Development of a CS activity assay for genome-wide RNAi screen in S2 cells. (A) TCA cycle and pyruvate metabolism. The biochemical reactions catalyzed by Citrate synthase, Pyruvate dehydrogenase, and α-ketoglutarate dehydrogenase are shown. (B) The effects of dsRNAs of the known mitochondria regulators on the normalized CS activity (Vmax/Ren) in S2 cells (n = 48). The value of CS is 0.22; 5 μg/mL dsRNA was used. **P < 0.01, ***P < 0.001. Error bars indicate the standard errors. (C) Dose response of the CS activity to Citrate synthase RNAi (n = 48). The normalized CS activities were reduced by RNAi in a dose-dependent manner. Seven dosages of Citrate synthase dsRNA were used, from CS1 to CS7, being 0.5, 1, 2, 4, 8, 16, 32 μg/mL/10⁶ cells. The relative Citrate synthase RNA expression levels after RNAi are shown (upper right). (D) The stability of the CS activity in S2 cells. The CS activity level was represented by the Vmax; 0.5 μg/mL dsRNA was used. Optical densities (OD) at 412 nm for cell lysates from lacZ RNAi and Citrate synthase RNAi are shown on the Y-axis. The X-axis shows time in seconds, and data were captured every 46 sec. (E) The linear range of the CS activity in S2 cells (n = 48). The Y-axis shows CS activity (Vmax). The X-axis shows the Renilla luciferase activity (RLU) of cell lysates. The Renilla luciferase activities are labeled next to each data point. Log scales are applied to both X- and Y-axes.

Rpd3(HDAC1) and HDAC6 modulate mitochondrial functions in vivo. (A) The CS activity in S2 cells transfected with Rpd3 and HDAC6 RNAi (n = 6). The lacZ RNAi acts as a negative control and Citrate synthase RNAi as a positive control. P-value was calculated against lacZ RNAi control group. (B) Rpd3 protein was highly reduced in the transgenic Rpd3 RNAi flies as shown by Western blot. Lysates from the Rpd3 RNAi flies and control siblings were blotted with the antibody against Rpd3 and Tubulin. The Tubulin levels were used as loading controls. (C) HDAC6 protein was highly reduced in the transgenic HDAC6 RNAi flies as shown by Western blot. Lysates from the transgenic HDAC6 RNAi and control siblings were blotted with the antibodies against HDAC6 and Tubulin. The Tubulin levels were used as loading controls. (D) The CS activity in the transgenic Rpd3 RNAi flies and control siblings (n = 8). (E) The CS activity in the transgenic HDAC6 RNAi flies and control siblings (n = 8). (F) The COX activity in the transgenic Rpd3 RNAi flies and control siblings (n = 8). (G) The COX activity in the transgenic HDAC6 RNAi flies and control siblings (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001. Error bars indicate the standard errors.

Whole genome RNAi screen. (A) The scatter plot shows the data from the first 12 duplicated plates of the primary screen. The X- and Y-axes represent NZ_1D for replica b and replica a, respectively. Each spot represents the result of one dsRNA. The Citrate synthase dsRNA concentration was used as 5 μg/mL. “Exp RNAi” indicates experimental RNAi, “CS RNAi” indicates control Citrate synthase RNAi. (B) The result of confirmation screen for the primary hits. The X-axis represents average fold change of normalized CS activity induced by a dsRNA (six replicates) against the lacZ RNAi controls. The Y-axis represents P-value. Hits were selected based on their P-values against lacZ RNAi controls. The hits that negatively affect CS activities (their RNAi result in an increase in CS activity) were selected if their P < 0.05. The hits that positively affect CS activities (their RNAi result in a decrease in CS activity) were selected if their P < 0.01. The Citrate synthase dsRNA concentration was the same as in A. (C) The distribution of the lacZ RNAi controls in the confirmation screen as shown in B. (D) The classification of the confirmed hits by their molecular functions. (E) The classification of the confirmed hits by their biological processes and pathways.

The CS activities in heterozygous mutants (male flies used except for A) with mutation in Citrate synthase (A, female flies), CG3249 (B), vimar (C), Src42A (D–F), smt3 (G,H), klumpfuss (I), barren (J,K). n = 8. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars indicate the standard errors.