Increased levels of pericardial Upd3 upregulate prc expression in fat cells of HSD-fed flies. (A and B) Increase in the reporter lacZ expression for upd3 (red) in the PCs of HSD-fed flies (B) as compared with flies reared on ND (A). Phalloidin (green) marks the heart and the alary muscles. Scale bars, 50 μm. (C) Quantification of the mean fluorescence intensity for upd3-lacZ expression in the PCs of flies reared on either ND or HSD. The dots represent the number of PCs quantified for each condition. (D) Increase in the levels of upd3 expression in the PCs of HSD-fed flies as compared with that observed upon rearing on ND. The transcript levels are normalized to that of the constitutive ribosomal gene rp49. (E) Changes in the levels of prc expression in the fat cells upon knocking down upd3 in the PCs of flies reared on either ND or HSD. The transcript levels are normalized to that of the constitutive ribosomal gene rp49. (F–H) Changes in Prc (red) accumulation around the heart in HSD-fed flies upon knocking down upd3 in the PCs (H) as compared with that observed in HSD-fed flies (G), and upon rearing them on ND (F). Scale bars, 50 μm. (I) Quantification of the mean fluorescence intensity for Prc accumulation around the second heart chamber in HSD-fed flies upon knocking down upd3 in the PCs. The dots represent the samples analyzed for each genotype. (J) Representative M-modes for heartbeats of adult flies showing the movement of the heart tube walls (y-axis) over time (x-axis). (K) Changes in arrhythmia index in the hearts of HSD-fed flies upon knocking down upd3 in the PCs. The dots represent the samples analyzed for each genotype. Genotypes are as mentioned. Data are represented as mean ± SD. P values (ns ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 1 × 10−3) were obtained by unpaired Student’s t test (two-tailed) with Welch’s correction (C and D) or by two-way ANOVA with Tukey’s multiple-comparison test (E) and one-way ANOVA with Tukey’s multiple-comparison test (I and K).
