Journal: Scientific Reports

Article Title: Discovery of uncompetitive inhibitors of SapM that compromise intracellular survival of Mycobacterium tuberculosis

doi: 10.1038/s41598-021-87117-x

Figure Lengend Snippet: Effect of SapM inhibitors 1 , 2 and 13 on Mtb H37Rv growth and intracellular burden. Efficacy of compounds at 1 and 40 µM on Mtb intracellular burden in THP-1 macrophages at 24 h ( A ) and 72 h ( B ) post infection. Fold change of Mtb burden was calculated from the average CFU/ml relative to the DMSO control. Statistical significance was evaluated by one-way ANOVA (Dunnett’s test) compared to DMSO control (**** p < 0.0001). Error bars in ( A,B ) indicate ± SD of two and three independent experiments, respectively. ( C ) Effect of compounds at 40 µM on the acellular growth of Mtb in Middlebrook 7H9 broth monitored over 9 days using optical density (OD 600 ). DMSO and the first-line antibiotic isoniazid (at 0.14 µg/ml) were used as negative and positive controls, respectively. Error bars indicate ± SD of triplicates. Statistical significance was evaluated by two-way ANOVA (Dunnett’s test) compared to DMSO control (**** p < 0.0001). ( D ) THP-1 macrophage viability at 72 h upon treatment with compounds at 10 and 40 µM. Percentage of viability was calculated relative to the control (RPMI media only). Dashed black line indicates 70% viability. Error bars indicate ± SD of three independent experiments.

Article Snippet: Mtb strain H37Rv was grown in Middlebrook 7H9 broth (BD Diagnostics) or on Middlebrook 7H10 agar, both supplemented with 0.05% Tween 80, 0.2% glycerol and 10% OADC (Oleic Albumin Dextrose Catalase from Becton Dickinson Microbiology Systems) at 37 °C in 5% CO 2 .

Techniques: Infection

Journal: Microorganisms

Article Title: LRRC25 Inhibits IFN-γ Secretion by Microglia to Negatively Regulate Anti-Tuberculosis Immunity in Mice

doi: 10.3390/microorganisms11102500

Figure Lengend Snippet: Increase in the transcription and translation of LRRC25 in H37Rv-infected BV2 cells. ( a ) The laser scanning confocal images GFP-H37Rv infects BV2 cells with MOIs of 1, 5, and 10 (n = 3). ( b ) Relative expression of LRRC25 mRNA after H37RV infection of BV2 cells for 4 h (n = 4), Student’s t test. ( c ) Western blot images of LRRC25 after H37RV infection of BV2 cells for 4 h (n = 3). ( d ) Cellular immunofluorescence image of LRRC25 H37RV-infected BV2 cells for 4 h. Scale bar, 100 μm. ( e ) H37Rv CFU count of mice brain tissue at 2 weeks, 4 weeks, and 6 weeks (n = 3), Kruskal–Wallis test. ( f ) HE staining images of the brain regions of mice infected with tuberculosis at 0, 2, 4, and 6 weeks (red arrows show the inflammatory cell infiltration areas. There were three mice in each group. Scale bar, 50 μm). ( g ) Immunofluorescence image of cerebellum of mice infected with H37Rv at 6 weeks; the nucleus is blue, IBA-1 is green, LRRC25 protein is red, and the scale is 50 μm (n = 3). The figure shows a representative result. *** p < 0.001.

Article Snippet: The Mtb standard strains H37Rv (ATCC27294) and GFP-H37Rv (recombinant Mtb -H37Rv strain stably expressing GFP constructed with vector PSC301 (31851, Addgene, Watertown, MA, USA)) used in this study were obtained from the Beijing Tuberculosis Clinical Sample Database of Beijing Chest Hospital affiliated with Capital Medical University, China.

Techniques: Infection, Expressing, Western Blot, Immunofluorescence, Staining

Journal: Microorganisms

Article Title: LRRC25 Inhibits IFN-γ Secretion by Microglia to Negatively Regulate Anti-Tuberculosis Immunity in Mice

doi: 10.3390/microorganisms11102500

Figure Lengend Snippet: Interference of the translation of LRRC25 using siRNA of LRRC25: L1, L2, and L3. ( a ) Relative expression of LRRC25 mRNA after transfection of BV2 cells for 48 h and 72 h (n = 4), One-way ANOVA. ( b ) Western blot images of LRRC25 after transfection of BV2 cells for 48 h and 72 h (n = 3). ( c ) Relative expression of LRRC25 mRNA in BV2 cells after being transfected for 48 h and 72 h and infected by GFP-H37Rv for 4 h at an MOI of 5 (n = 4), Student’s t test. ( d ) Western blot images of LRRC25 in BV2 cells after being transfected for 48 h and 72 h and infected by H37Rv for 4 h at an MOI of 5 (n = 3). ( e ) Histogram and percentage statistics of uninfected BV2 cells in flow cytometry (n = 3), One-way ANOVA, Ex post facto comparison -LSD. ( f ) Confocal images of infected BV2 cells. Among them, the nucleus was stained and marked with Hoechst (blue), and GFP-H37Rv was marked with green fluorescent protein (green). Scale bar, 50 μm. BV2 cells were transfected with L1 for 72 h and infected with GFP-H37Rv for 4 h at an MOI of 5. GA is the abbreviation for GAPDH, and NC is the abbreviation for Negative Control. *** p < 0.001.

Article Snippet: The Mtb standard strains H37Rv (ATCC27294) and GFP-H37Rv (recombinant Mtb -H37Rv strain stably expressing GFP constructed with vector PSC301 (31851, Addgene, Watertown, MA, USA)) used in this study were obtained from the Beijing Tuberculosis Clinical Sample Database of Beijing Chest Hospital affiliated with Capital Medical University, China.

Techniques: Expressing, Transfection, Western Blot, Infection, Flow Cytometry, Comparison, Staining, Negative Control

Journal: Microorganisms

Article Title: LRRC25 Inhibits IFN-γ Secretion by Microglia to Negatively Regulate Anti-Tuberculosis Immunity in Mice

doi: 10.3390/microorganisms11102500

Figure Lengend Snippet: LRRC25 prevents BV2 cells from releasing ISG15 and IFN-γ. ( a ) Relative expression of IL-1β mRNA of BV2 and secretion level of IL-1β in the cell supernatant of each group. ( b ) Levels of IFN-α, IFN-γ, and ISG15 in the cell culture supernatant before and after H37Rv infection of BV2 cells. ( c ) Relative expression of ISG15 mRNA and the expression of ISG15 in the cell supernatant in each group. ( d ) Expression of IFN-γ in the cell supernatant in each group. ( e ) Expression of IFN-α in the cell supernatant in each group. Bar graphs show the mean ± SD from four independent experiments (n = 4). Student’s t test for figure ( b ) and Two-way ANOVA test for figure ( a , c , d ). BV2 cells were transfected with L1 for 72 h and infected with GFP-H37Rv for 4 h at an MOI of 5. ** p < 0.01, and *** p < 0.001.

Article Snippet: The Mtb standard strains H37Rv (ATCC27294) and GFP-H37Rv (recombinant Mtb -H37Rv strain stably expressing GFP constructed with vector PSC301 (31851, Addgene, Watertown, MA, USA)) used in this study were obtained from the Beijing Tuberculosis Clinical Sample Database of Beijing Chest Hospital affiliated with Capital Medical University, China.

Techniques: Expressing, Cell Culture, Infection, Transfection