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. Author manuscript; available in PMC: 2020 Jan 9.
Published in final edited form as: J Am Chem Soc. 2018 Dec 20;141(1):191–203. doi: 10.1021/jacs.8b07911

Figure 3.

Figure 3.

Identifying a selective inhibitor of PKN3. (A) Structures of JZ128 and related compounds. (B) Summary of results for in vitro kinase assays for PKN3. (C) Live PC3 cell treatments with JZ128. After lysis samples were treated with THZ1-DTB or JZ128-DTB, followed by streptavidin pulldown and Western blot. CCNK was used as a proxy for CDK12/13. (D) Live PC3 cell treatments with JZ128-R. After lysis samples were treated with JZ128-DTB, followed by streptavidin pulldown and Western blot. (E) Similar to Figure 2D, dual FLAG-HA tagged PKN3 WT and PKN3 C840S constructs were expressed in PC3 cells lacking endogenous PKN3. Lysates were treated with JZ128-DTB followed by streptavidin pulldown or HA immunoprecipitation and Western blot. In = 1% Input, Avi = Streptavidin pulldown, HA = HA pulldown. Blot lanes were rearranged for clarity. (F) Confluent PC3 PKN3 KO cells expressing PKN3 rescue constructs were serum starved and treated with DMSO, JZ128, or reversible control. A wound was scored across the culture and monitored for 24 h. Shown is the mean ratio of remaining wound area after 24 h. normalized to the mean wound area of the DMSO condition for each cell line with individual replicate values shown as colored circles. See Figure S4 for representative raw images. p-values determined by unpaired 2-sided t test. (n = 6–9 replicates) NS: p-value >0.05, **: p-value = 0.0015, ***: p-value = < 0.0009. Error bars show one standard deviation from the mean.