biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicsHan-Gyu Choitranscription profiling by arrayMus musculusMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16005This study investigates the molecular mechanism underlying the synergistic anti-mycobacterial effect of IFN-γ and IL-17. We performed microarray analysis on bone marrow-derived macrophages (BMDMs) from C57BL/6J mice infected with Mycobacterium tuberculosis H37Rv and treated with infection control, IFN-γ (1, 10, 100 ng/mL), IL-17 (10, 100 ng/mL), or IFN-γ/IL-17 combination (1 ng/mL each) for 24 hours. The study identified coronin-1A as a key molecule whose retention on phagosomes is inhibited by IFN-γ/IL-17 treatment via LRG47-dependent STAT1/STAT3 pathway activation. These findings provide insights into cytokine-based adjunctive therapy for tuberculosis treatment.biostudies-arrayexpressGrowth Protocol - Bone marrow-derived macrophages (BMDMs) were isolated from 6-7 week-old female C57BL/6J mice and cultured for 6 days in Dulbecco's modified eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 50 ng/mL mouse macrophage colony stimulating factor (M-CSF). Cultured BMDMs were infected with Mycobacterium tuberculosis H37Rv at a multiplicity of infection (MOI) of 1:1 (bacteria to cells) for 4 hours at 37°C in 5% CO2. After infection, cells were washed with phosphate-buffered saline (PBS) and treated with amikacin (200 μg/mL) for 2 hours to eliminate extracellular bacteria.Sample Treatment - Following Mtb infection and amikacin treatment, BMDMs were treated with recombinant mouse IFN-γ (at concentrations of 1, 10, or 100 ng/mL), recombinant mouse IL-17 (at concentrations of 10 or 100 ng/mL), or a combination of IFN-γ and IL-17 (1 ng/mL each) in complete DMEM. PBS-treated cells served as infection-only controls. Cells were incubated with cytokines for 24 hours at 37°C in 5% CO2 before RNA extraction.Sample Collection - Bone marrow cells were collected from the femurs and tibias of 6-7 week-old female C57BL/6J mice (Japan SLC Inc., Shizuoka, Japan). Mice were euthanized by CO2 asphyxiation according to institutional animal care guidelines (IACUC protocol 202009A-CNU-132). Femurs and tibias were aseptically removed, and bone marrow was flushed out using sterile phosphate-buffered saline (PBS). Cells were collected by centrifugation, red blood cells were lysed using ACK lysis buffer, and viable cells were counted using trypan blue exclusion. Bone marrow cells were then differentiated into macrophages by culturing in DMEM supplemented with 10% FBS and 50 ng/mL M-CSF for 6 days. On day 6, adherent BMDMs were harvested for infection experiments.Scaning - Following hybridization and washing, Affymetrix Clariom S Arrays were scanned using the Affymetrix GeneChip Scanner 3000 7G with autoloader. Scanning was performed at 0.7 μm resolution using the default settings optimized for the Clariom S platform. The scanner generated DAT image files, which were automatically converted to CEL files containing probe cell intensity values using the Affymetrix GeneChip Command Console (AGCC) software version 4.0 or higher. Quality control metrics including average signal intensity, background noise, and percentage of present calls were evaluated for each array. Arrays with quality control parameters outside acceptable ranges were excluded from further analysis. CEL files were used for downstream data processing and normalization. ```Nucleic Acid Extraction - Total RNA was extracted from treated BMDMs using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. RNA quality was assessed using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano Chip (Agilent Technologies). RNA concentration was determined using an ND-2000 Spectrophotometer (Thermo Inc.). Only samples with RIN > 7.0 and 260/280 ratio between 1.8-2.0 were used for microarray analysis.Hybridization - Fragmented biotin-labeled cRNA (5.5 μg) was hybridized to the Affymetrix Clariom S Array, mouse (Thermo Fisher Scientific) at 45°C for 16 hours with rotation at 60 rpm in an Affymetrix GeneChip Hybridization Oven 645. Arrays were washed and stained using the Affymetrix GeneChip Fluidics Station 450 according to the standard Affymetrix protocol. Arrays were scanned using the Affymetrix GeneChip Scanner 3000 7G to generate CEL files.Labeling - Total RNA was amplified and biotin-labeled using the Affymetrix GeneChip WT Plus Reagent Kit according to the manufacturer's protocol. Briefly, 100 ng of total RNA was reverse transcribed to synthesize first-strand cDNA, followed by second-strand cDNA synthesis. The double-stranded cDNA was transcribed in vitro to generate amplified and biotin-labeled antisense cRNA. The labeled cRNA was purified and quantified before fragmentation for hybridization.MIAME ScoreRaw DataOrganizationAssays and DataMAGE-TAB FilesArray DesignsHan-Gyu ChoiData Transformation - CEL files were imported and analyzed using Affymetrix Transcriptome Analysis Console (TAC) Software. Signal values were background-corrected, normalized using the RMA (Robust Multi-array Average) algorithm, and log2-transformed. Gene-level expression values were generated using the SST-RMA algorithm. Probe sets with detection p-value < 0.05 were considered as reliably detected.falseIFN-γ and IL-17 synergistic anti-mycobacterial response via coronin-1A inhibitionThis study investigates the molecular mechanism underlying the synergistic anti-mycobacterial effect of IFN-γ and IL-17. We performed microarray analysis on bone marrow-derived macrophages (BMDMs) from C57BL/6J mice infected with Mycobacterium tuberculosis H37Rv and treated with infection control, IFN-γ (1, 10, 100 ng/mL), IL-17 (10, 100 ng/mL), or IFN-γ/IL-17 combination (1 ng/mL each) for 24 hours. The study identified coronin-1A as a key molecule whose retention on phagosomes is inhibited by IFN-γ/IL-17 treatment via LRG47-dependent STAT1/STAT3 pathway activation. These findings provide insights into cytokine-based adjunctive therapy for tuberculosis treatment.2025-11-11T00:00:00Z2026-05-27T16:42:29.15Z2025-11-06T12:31:47.59ZE-MTAB-16005EFO_0002768EFO_0002944EFO_0003814EFO_0003813EFO_0003789EFO_0005518EFO_0003816EFO_0003815EFO_0003969