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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 4. Lys 258 acetylation is required for optimal ACO2 enzyme activity.

Figure 4.

(A) PC3 cells were transfected with myc-ACO2-WT or myc-ACO2-K258R for 48 hours (n=4, each group). Immunofluorescence staining was performed with anti-myc (green) while being counterstained with mitotracker (red) and DAPI (blue) followed by confocal microscopy. Scale bars, 10 μm.

(B) Ectopic protein expression of ACO2-WT and ACO2-K258R were analyzed by immunoblotting with ACO2 and myc antibodies, and β-actin as a housekeeping loading control.

(C) Lentivirus expressing myc-ACO2-WT or myc-ACO2-K258R was transduced into C4-2-ACO2-KD cells. Re-expression was confirmed by immunoblotting with ACO2 and myc antibodies using β-actin as a housekeeping loading control.

(D) C4-2-ACO2-KD cells were transduced with myc-ACO2-WT or myc-ACO2-K258R for 4 days (n=9, each group) and counted on days 1, 5, and 7. Data are shown in mean ± S.D. **** P=0.0001, two-way ANOVA with Dunnett’s multiple comparisons test.

(E) Mitochondrial fractions from WT, KD, and KD cells re-expressing myc-WT-ACO2 or myc-ACO2-K258R were immunoblotted with Tom20, ACO2 and myc antibodies.

(F) Mitochondrial fractions of C4-2 cells expressing either WT (n=3) or K258R (n=6) were used to measure ACO2 enzyme activity. The activity was normalized to the protein concentration. Boxes are as in Figure. 2D. **P=0.0021, unpaired t-test two-tailed.