Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2022 Apr 20;604(7907):749–756. doi: 10.1038/s41586-022-04638-9

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Extended Data Fig. 2 — a, Chemical structure of RP-6421, an analog of RP-6306 b, Dose-response of RP-6421 on PKMYT1 catalytic activity as measured with ADP-Glo kinase assay. c, d, Cell culture of FT282-hTERTTP53^R175H CCNE1-high cells were treated with the indicated doses of RP-6306 and AZD-1775 for 24 h. Left, cellular extracts were prepared and immunoprecipitated (IP) with agarose-coupled CDK1 (c) or CDK2 (d) antibodies. Immunoprecipitates were subjected to in vitro kinase assays using [γ³²P]-ATP and recombinant histone H1 as a substrate. Reactions were resolved by SDS-PAGE and gels were imaged using a phosphor screen. A sample of each immunoprecipitate was immunoblotted (IB) with CDK1 or CDK2 antibodies. CDK1i (RO-3306) or CDK2i (PF-06873600) was added to indicated in vitro reactions. Right, quantitation ³²P-H1 band intensity. Data are shown as mean ± s.d. (n = 3) and P value was determined by one-sided sum-of-squares f-test. e, HCC1569 cells were treated with the indicated doses of RP-6306 for 2 h and subjected to the AlphaLISA assay using CDK1-pT14, CDK1-pY15 and total CDK1 antibodies. Data are shown as mean ± s.d. (n = 3). f, FT282-hTERT TP53^R175H parental and CCNE1-high cells were treated with the indicated doses of RP-6306 and AZD-1775 for 4 h. Whole cell lysates were prepared and immunoblotted with antibodies against CDK1-pT14, CDK1-pY15, CDK1 and vinculin as a loading contol. Representative of two immunoblots. g,h,k, EC50 determination for growth inhibition for the parental and CCNE1-high cells in the (g) RPE1-hTERT TP53 ^−/− Cas9 (RPE1) (h) FT282 TP53^R175H (FT282) and (k) indicated cancer cell lines treated with the indicated compounds. Growth was monitored with an Incucyte for up to 6 population doublings. Data are shown as mean ± s.d. (n = 3). In (k) cell lines are also grouped according to their CCNE1 or BRCA status and the red bar indicates the mean of each grouping. i, Whole cell lysates of FT282-hTERT TP53^R175H parental and CCNE2-2A-GFP expressing cells were immunoblotted with cyclin E2 and KAP1 specific antibodies. Representative of two immunoblots. j, Clonogenic survival of FT282-hTERT TP53^R175H CCNE2-2A-GFP (CCNE2) and WT parental cells treated with RP-6306. Shown on top are representative images of plates stained with crystal violet and bottom is the quantitation. Data are shown as mean ± s.d. (n = 3). For gel source data, see Supplementary Fig. 1.

Source data