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. 2020 Sep 21;11(11):2305–2311. doi: 10.1021/acsmedchemlett.0c00423

Figure 3.

Figure 3

TM-P4-Thal inhibits both SIRT2 deacetylase and defatty-acylase activities in cells. (A) Immunofluorescence images of acetyl α-tubulin (K40) in MCF7 cells treated with DMSO (control), TM-P4-Thal (5, 10 μM), and TM (5, 10 μM) for 12 h. (B) Normalized Ac-α-tubulin fluorescence intensity level of 5 μM TM-P4-Thal and TM for 12 h, using ImageJ software for quantification. (C) Immunoblot with pan-Ras antibody to show the stable overexpression of Flag-tagged K-Ras4a-G12V in HEK 293T cells. (D) Immunoblots for SIRT1, SIRT2, and SIRT3 after treating HEK 293T cells with 1 μM of TM-P4-Thal or 10 μM of TM in the presence of 50 μM of Alk-14 for 24 h. (E) Alk14 labeling and in-gel fluorescence to detect lysine fatty acylation of K-Ras4a in HEK 293T cells. Cells were treated with 1 μM TM-P4-Thal or 10 μM TM in the presence of 50 μM Alk-14. (F) Quantified relative lysine fatty acylation level of K-Ras4a from part E, using ImageJ for quantification.