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Establishment of Dual-Specific Phosphatase Overexpressed/Interfered RAW264.7 Cell Strain and Its Effect on Autophagy
Luo Jia, Zeng Jin, Yang Yi, Wang Yujiong*
Key Lab of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China, Ningxia University, Yinchuan 750021, China
Abstract: To study the effect of dual-specificity phosphatase 5 (DUSP5) on the autophagy of macrophages， they established DUSP5 overexpressed/interfered RAW264.7 cell strain by transfecting lentivirus vector. Besides， rapamycin (Rapa) and bafilomycin A1 (Baf A1) were used to stimulate the cell strains. Western blot， realtime fluorescence quantitative PCR (qRT-PCR)， Monodansylcadaverine (MDC) staining and immunofluorescence were all applied to this research. The results showed that RAW264.7 transfected with DUSP5 overexpression/ interference lentivirus could significantly increase/inhibit (P<0.01) the expression of DUSP5 mRNA and protein. Overexpressing DUSP5 inhibited the expression of autophagy-related proteins Beclin1 and LC3II and the formation of intracellular autophagosomes after Rapa stimulation， however， suppressing DUSP5 expression had reversed result. The expression of LC3II and the number of autophagosomes were significantly increased after blocking autophagy flux of DUSP5 interfered RAW264.7 cell strain with Baf A1. In a conclusion， DUSP5 plays a role in the regulation of macrophages autophagy， it may inhibit autophagy by interrupting autophagosomes formation. This finding will provide a new idea for further exploring the regulation mechanism of macrophage autophagy.
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