Fig 4 -

nazo mutants show diminished organismal triglyceride stores along with increased sensitivity to starvation and oxidative stress (A) Confocal images of fat-body of 40-day old male adults of indicated genotypes, stained with BODIPY 493/503 dye (BODIPY 493/503 dye–green and DAPI–blue; Scale bar = 10μm) (B) Quantification of percentage area covered by lipid droplets in each fat body (N = 5–6 males; Student’s t-test, p-value ***p NP-Gal4::UAS-NazoMyc; nazo/nazo. 6 males per genotype per biological replicates were used in triplicates. (Student’s t-test **** p-value = 2O2, counted every 12 hours (N = 80),Student’s t-test, p-value ***p 2O2 quantification shows that nazo mutants along with CG3740 have increased H2O2 levels relative to WT flies. 8 males per genotypes per biological replicates were used in quadruplets. (Student’s t-test **** p-value = < 0.0001. * = 0.0188).</p

S3 Fig -

(A) Guts from 20-day-old males with relish mutation or ubiquitous knockdown of dSTING by Actin-Gal4 driven dSTING RNAi do not show gut lipid droplet depletion on BODIPY 493/503 dye staining (Scale bar 10μm). (B) Quantification of the amount of food consumed by adult males in 24-hour period laced with Blue-1 food dye color shows that nazo mutants do not have feeding defects. (N = 20, Student t-test p-value ns = not significant) (C) Quantification of gut fluorescence in flies of indicated genotypes fed with fluorescent oleic acid reveals that that nazo mutants are not defective in absorption of fluorescent labeled oleic acid (N = 20, Student t-test, p-value ns = not significant). (D) Quantification of whole-body glucose from males of indicated genotypes. 8 males per genotype per biological replicates were used in triplets. (Student t-test p-value ns = not significant) (E) Nazo microRNA leads to efficient depletion of Nazo transcript. Relative expression of nazo transcripts in guts of flies ubiquitously expressing NazomircroRNA under actin-Gal4 driver. The three transgenic lines represent different insertions of the same microRNA transgene. (Student’s t-test p-value **** = (TIF)</p

S8 Fig -

Confocal images of guts from 20-day old flies harboring a GFP trap in plin-2 (LSD-2) locus stained with GFP (green), Lipid-TOX-Red, and DAPI shows reduced levels of Perilipin-2 upon Nazo depletion (Scale bar = 10μm). (TIF)</p

Drosophila c19orf12 mutants show reduced lifespan, functional deficits, and dysregulation of lipid metabolism genes.

(A) Schematic of Drosophila Nazo and CG3740 genomic locus with deletion (open reading frame in dark blue, 5” and 3” untranslated regions in light blue, extent of deletion indicated by line) (B) Lifespan of males of indicated genotypes (N = 60; WT vs CG3740 p–ns, WT vs nazo pN  =  60 for each genotype, ***p t-test). (D) Lipid metabolism genes upregulated or downregulated (with indicated fold change) in guts of 20-day old nazo/CG3740 mutant males relative to WT flies.</p

GO molecular function analysis of upregulated and downregulated genes from the gut of WT vs <i>nazo/CG3740</i> double mutants.

GO molecular function analysis of upregulated and downregulated genes from the gut of WT vs nazo/CG3740 double mutants.</p

S1 Fig -

(A) Relative expression of Nazo transcripts in guts from WT and nazo mutant flies (Student’s t-test **** p-value = rp49 (control), nazo and CG3740 transcripts in gut extracts (D) Relative expression of human c19orf12 in indicated human tissue (image spliced from RNA expression consensus dataset from human protein atlas). (E) Confocal images of whole guts from 20-day old males of indicative genotypes stained with BODIPY 493/503 dye–green and DAPI–blue: Scale bar = 5μm. There are two LD rich regions in male guts both of which exhibit lipid droplets depletion in 20-day old nazo mutant males. (F) Confocal images of 20 Day old female guts stained with BODIPY 493/503 dye–green and DAPI–blue: Scale bar = 10μm. (G) Quantification of the percentage area occupied by lipid droplets midguts of flies of indicated genotypes (N = 5–6 females, Student’s t-test ** p-value = 0.0074). (TIF)</p

S5 Fig -

(A) Relative expression of nazo transcript in the guts of WT, nazo mutant and UAS-nazoMyc rescue flies (NP-Gal4, UAS-Nazomyc; nazo/nazo); Student’s t-test p-value **** = nazo mutant guts (N = 11) (D) Quantification of the proportion of Smurf male flies in wildtype and nazo mutant males show no significant difference in the gut integrity. (N = 10 males in triplicates) (E) Confocal images of R4/5 regions of the male guts of indicated genotypes and ages stained with Armadillo (Green), Sox21a (Red) and DAPI (Blue) (arrow heads: stem cells and enteroblasts; arrows: enterocytes). (F) Quantification of the number of pH3+ve cells in the guts of wildtype and nazo mutant males at indicated ages. Each data point represents a single gut. (TIF)</p