Supplementary Figure 2: Secreted HRG-7 exhibits a tissue specific localization pattern.
From: Inter-organ signalling by HRG-7 promotes systemic haem homeostasis
(a) ZnMP staining in worms fed dsRNA against vector control, hrg-4, hrg-1, or hrg-7. Worms were exposed to RNAi from L1 to L4 larval stages, then pulsed with 10 mM ZnMP overnight. ZnMP staining was quantified using the region of interest function of Simple PCI. ZnMP intensity is presented as fold change compared to vector. Graph represents the mean and SEM of three biological independent experiments. N = 10 worms per treatment per experiment. ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05, when compared to control worms (one-way ANOVA). See Supplementary Table 3 for statistics source data. (b) mCherry expression in the anterior (left panel) and posterior (right panel) of strain IQ7670 (Pvha−7::HRG-7::mCherry). Scale bar = 20 μm. Images are representative of three independent experiments. (c) Coexpression of the coelomocyte marker Punc-122::GFP and Pvha−6::HRG-7::mCherry. Image depicts a middle coelomocyte. Scale bar = 20 μm. Images are representative of three independent experiments. (d) Left panels: mCherry expression in the gonad and coelomocytes of IQ8333 (Pvha−6::HRG-3::mCherry). No mCherry is observed in the anterior of the worm. Right panels: mCherry expression in the intestine, coelomocytes, and anterior of strain IQ8330 (Pvha−6::HRG-3-SP::mCherry). In the anterior of the worm, mCherry is non-specifically diffused throughout the pseudocoelomic fluid. Scale bar = 20 μm. Images are representative of three independent experiments. (e) Representative images of tissue specific expression of GFP/YFP markers Pceh−22::GFP (pharyngeal muscle), Pdpy−7::YFP (hypodermis), and Pmyo−2::GFP (body wall muscle) crossed into strain IQ7670 (Pvha−6::HRG-7::mCherry). ∗ indicates body wall muscle (BWM). Arrow indicates pharyngeal muscle (PM). Arrowhead indicates hypodermis (H). Scale bar = 20 μm. Images are representative of three independent experiments. (f) GFP fluorescence quantified from IQ6015 [(Phrg−1::GFP; lin-15b(n744)] fed dsRNA against control, hrg-4, mrp-5, or hrg-7 alone or in combination. GFP was quantified using COPAS BioSort. GFP is presented as fold change compared to vector. Graph represents the mean and SEM of three biological independent experiments. N = 120 worms per treatment per experiment. ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05 (one-way ANOVA). See Supplementary Table 3 for statistics source data.
