A homogeneous time-resolved fluorescence screen to identify SIRT2 deacetylase and defatty-acylase inhibitors
Fig 4
Inhibition of SIRT2 by 1 in cells.
(A) Immunofluorescence images of acetylated alpha-tubulin in A549 cells showing enhanced fluorescence in cells treated with 25 μM of SirReal2 (positive control) or 100 μM of 1. The media for the control contained 2% DMSO, and the scale bar is 10 μm. (B) Comparison of GFP fluorescence intensities from acetylated alpha-tubulin staining from the images in panel A. GFP fluorescence intensities from individual cells were measured and normalized to the DAPI intensities of the same cells, and the intensities were compared with a one-way ANOVA followed by Dunnett’s multiple comparison test (***p < 0.001). (C) Western blot showing knockout of SIRT2 in A549 cells using CRISPR/Cas9. The alpha-tubulin blot, which was performed on a separate membrane, was used as a loading control. (D) Immunofluorescence images of A549 SIRT2-KO cells showing increased acetylated alpha-tubulin levels in cells without the protein, and the ligands SirReal2 and 1 had no effect on the levels of acetylated alpha-tubulin in SIRT2-KO cells. Experiments were performed identical to panel A. (E) Comparison of GFP fluorescence intensities from acetylated alpha-tubulin staining from the images in panel D. These were quantified and compared as described above for panel B. (F) SirReal2 treatment for 72 hours did not cause toxicity in A549 cells, as measured with an MTT assay. The control (Ctrl) cells were treated with 0.7% DMSO to be consistent with the drug treated cells. (G) Treatment of A549 cells with 1 for 72 hours did not cause toxicity, which was determined with an MTT assay as in panel F.