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A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening
Published on: January 17, 2014
[ASAP1 knockdown reduces migration of RAW264.7 cells infected with Mycobacterium tuberculosis]
Jia Cui1, Da Wen2, Yuhuan Wang2
1Institutes of Biomedical Sciences, Shanxi University, Taiyuan 030006; Department of Microbiology, Changzhi Medical College, Changzhi 046000, China.
Abstract:
Objective To investigate the role of ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) in the infection of murine macrophages RAW264.7 with Mycobacterium tuberculosis, and the function of ASAP1 in macrophage migration. Methods The expression of ASAP1 was down-regulated in RAW264.7 cells using siRNA, and then the cells were infected with H37Ra. The intracellular bacterial load was determined by fluorescence confocal microscopy and colony forming unit (CFU), and the migration ability of the cells were evaluated by TranswellTM assay. Results Compared with the control cells, the intracellular bacterial load was significantly enhanced in ASAP1-knockdown cells, whereas the migration ability of cells was reduced after knockdown of ASAP1 expression. Conclusion ASAP1 is important for RAW264.7 cell migration, and down-regulated ASAP1 expression may impair the migration ability of RAW264.7 cells.
Insights
ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) is crucial for macrophage migration and controlling Mycobacterium tuberculosis infection. Reduced ASAP1 impairs macrophage movement and increases bacterial load.
Area of Science:
- Immunology
- Cell Biology
- Microbiology
Background:
- Mycobacterium tuberculosis (M.tb) infection impacts macrophage function.
- ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) is a protein involved in cellular processes.
- The specific role of ASAP1 in macrophage response to M.tb is not fully understood.
Purpose of the Study:
- To investigate the role of ASAP1 in murine macrophage RAW264.7 infection with Mycobacterium tuberculosis.
- To determine the function of ASAP1 in macrophage migration.
Main Methods:
- ASAP1 expression was reduced in RAW264.7 cells using siRNA.
- Cells were infected with H37Ra strain of M.tb.
- Intracellular bacterial load was assessed via fluorescence confocal microscopy and colony-forming unit (CFU) assays.
- Macrophage migration ability was evaluated using Transwell assays.
Main Results:
- Knockdown of ASAP1 significantly increased intracellular bacterial load in RAW264.7 cells.
- Reduced ASAP1 expression led to decreased migration ability of the macrophages.
- ASAP1 plays a role in regulating macrophage response to M.tb infection.
Conclusions:
- ASAP1 is essential for RAW264.7 cell migration.
- Down-regulation of ASAP1 expression impairs macrophage migration, potentially affecting host defense against M.tb.
