Figure 6. PDF modulates EYA protein but not eya mRNA expression.

(A) eya mRNA daily oscillations in Pdf-null mutants (yw ; ; Pdf⁰¹ flies, red dots, RAIN p = 0.833) and in flies expressing a Pdf genomic rescue (yw ; Pdf{WT} ; Pdf⁰¹, white dots, RAIN p = 0.0078) in 12:12 LD cycles at 25°C. (B) Representative western blot and (C) quantification of EYA levels in whole-head lysates from Pdf null-mutant (red dots, RAIN p = 0.677) and in the genomic rescue (white dots, RAIN p = 0.039) at ZT0, 4, 8, 12, 16 and 20 in 12:12 LD 25°C cycles. (D) Representative western blot and (E) quantification of EYA levels from whole head lysates of yw (genetic control, white boxes), Pdf-null mutants (red boxes) and the genomic rescue (grey boxes) at ZT4 and ZT16 in 12:12 LD 25°C condition. (F) Representative western blot and (G) quantification of EYA levels in head lysates of flies where the PDF receptor was knocked down in eya+ cells (eya > Pdfr-RNAi, red boxes) and controls (eya / + and Pdfr-RNAi / +, white and grey boxes, respectively) at ZT4 and ZT16. (H) Representative western blot and (I) quantification of EYA levels in flies expressing a membrane-tethered version of PDF in eya+ cells (eya > t-PDF, red boxes) compared to a scrambled peptide as control (eya > t-SCR, white boxes) at ZT4 and ZT16. Data were analyzed using a two-way ANOVA followed by Tukey’s post hoc test in B and C, and šídák’s multiple comparison test in E, G and I. HSP70 was used for normalization in all cases. n > 3 biological replicates, each consisting of around 50 flies for each genotype.