Exosomal miR-224 contributes to hemolymph microbiota homeostasis during bacterial infection in crustacean
Fig 5
Role of HSP70 in exosomal miR-224-mediated hemolymph microbiota homeostasis.
(A) The participation of HSP70 in miR-224-mediated ALFs regulation in mud crabs. AMO-miR-224 was co-injected with HSP70-siRNA into V. parahaemolyticus-challenged mud crabs, followed by analysis of the expression levels of ALFs using qPCR. Data were presented relative to the value of Vp group, which was treated as standard “1”. (B-C) The involvement of HSP70 in miR-224-mediated ROS production. The level of ROS in mud crab hemocytes was determined using fluorescence microscopy, Scale bar, 50 μm (B) and microplate reader (Data were presented relative to the value of Vp group, which was treated as standard “1”) (C). (D) The effect of the indicated exosomes on HSP70 expression. Isolated exosomes from mud crabs treated with PBS and V. parahaemolyticus were injected into mud crabs for 48 h, followed by determination of HSP70 protein level using Western blot analysis, tubulin was used as an internal reference. (E-F) The effect of HSP70 silencing on exosome-mediated hemolymph microbiota homeostasis. Hemolymph was collected from mud crabs with the indicated treatments and subjected to 16S rDNA sequencing, then the bacterial cell count (E) and species (F) were analyzed. The data of Vp+Exosome-PBS and Vp+Exosome-Vp groups were from Fig 2D. All the data are the average from at least three independent experiments, mean ± s.d. (*, p<0.05; **, p<0.01).