Figure 4. The role of Mesh-Arrestin-ERK/JNK-MAPK signaling cascade in DmDuox regulation in Drosophila.

(a–b) Regulation of the DmDuox gene in the guts of DmERK (a) and DmJNK (b) RNAi flies.

(c) Regulation of ROS activity in the guts of DmERK and DmJNK RNAi flies.

(d) Enhancement of the gut microbiome in the guts of DmERK and DmJNK RNAi flies.

(e–f) Silencing DmArrestins impaired expression of the DmDuox gene (e) and ROS activity (f) in the Drosophila guts.

(g) Increasing the burden of gut microbiome in the guts of DmArrestins RNAi flies.

(h–i) Assessing the role of the “Arrestin-ERK/JNK” cascade in AaMesh-mediated Duox expression in Drosophila. Both DmArrestin-1 and DmArrestin-2 were silenced by dsRNA transfection in the pAc-DmMesh-trnasfected Drosophila S2 cells. (h) The phosphorylation of DmERK (p-ERK) and DmJNK (p-JNK) was detected by western blotting. (i) The abundance of the DmDuox gene was determined by SYBR Green qPCR and normalized by Drosophila actin (CG12051).

(a–g) GFP RNAi flies served as a negative control.

(a–b, e) The gene expression was determined by SYBR Green qPCR and normalized against D. melanogaster actin (CG12051). The qPCR primers are described in Supplementary Table 6. One dot represents one fly gut. The horizontal line represents the mean value of the results.

(c, f) The ROS activity was detected by a H₂O₂ assay. The data are presented as the mean ± S.E.M.

(d, g) The burden of gut microbes was determined by a CFU assay.

(a–g, i) The data were analyzed using the non-parametric Mann-Whitney test.

(a–i) The results were reproduced by at least 3 independent experiments.