Characterization of a highly selective USP9X inhibitor. (A) Chemical structure of FT709. (B) In vitro potency of FT709 against USP9X. Activity is monitored by a fluorescence increase following cleavage of a Ub-rhodamine substrate. (C) BxPC3 cell-based potency of FT709 for reduction of CEP55 measured using a MSD ELISA assay. Graphs in B and C show the average of two experiments with error bars indicating the range. (D–F) Cell lysates (D) or intact MCF7 cells (E) were incubated with FT709 (30 min at 25°C for cell extracts, 3 h at 37°C for cells) at the indicated concentrations. Cells were lysed, and extracts were incubated with 0.1 µg HA-UbC2Br probe for 5 min at 37°C, followed by SDS-PAGE analysis. Samples were immunoblotted with USP9X and HA antibodies as indicated. Arrow indicates HA-probe labeled band corresponding to the USP9X~Ub probe adduct. Modification of USP9X with a ubiquitin probe (USP9X~Ub) was lost with increasing concentrations of inhibitor. (F) Quantitation of Western blots shown in D and E. (G and H) HA-based immunoprecipitation of HA-UbC2Br probe–labeled DUBs from cell lysates incubated first with DMSO, 1 or 10 µM FT709, for 1 h at 37°C. Immunoprecipitated proteins were eluted and either analyzed side by side with total lysate samples by immunoblotting (TL, total lysate; EL, eluate) or subjected to mass spectrometry–based quantification in three technical replicates. Differences in DUB-probe binding were quantified for 21 identified DUBs and normalized relative to DMSO control (error bars represent standard deviation of three technical replicates). See Fig. S2 for uncropped immunoblots. conc, concentration.
Figure 3.

Characterization of a highly selective USP9X inhibitor. (A) Chemical structure of FT709. (B) In vitro potency of FT709 against USP9X. Activity is monitored by a fluorescence increase following cleavage of a Ub-rhodamine substrate. (C) BxPC3 cell-based potency of FT709 for reduction of CEP55 measured using a MSD ELISA assay. Graphs in B and C show the average of two experiments with error bars indicating the range. (D–F) Cell lysates (D) or intact MCF7 cells (E) were incubated with FT709 (30 min at 25°C for cell extracts, 3 h at 37°C for cells) at the indicated concentrations. Cells were lysed, and extracts were incubated with 0.1 µg HA-UbC2Br probe for 5 min at 37°C, followed by SDS-PAGE analysis. Samples were immunoblotted with USP9X and HA antibodies as indicated. Arrow indicates HA-probe labeled band corresponding to the USP9X~Ub probe adduct. Modification of USP9X with a ubiquitin probe (USP9X~Ub) was lost with increasing concentrations of inhibitor. (F) Quantitation of Western blots shown in D and E. (G and H) HA-based immunoprecipitation of HA-UbC2Br probe–labeled DUBs from cell lysates incubated first with DMSO, 1 or 10 µM FT709, for 1 h at 37°C. Immunoprecipitated proteins were eluted and either analyzed side by side with total lysate samples by immunoblotting (TL, total lysate; EL, eluate) or subjected to mass spectrometry–based quantification in three technical replicates. Differences in DUB-probe binding were quantified for 21 identified DUBs and normalized relative to DMSO control (error bars represent standard deviation of three technical replicates). See Fig. S2 for uncropped immunoblots. conc, concentration.

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