Figure 3.
ROS-independent activation of JNK signaling triggers pericardial upd3 expression. (A) Diagram showing the signaling cascade that involves ROS, Ask1, and JNK/p38 in activating upd3 expression in the adult PCs during normal physiological conditions. (B and C) Drop in the levels of ROS (DHE staining; red) in the PCs of HSD-fed flies (C) as compared with those reared on ND (B). (D) Quantification of the mean fluorescence intensity for DHE staining in the PCs of flies reared on either ND or HSD. The dots represent the number of PCs analyzed for each genotype. (E and F) Drop in the levels of phosphorylated p38 (red) in the PCs of HSD-fed flies (F) as compared to those reared on ND (E). Phalloidin (green) marks the cardiac tube and the alary muscles. (G) Quantification of the mean fluorescence intensity for phosphorylated p38 levels in the PCs of flies reared on either ND or HSD. The dots represent the number of PCs analyzed for each genotype. (H–I′) Increase in the expression of TRE-dsRed (red; reporter for JNK pathway) in the PCs of HSD-fed flies (I and I′) as compared to those reared on ND (H and H′). Phalloidin (green) marks the cardiac tube and the alary muscles. H and I show TRE-dsRed expression in grayscale; H′ and I′ represent the merged images of the two colors. (J) Quantification of the mean fluorescence intensity for TRE-DsRed expression in the PCs of flies reared on either ND or HSD. The dots represent the number of PCs analyzed for each genotype. (K) Changes in the level of upd3 expression in the PCs of flies reared on ND or HSD upon attenuating JNK signaling. (L–N′) Changes in TRE-dsRed expression (red) in the PCs of HSD-fed flies upon PC-specific attenuation of Ask1 (N and N′) as compared with that observed in HSD-fed flies (M and M′), and upon rearing them on ND (L and L′). Phalloidin (green) marks the cardiac tube and the alary muscles. L, M, and N show TRE-dsRed expression in grayscale; L′, M′, and N′ represent the merged images of the two colors. (O) Quantification of the mean fluorescence intensity for TRE-DsRed expression in the PCs of HSD-fed flies upon attenuating Ask1. The dots represent the number of PCs analyzed for each genotype. (P) Changes in the level of upd3 expression in the PCs of flies reared on ND or HSD upon PC-specific attenuation of Ask1. 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 (D, G, and J) or by two-way ANOVA with Tukey’s multiple-comparison test (K, O, and P). Scale bars, 50 μm.

ROS-independent activation of JNK signaling triggers pericardial upd3 expression. (A) Diagram showing the signaling cascade that involves ROS, Ask1, and JNK/p38 in activating upd3 expression in the adult PCs during normal physiological conditions. (B and C) Drop in the levels of ROS (DHE staining; red) in the PCs of HSD-fed flies (C) as compared with those reared on ND (B). (D) Quantification of the mean fluorescence intensity for DHE staining in the PCs of flies reared on either ND or HSD. The dots represent the number of PCs analyzed for each genotype. (E and F) Drop in the levels of phosphorylated p38 (red) in the PCs of HSD-fed flies (F) as compared to those reared on ND (E). Phalloidin (green) marks the cardiac tube and the alary muscles. (G) Quantification of the mean fluorescence intensity for phosphorylated p38 levels in the PCs of flies reared on either ND or HSD. The dots represent the number of PCs analyzed for each genotype. (H–I′) Increase in the expression of TRE-dsRed (red; reporter for JNK pathway) in the PCs of HSD-fed flies (I and I′) as compared to those reared on ND (H and H′). Phalloidin (green) marks the cardiac tube and the alary muscles. H and I show TRE-dsRed expression in grayscale; H′ and I′ represent the merged images of the two colors. (J) Quantification of the mean fluorescence intensity for TRE-DsRed expression in the PCs of flies reared on either ND or HSD. The dots represent the number of PCs analyzed for each genotype. (K) Changes in the level of upd3 expression in the PCs of flies reared on ND or HSD upon attenuating JNK signaling. (L–N′) Changes in TRE-dsRed expression (red) in the PCs of HSD-fed flies upon PC-specific attenuation of Ask1 (N and N′) as compared with that observed in HSD-fed flies (M and M′), and upon rearing them on ND (L and L′). Phalloidin (green) marks the cardiac tube and the alary muscles. L, M, and N show TRE-dsRed expression in grayscale; L′, M′, and N′ represent the merged images of the two colors. (O) Quantification of the mean fluorescence intensity for TRE-DsRed expression in the PCs of HSD-fed flies upon attenuating Ask1. The dots represent the number of PCs analyzed for each genotype. (P) Changes in the level of upd3 expression in the PCs of flies reared on ND or HSD upon PC-specific attenuation of Ask1. 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 (D, G, and J) or by two-way ANOVA with Tukey’s multiple-comparison test (K, O, and P). Scale bars, 50 μm.

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