GSK3β knockdown modulates the transcription of immune genes in Ae. fluviatilis embryonic cell line.A, schematic representation of experimental design: wAflu1 cells were treated with dsGSK3β. B, GSK3β transcript fragment was amplified by qRT-PCR using cDNA from wAflu1 cell lines. mRNA levels are expressed as mean ± SD (n = 3). C, Western blotting and densitometry for anti-phospho-GSK3β and anti-β-tubulin from two independent replicates show the effect of dsGSK3β in phosphor-GSK3β content. Data were normalized by the dsGFP results, and expressed in arbitrary units (A.U.), where each bar shows the mean ± SD. D, glycogen content was analyzed in three independent biological samples with three experimental replicates each, using the t test. Asterisks indicate significant differences (∗∗∗∗p < 0.0001). E, WSP gene was quantified by qPCR using DNA from wAflu1 cell line. Relative quantification (WSP/RP49) is expressed as mean ± SD (n = 3). F, G, REL2 (F) and gambicin (G) transcript fragments were amplified by RT-PCR using cDNA from wAflu1 cell lines. mRNA levels are expressed as mean ± SD (n = 3). Quantitative PCR results were analyzed using the t test; asterisks indicate significant differences (∗∗∗∗p < 0.0001). GSK3β, glycogen synthase kinase β; REL2, Relish 2; WSP, Wolbachia surface protein.