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. Author manuscript; available in PMC: 2021 Jul 1.
Published in final edited form as: Cancer Res. 2020 Oct 28;81(1):50–63. doi: 10.1158/0008-5472.CAN-20-1708

Figure 2. ACO2 regulates mitochondrial programming to promote citrate synthesis.

Figure 2.

(A) C4-2-WT and C4-2-ACO2-KD cells (n=5, each group) were used to measure oxygen consumption rates (OCR) sequentially before and after the addition of oligomycin (2 μM), FCCP (1 μM), and antimycin A (1 μM) by XF24 Extracellular Flux Analyzer. Data are shown in mean ± S.D. Each time point is statistically significant. **** P<0.000001, two-way ANOVA with Sidak’s multiple comparisons test.

(B) Acute ACO2 knockdown was induced by lentivirus pGIPZ-shNT or pGIPZ-shACO2 for 4 days in C4-2 cells (n=3, each group) and cultured overnight in regular RPMI media with or without 2mM glutamine. Maximal OCR rates were measured. Data are shown as mean ± S.D. * P=0.007012, two-way ANOVA with Sidak’s multiple comparisons test.

(C) C4-2-WT and C4-2-ACO2-KD cells were stained with Oil O Red followed by microcopy (100x oil immersion). Scale bars, 40 μm.

(D) Percentage distribution of citrate C13 isotopologues in C4-2-WT and C4-2-ACO2-KD cells from L-[U-C13] glutamine after 24 hours of labeling (n=3, biological cell sample). Data are shown as mean ± S.D. *P < 0.05, **P < 0.001 and ****P < 0.0001 by 2-way ANOVA with Tukey’s multiple comparisons test.

(E) Glutamine (m+5), α-ketoglutarate (m+5), and malate (m+3) labeling from L-[U-C13]glutamine in C4-2-WT and C4-2-ACO2-KD cells are shown (n=3, biological cell sample). *P < 0.05 and ***P < 0.001 by 2-way ANOVA with Tukey’s multiple comparisons test.

(F) Mitochondrial fraction from C4-2-WT (n=18) and C4-2-ACO2-KD (n=6, biological sample) cells were used for ACO2 activity. Boxes represent the twenty-fifth and seventy-fifth percentiles, lines represent median, whiskers showing minimum and maximum points, and plus symbol indicates the mean. ** P=0.0028, unpaired t-test two-tailed.

(G-H) Heatmap showing the relative abundance of key TCA-cycle metabolites and selected lipids (Stearic acid, oleic acid, and palmitic acid) in (G) C4-2-WT, C4-2-ACO2-KD1 and C4-2-ACO2-KD2 cells, and (H) 22Rv1-WT, 22Rv1-ACO2-KO1 and 22Rv1-ACO2-KO2 cells. Metabolite abundance was normalized and calculated as log2 fold change compared to WT cells. Rows represent metabolites that are clustered based on euclidean distance. Columns represent WT and two different ACO2-depleted groups with n=3, biological cell samples. Statistical testing between different groups was performed using Dunnett's multiple comparisons test.