SuPPLEMENTAL FIGURES
Supplementary Fig. 1. Additional phenotypic analysis of flies with reduced O-GlcNAcase activity. A Dose-response test for the O-GlcNAcase inhibitor NButGT. Lysates of mock-fed larvae (CTL) and NButGT-fed larvae were prepared for Western blot by CTD110.6 to analyze O-GlcNAcylation of total proteins. β-tubulin was used as a loading control. B 200 μl of 10 mM glucose solution added to the food surface did not significantly change the body weight of adult flies. Larvae were cultured from the first instar to the wandering third instar stages on fly food supplemented with glucose at concentrations equivalent to the NButGT solution as described in Fig. 1A and B. The body weight of adult flies was measured (n = 184 ♂ and n = 162 ♀ for control group and n = 100 ♂ and n = 118 ♀ for experimental group). Data are mean ± S.E. C NButGT-fed flies eclosed faster than mock-fed flies. The average time from egg deposition to eclosion was plotted (n = 454 for the control and n = 578 for NButGT). Data are mean ± S.E. *P < 0.0001. D Knock-down efficiencies of the two OGA RNAi transgenes. The relative amounts of OGA mRNA in larvae of indicated genotypes were measured using real-time PCR. Total RNAs were extracted from the third instar larvae of indicated genotypes using Trizol (Invitrogen). RNAs were reverse transcribed into oligo-dT primed cDNA (Reverse Transcription Kit, Promega), and the cDNAs were used as templates for real-time PCR analysis. SYBR Green Master Mix (Applied Biosystems) was used, and the experiments were repeated four times using independently prepared RNA samples. Rp49 was used as a control. Data are mean ± S.E.
Supplementary Fig. 2. Knock-down of OGT decreases the size of the wing imaginal disc. The areas of the male larval wing discs dissected from UAS-only (n = 32) and Act>OGT RNAiII (n = 50) animals were measured. Data are mean ± S.E. *P <0.0001. Scale bar represents 100 µm.
Supplementary Fig. 3. Knock-down of OGA increases cell size. A and B Quantification of the cell size in the wing. A Act>OGA RNAiX flies (n = 15) have fewer hair cells compared to the UAS-only control flies (n = 13). Wing hair cells were counted in an area of 80 × 160 µm between vein 2 and vein 3 of the female adult wing. Scale bar represents 50 µm. C-I’ Post-mitotic expression of UAS-OGA RNAiX transgene increases cell size in the fat body. OGA RNAiX was expressed post-mitotically in a small patch of cells using the flip-out technique after a 10 min heat shock (34°C) 48 h after egg laying. Fat bodies of female third instar larvae were dissected and the average cell size was quantified by measuring the area of OGA RNAiX-expressing cells (green, GFP-positive) and dividing it by the cell number. These values were compared with that of UAS-only (GFP-negative) cells. Cell boundaries were visualized by phalloidin-rhodamine (red), and nuclei with DAPI (blue). C Quantification of the cell size. The average size of OGA RNAiX-expressing cells was 130.1% of the size of the control cells. Data are mean ± S.E. *P <0.01. The size of fat body cells is often dissimilar even in the same lobe. Therefore, we also examined the average size of GFP-positive cells and compared it with that of the immediate neighboring cells that were size-matched GFP-negative cells. The GFP-positive cells were still significantly larger than the neighboring cells (115.4% control; data not shown). D-I’ Six different samples used for the analysis in C are shown. D, E, F, G, H, and I Areas marked by white lines, which include both GFP-positive and GFP-negative cells, were analyzed. D’, E’, F’, G’, H’, and I’ Areas marked by green lines, which include GFP-positive cells only, were analyzed. Scale bar represents 50 µm.
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