Fig. 2. Lysosomal genes are significantly up-regulated and endolysosomes are expanded in macrophages isolated from rraga mutants. (A) Experimental schematic for RNA-seq. Macrophages were isolated using FACS from N = 90 larvae for each biological replicate RNA sample from rraga mutants or wild-type siblings. Four biological replicate samples of each genotype were processed for RNA-seq. (B) Volcano plot showing significantly differentially up-regulated and down-regulated genes in the macrophages of rraga mutants relative to wild-type siblings. (C) GO (Cellular Component) enrichment analysis showing an up-regulation of lysosome-associated terms in macrophages from rraga mutants. (D) Imaging and quantification of LAMP1 or LAMP2-mCherry transgene expression in rraga mutants and wild-type siblings at 4 dpf. Scale bars, 20 μm. (E) Imaging and quantification of mCherry-Rab5 or Rab7 transgene expression in rraga mutants and wild-type siblings at 4 dpf. Scale bars, 20 μm. (F) LysoSensor Green labeling and quantification of LysoSensor Green intensity at 4 dpf. Scale bars, 50 μm. Graphs in (D) to (F) show mean + SD; significance was determined using parametric unpaired t test. (G) DQ-BSA labeling at 4 dpf and quantification of DQ-BSA intensity inside macrophages. Scale bars, 50 μm. Graph shows mean + SD; significance was determined using nonparametric Mann-Whitney U test. The number of animals analyzed for each experiment is listed in table S1; all the panels (except RNA-seq) are representative of at least two independent experiments. A.U., arbitrary units.
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