Journal: Scientific Reports

Article Title: BORC complex specific components and Kinesin-1 mediate autophagy evasion by the autophagy-resistant Mycobacterium tuberculosis Beijing strain

doi: 10.1038/s41598-023-28983-5

Figure Lengend Snippet: Depletion of BORCS5-6 and Kinesin-1 reverts the BJN’s ability to resist autophagic elimination in BMDMs. ( a ) BMDMs were transfected with siRNAs, as in Fig. . Successful knockdown was determined by qRT-PCR. Expression levels of the target genes were normalised to that of the housekeeping gene, Gapdh . Data are means ± SEM from at least three independent experiments; ****p < 0.0001, relative to the scrambled siRNA control set to 1.0 determined by one-way ANOVA with Tukey’s multiple comparison test. ( b,c ) BMDMs with decreased expression of BORCS5-8 and Kinesin-1 were infected with the mCherry-expressing H37Rv or BJN and induced to undergo autophagy by starvation, as in Fig. . High-content imaging was then used to assess the number of intracellular mycobacteria per cell. Percent mycobacterial survival was calculated and compared ( b ). Data are means ± SEM from at least three independent experiments; ns, non-significant and ****p < 0.0001, all relative to the full control set of 100% determined by one-way ANOVA with Tukey’s multiple comparison test. Representative images are displayed in ( c ). Bar 10 µm.

Article Snippet: The lysosome distribution in mycobacteria-infected BMDMs was automatically determined by the Columbus image analysis server (PerkinElmer, USA), as described above, but analysed for the number of Lamp1 + lysosomes in each cytoplasmic subregion in the infected cells.

Techniques: Transfection, Knockdown, Quantitative RT-PCR, Expressing, Control, Comparison, Infection, Imaging

Journal: Scientific Reports

Article Title: BORC complex specific components and Kinesin-1 mediate autophagy evasion by the autophagy-resistant Mycobacterium tuberculosis Beijing strain

doi: 10.1038/s41598-023-28983-5

Figure Lengend Snippet: BORCS5-8 and Kinesin-1 suppress lysosome delivery to the BJN phagosomes in BMDMs. ( a,b ) BMDMs deficient in BORCS5-8 and Kinesin-1 expression were infected with the mCherry-expressing H37Rv or BJN and induced to undergo autophagy by starvation, as in Fig. . Cells were fixed and stained for lysosomes using an anti-Lamp1 antibody followed by nuclear staining with Hoechst. Percent mycobacteria-Lamp1 colocalisation was then analysed by high-content image analysis. Data are means ± SEM from at least three independent experiments; ns, non-significant and ****p < 0.0001, all relative to the full control set of 100% determined by one-way ANOVA with Tukey’s multiple comparison test ( a ). Representative images are displayed in ( b ). Bar 5 µm.

Article Snippet: The lysosome distribution in mycobacteria-infected BMDMs was automatically determined by the Columbus image analysis server (PerkinElmer, USA), as described above, but analysed for the number of Lamp1 + lysosomes in each cytoplasmic subregion in the infected cells.

Techniques: Expressing, Infection, Staining, Control, Comparison

Journal: Scientific Reports

Article Title: BORC complex specific components and Kinesin-1 mediate autophagy evasion by the autophagy-resistant Mycobacterium tuberculosis Beijing strain

doi: 10.1038/s41598-023-28983-5

Figure Lengend Snippet: BORCS5-8 and Kinesin-1 dampen lysosome relocation to the perinuclear area in the BJN-infected BMDMs. ( a,b ) BORCS5-8- and Kinesin-1-depleted BMDMs were infected with the mCherry-expressing H37Rv or BJN for 15 min and chased for 1 h, as in Fig. . Autophagy was then induced by starvation for 24 h. Cells were stained with anti-Lamp1 antibody and Hoechst and then processed for high-content image analysis. The number of Lamp1 + lysosomes in each cytoplasmic subregion of the mycobacteria-infected BMDMs was quantified. The percentage of perinuclear Lamp1 + lysosomes (0–4 µm distance from the nucleus) and peripheral Lamp1 + lysosomes (4 µm from the nucleus and cell boundary) were then calculated and compared. Data are means ± SEM from at least three independent experiments; ns, non-significant and ****p < 0.0001, all relative to the full control determined by one-way ANOVA with Tukey’s multiple comparison test ( a ). Representative images with a line specifying the perinuclear region (0–4 µm distances from the nucleus) are shown in ( b ). Bar 5 µm.

Article Snippet: The lysosome distribution in mycobacteria-infected BMDMs was automatically determined by the Columbus image analysis server (PerkinElmer, USA), as described above, but analysed for the number of Lamp1 + lysosomes in each cytoplasmic subregion in the infected cells.

Techniques: Infection, Expressing, Staining, Control, Comparison

Journal: Scientific Reports

Article Title: BORC complex specific components and Kinesin-1 mediate autophagy evasion by the autophagy-resistant Mycobacterium tuberculosis Beijing strain

doi: 10.1038/s41598-023-28983-5

Figure Lengend Snippet: BORCS5-8 and Kinesin-1 are not involved in the BJN delivery to the autophagosomes. ( a,b ) BMDMs deficient in BORCS5-8 and Kinesin-1 expressions were infected with the mCherry-expressing H37Rv or BJN (MOI = 10) for 15 min and chased for 1 h followed by autophagy induction by starvation for 2 h. Cells were fixed and stained with anti-LC3B antibody and Hoechst. Percent mycobacteria-LC3B colocalisation was then analysed by high-content image analysis. Data are means ± SEM from at least three independent experiments; ****p < 0.0001, relative to the full control set of 100% determined by one-way ANOVA with Tukey’s multiple comparison test ( a ). Representative images are displayed in ( b ). Bar 5 µm.

Article Snippet: The lysosome distribution in mycobacteria-infected BMDMs was automatically determined by the Columbus image analysis server (PerkinElmer, USA), as described above, but analysed for the number of Lamp1 + lysosomes in each cytoplasmic subregion in the infected cells.

Techniques: Infection, Expressing, Staining, Control, Comparison

Journal: Molecular Therapy

Article Title: Characterisation of a Novel Fc Conjugate of Macrophage Colony-stimulating Factor

doi: 10.1038/mt.2014.112

Figure Lengend Snippet: Network analysis of liver transcripts post pig CSF1-Fc treatment. Expression data for mouse livers +/- CSF1-Fc ( n = 3) was analyzed alongside that of BMDM expression data (+/- LPS). ( a ) A network graph of transcript-to-transcript Pearson correlation relationships was filtered to show relationships of r ≥ 0.96, resulting in a graph of 2,555 nodes (transcripts) connected by 265,108 edges (Pearson correlation relationships). The graph was then clustered using the MCL clustering algorithm into groups of co-expressed genes. Nodes with the same color belong to the same cluster of co-expressed genes and tend to be highly connected within the network. ( b ) Expression data for five clusters with each point on the graph representing an individual mouse.

Article Snippet: Partek Inc.) Expression data from the liver +/− CSF1-Fc study was analyzed alongside that of BMDM data generated from the same strain of mice (C57BL/6) (see BMDM method below).

Techniques: Expressing