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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 5. SIRT3 functions as an ACO2-deacetylase.

Figure 5.

(A) HEK293T cells were transduced with WT-SIRT3-Flag or HA-SIRT3-H248Y plasmid and immunoblotted for ACO2, HA, Flag, Actin, and Ac-K in input depicting WT-SIRT3-Flag or HA-SIRT3-H248Y expression. Transfected cell lysates were used for endogenous ACO2 and IgG (control) immunoprecipitation, followed by immunoblotting for ACO2 and Ac-K.

(B) Adv-GFP (control) and Adv-SIRT3 were re-expressed in TRAMP-C2 cells followed by immunoblotting for ACO2, SIRT3, Ac-K, and Actin for inputs. RWPE-1 (normal prostate cell line) was used to titer the SIRT3 re-expression level in TRAMP-C2 cells. Endogenous ACO2 or IgG (control) was immunoprecipitated, followed by immunoblotting for ACO2 and Ac-K.

(C) Schematic representation showing SIRT3 regulating acetylation-status of ACO2.

(D) C4-2 cells were transduced with Adv-GFP or Adv-SIRT3 for 8 days followed by immunoblotting with SIRT3 and β-actin antibodies. Actin was used as a loading control.

(E) C4-2 cells transduced with Adv-GFP or Adv-SIRT3 for 3 days, were used for viability assay (n=12, each group). Reading was taken at the end of 5 days. Data are shown in mean ± S.D. **** P<0.000001, unpaired t-test two-tailed.

(F) mRNA expression of SIRT3, SRC-2 (gene name: NCOA2), and AR from GSE35988 dataset using GPL9128 platform consisting of Normal n=28, Primary tumor n=59, and Met n=32. Expression of SIRT3, SRC-2, and AR in Primary vs Met, P<0.001.