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. Author manuscript; available in PMC: 2020 Jun 20.
Published in final edited form as: Cell Chem Biol. 2019 Mar 28;26(6):804–817.e12. doi: 10.1016/j.chembiol.2019.02.015

Figure 4:

Figure 4:

Characterization of the target engagement and cellular effects of FMF-04-159-2 and FMF-04-159-R in HCT116 cells. (A) Target engagement of FMF-04-159-2 and FMF-04-159-R with 4 h treatment at the indicated concentration, assessed using biotin-FMF-03-198-2 pull down, followed by immunoblotting. (B) Target engagement of FMF-04-159-2 and FMF-04-159-R, treated for 4 h at 1 μM before washout for the indicated time, assessed using biotin-FMF-03-198-2 pull down followed by immunoblotting. (C) Immunoblot for known phospho-substrates of targets of FMF-04-159-2 identified by KiNativ. HCT116 cells were treated for either 6h or 4 h followed by 2 h compound washout with the indicated inhibitors. (D) Double thymidine-synchronized HCT116 CDK14 knockout cellsexpressing WT or C218S CDK14 were released and allowed to progress through the cell cycle, subject to treatment with 1 μM FMF-04-159-2 or FMF-04-159-R for 4 h windows, followed by compound washout. Cells were collected at indicated times after release. (E) FACS PI cell cycle analysis of 24 h treatment of HCT116 CDK14 knockout cells expressing WT or C218S CDK14. Reported as mean ± standard error for percentage of cells in each cell cycle phase from n=3 biological replicates. See also Figure S4.