Figures and data
Reduced longevity response to DR in ClkJrk mutant flies
(A-B) Survival curves of wild-type control flies (iso31) and ClkJrk homozygous mutant flies in 1%, 5%, 10%, 15%, and 20% Sucrose-Yeast (SY) diets (total dilution). (C) Mean lifespan plots of iso31 and ClkJrk flies across different concentrations of SY diets. Basic survival parameters from the Kaplan-Meier method for each diet and genotype are in Table S1. P values represent diet*genotype interaction effects from Cox proportional hazards analysis. Independent replication of the experiment is presented in figure S2 and Table S1.
Reduced fecundity response to diets in ClkJrk mutant flies
(A & B) Cumulative and daily average number of eggs produced per fly for 7 days in wild-type control flies (iso31) and ClkJrk homozygous mutant flies in 5%, 10%, 15%, and 20% Sucrose-Yeast (SY) diets. (C) Cumulative number of eggs produced per fly over 7 days. ** p < 0.01 by t-test for specified pair-wise comparison. All error bars represent SEM.
Effects of DR on circadian transcriptome in the abdominal fat body
(A) Lifespan extension by DR. Samples for RNA-Seq analysis were collected after ~5 days under either control or DR diets. (B) Number of rhythmic genes (0.25 < FDR from Boot eJTK analysis and fold change in TPM ≥ 1.5). (C) Re-organization of circadian transcriptome by DR. Heatmap represents relative expression (Z-score, yellow=high, blue=low expression) of rhythmic genes in each group across 48 h at 2 h intervals (2 replicates of 12 samples for 24 h). Genes in the top panels are rhythmic in both control and DR diets (common); those in the middle panels are rhythmic in control diet (left) but arrhythmic in DR diet (right); those on the bottom panels are rhythmic in DR diet (right) but arrhythmic in control diet (left). (D) DR failed to affect the expression pattern of core clock genes. (E) Effect of DR in overall time-averaged expression of rhythmic genes in each group. Time-averaged expression in control and DR was compared by t-test with Benjamini-Hochberg correction (Padj ≤ 0.05). TPMs of rhythmic genes in each group were averaged across all time points and were normalized to those in control diet. (F) Increased overall expression in the common rhythmic genes by DR. Heat map represents relative expression (Z-score) of common rhythmic genes across all time points from both control and DR diets. (G) Examples of common rhythmic genes with increased expression by DR.
Oscillation of proteasome subunits by DR
(A) Identification of the co-expression module enriched with proteasome subunit genes by DR. The “proteasome module” is enlarged at the bottom right of the dendrogram. Gene co-expression networks under the DR diet were built using the WGCNA/r package. A topological overlap matrix (TOM) was then computed to evaluate the neighborhood similarities between genes and to classify network genes into modules using hierarchical clustering and dynamic tree cut (methods). (B) Gene ontology (GO) analysis for the genes in the proteasome module. Enrichment scores in P value were corrected with Benjamini-Hochberg approach with 0.01 as threshold. (C) Physical interaction map among the genes in the proteasome module. Physical interaction was analyzed in the esyN network builder (www.esyN.org) and visualized using the Cytoscape program. (D) Increased overall expression of proteasome subunits by DR. Time-averaged expression in control and DR was compared by t-test with Benjamini-Hochberg correction (Padj ≤ 0.05). All error bars represent SEM.
Reduced longevity response to DR by prosβ3 and rpn7 knockdown in the abdominal fat body.
(A-B) Survival curves of the flies with prosβ3 and rpn7 knockdown (+RU) in the adult abdominal fat body (S106-GeneSwitch (GS) driver) and their controls (- RU) in control and DR diets. Survival curves were pooled from 3~4 independent trials (See also Fig. S6 and Table S1 for survival curves and detailed statistical analysis for the independent trials). p < 0.0001 and p = 0.0169 for prosβ3 and rpn7, respectively for the gene*diet interaction effects from Cox proportional hazards analysis.
