Skip to main content
. 2016 Feb 25;59(10):4462–4475. doi: 10.1021/acs.jmedchem.5b01865

Figure 4.

Figure 4

FRAP assay using U2OS cells transfected with GFP–BRD9. (a) Recovery half times of wild-type (wt) cells treated with DMSO in the absence or presence of 2.5 μM SAHA or treated with 1 at 1 μM and SAHA as indicated. In addition, cells expressing GFP–BRD9 with a BD-inactivating mutation (N100F) were analyzed. Significant differences relative to cells treated with SAHA (p < 0.0001) are shown by ****. (b) Time dependence of fluorescence recovery in the bleached area of cells expressing wt or mutant GFP–BRD9 with the corresponding treatments shown in (a). (c) Recovery half times of cells expressing wt GFP–BRD9 treated with various concentrations of DMSO and 2 in the presence or absence of SAHA as indicated. Cells expressing the GFP–BRD9 mutant (N100F) were treated as indicated. Significant differences relative to cells treated with SAHA (p < 0.0001) are shown by ****. (d) Time dependence of fluorescence recovery in the bleached area of cells expressing wt or mutant GFP–BRD9 with the corresponding treatments shown in (c). Curves represent averaged data of at least 20 replicates. 1 shows potency (100% inhibition) at 1 μM in the BRD9 FRAP assay. 2 shows potency (∼90% inhibition) at 0.1 μM in the BRD9 FRAP assay. Both compounds showed no toxicity in U2OS cells after 24 h. The N100F construct is a negative control BRD9 mutant in which Asn100 is replaced by Phe100 and therefore acetylated histone cannot bind because of the lack of interaction to the anchor Asn and because of steric hindrance. SAHA is added to the mixture to increase the signal-to-noise ratio by inhibiting the deacetylation of histones.