin-fixed, pelvic bone specimens using the mouse on mouse (MOM) immunodetection kit (Vector Laboratories, Burlingham, CA, USA) according to the manufacturer’s protocol. A mouse monoclonal antibody against human CD45 (Dako, Glostrup, Denmark), diluted 1:50, was applied for 60 min at room temperature. MOM biotinylated anti-mouse IgG was used as secondary antibody. As peroxidase substrate, 3-amino-9-ethylcarabzole was applied. Slides were counterstained in Mayer´s hematoxylin. Pictures were taken at a magnification of 60 x. TPSAB1/TPSB2 mRNA levels were measured by quantitative real-time PCR (qPCR) in BM cells flushed from long bones of NSGSCF mice as follows: RNA was isolated using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase and random primers (Invitrogen, Carlsbad, CA). Primers for TPSAB1/TPSB2 (forward: 5'- CGG GAA CAC CCG GAG GGA CT -3'and reverse: 5' GCC TGC AGC CAG GTG CCA TT-3') and ACTB/Actb (forward: 5'- TCG ACA ACG GCT CCG GCA TG-3' and reverse: 5'- CCT CTC TTG CTC TGG GCC TCG TC-3') were applied. qPCR was performed on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) using iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and plasmid standards [11]. TPSAB1/TPSB2 mRNA copy numbers were normalized to ACTB/Actb mRNA copy numbers and expressed as percent of ACTB/Actb. ´Detected expression´ of TSPAB1/TPSB2 mRNA was defined by >0.05% TSPAB1/TPSB2 mRNA relative to ACTB/Actb mRNA levels. To detect human KIT DNA in these samples, droplet digital PCR (ddPCR) was performed as follows: genomic DNA was extracted from FFPE pelvic mouse specimens using QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, DE). DNA concentrations were measured using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Approximately 280 ng of DNA per sample were used for ddPCR. DNA was pretreated with uracil DNA glycosylase (UDG, New England Biolabs, Ipswich, MA) for 20 min at 37 °C. ddPCR was performed with the Bio-Rad’s ddPCR Mutation Detection Assay (FAM + HEX) for human KIT wild-type. The reaction mixture additionally contained ddPCR Supermix for Probes (no dUTP, Bio-Rad, Hercules, CA) and HindIII-HF (New England Biolabs) restriction enzyme. The samples were fractionated into approximately 20000 droplets using a QX200 droplet generator. The PCR was then performed on a Gradient T100 Thermal Cycler (Bio-Rad) using the following protocol: 95 °C for 600 s (ramp rate 2 °C/second), 40 cycles at 94 °C for 30 s and 55 °C for 60 s (ramp rate 2 °C/second), 98 °C for 600 s and 4 °C (ramp rate 1 °C/second) until measurement. A QX200 droplet reader (Bio-Rad) was used for analysis.