Figure 5. Loss of E2F/Dp impairs carbohydrate metabolism.

(A) Overview of tandem-mass tag-mass spectrometry (TMT-MS) profiles generated from third instar larval fat body in Dp-/- mutant and wild-type (WT) animals and from thoracic muscles in Mef2>Dp-RNAi and control animals staged at pharate; 6578 identified proteins in fat bodies and 5730 identified proteins in muscles. (B) TMT-MS intensities showing protein levels of Dp (Uniprot Q24318), E2F2/DP target protein Arp53 (Uniprot Q9VF26), SpnE (Uniprot P455891), and dATM (Uniprot Q5EAK6) in fat bodies and muscles. Data are represented as individual intensity value for each replicate, n = 3 per genotype in fat body and n=4 per genotype in muscles. (C) Volcano plots indicating proteins that are differentially expressed between larval WT and Dp-/- mutant fat bodies (left panel), and Mef2>mCherry-RNAi and Mef2>Dp-RNAi pharate muscles (right panel). Significant changes are shown in red (false discovery rate [FDR] < 0.05 and abs [fold change]>1.5). The x-axis is the log₂ of the fold change and the y-axis is the negative log₁₀ of the adjusted p-value. (D) DAVID functional annotation clustering analysis of proteomic changes in Dp-depleted tissue compared to WT that are in common between fat body and muscles. Upregulated (top panel, 190 proteins) and downregulated (bottom panel, 88 proteins) were analyzed separately. Dashed line indicates FDR=0.05. Only significant terms (FDR<0.05) are displayed. The categories GO term biological processes, KEGG pathway, and Up keywords are shown. (E) Overview of targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) metabolomic profiles generated from whole third instar larvae. Dp-/- mutant animals were compared to two different WT animals (Canton-S and w¹¹¹⁸). (F) KEGG pathway enrichment was done on metabolites that were significantly reduced in Dp-/- mutant compared to both controls. Only significant terms are shown (FDR<0.05). (G) Schematic of the flow of glycolysis toward pyruvate. Metabolites that are significantly reduced in Dp-/- mutant compared to controls are shown in blue. (H) Normalized levels of the metabolites D-glyceraldehyde 3-phosphate, 2,3-diphosphoglycerate, 3-phosphoglycerate, and phosphoenolpyruvate in Dp-/- mutant compared to controls w¹¹¹⁸ and Canton-S. Data are represented as box plot, which extends from 25 to 75 percentiles; line marks median; whiskers extend to 25/75% plus 1.5 times the interquartile range. Values outside the whiskers are outliers. Welch’s ANOVA test, *p<0.05, *** p<0.001. n=6 per genotype. Full genotypes are (A–D) Dp^a2/Dp^a3, w¹¹¹⁸, UAS-Dp[GD4444]-RNAi,Mef2-GAL4 and Mef2-GAL4/UAS-mCherry-RNAi (E–H) Dp^Exel7124/Dp^a3, w¹¹¹⁸ and Canton-S.

Figure 5—figure supplement 1. E2F/Dp-deficient muscles and fat bodies undergo severe changes in their proteome and metabolome.

(A) Western blot analysis of fat body (left panel) and muscle (right panel) protein lysates using the antibodies anti-Dp and anti-beta-actin as a loading control. n = 3 replicates per genotype for fat body and n=4 replicates per genotype for muscles. (B) Correlation score heatmap plots of log two median protein intensities across replicates from fat body (left panel) and muscle (right panel). (C) Correlation of log₂FC between Dp-depleted tissue and wild type in fat body compared to muscles. Pearson correlation test, R=0.172, N=5024 pairs of proteins shared in both tissues. (D) Venn diagrams: 5024 identified proteins are shared across fat body and muscle proteomes; 556 and 844 proteins are up in Dp-depleted compared to control in fat body and muscles, respectively, and 456 and 1208 proteins are down in Dp-depleted compared to control in fat body and muscles, respectively. (E) Heatmap representation for hierarchically clustering of differentially expressed proteins (rows) between Dp-depleted tissue and control samples (column) in fat body (left panel) and muscle (right panel). n = 3 replicates per genotype for fat body and n=4 replicates per genotype for muscles. Normalized protein levels were clustered in upregulated as FC≥1.5 (red) and downregulated as FC ≤ −1.5 (blue), moderated t-test, false discovery rate (FDR)<0.05. (F) Enrichment of KEGG pathway among proteomic changes that are significantly increased (upregulated, FDR<0.05 and FC≥1.5, top panel) and reduced (downregulated, FDR<0.05 and FC ≤ −1.5, bottom panel) in Dp-depleted tissues compared with control tissue. Fat body is on the left and muscle on the right. (G) Heatmap representation showing hierarchically clustering of normalized metabolites (row) between Dp-/- mutant and both control samples (Canton-S and w¹¹¹⁸, column) in third instar larva. Only significant changes to both controls are displayed, ANOVA test, FDR<0.05. n=6 replicates per genotype. Full genotypes are (A–F) Dp^a2/Dp^a3,w¹¹¹⁸, UAS-Dp[GD4444]-RNAi,Mef2-GAL4 and Mef2-GAL4/UAS-mCherry-RNAi (G–H) Dp^Exel7124/Dp^a3, w¹¹¹⁸ and Canton-S.