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

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© 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/.

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Extended Data Fig. 4 — a, Representative QIBC micrographs of HCC1569 cells treated with increasing doses of RP-6306. The DAPI (cyan) and γH2AX (magenta) channels are merged. b, QIBC quantitation of pan-γH2AX staining as a function of time after addition of RP-6306 (500 nM) in HCC1569 cells. Data are shown as mean ± s.d. (n = 3). c, Clonogenic survival assays of the indicated FT282-hTERT TP53^R175H Cas9 cell lines transduced with lentivirus expressing CDK1-T14A-GFP, CDK1-Y15F-GFP or CDK1-T14A/Y15F-GFP relative to WT CDK1-GFP. Data are shown as mean ± s.d. (n = 3). d, QIBC quantitation of pan-γH2AX staining in FT282-hTERT TP53^R175H CCNE1 cells treated with RP-6306 (500 nM) as a function of CDK1 inhibitor RO-3306 dose. Data are shown as mean ± s.d. (n = 3). e, f, FT282-hTERT TP53^R175H CCNE1 cells transfected with either non-targeting siRNA (siCTRL) or siRNA targeting cyclin B (siCCNB1) were treated with RP-6306 (500 nM). Representative QIBC micrographs with DAPI (cyan) and γH2AX (magenta) channels merged are shown in (e). Immunoblot analysis of cyclin B levels in lysates prepared from DMSO-treated cells is shown in (f). Tubulin was used as a loading control and are representative of three immunoblots g, QIBC quantitation of pan-γH2AX staining in FT282-hTERT TP53^R175H CCNE1 treated with RP-6306 (500 nM) as a function of the dose of dinaciclib or (left) PF-06873600 (right). Data are shown as mean ± s.d. (n = 3). h, Representative QIBC micrographs of FT282-hTERT TP53^R175H (WT) and CCNE1-high (CCNE1) cells transfected with the indicated siRNA and stained with DAPI (cyan) and cyclin B-pS126 antibody (magenta). The channels are merged and image represents one replicate. i, Representative QIBC micrographs of FT282-hTERT TP53^R175H (WT) and CCNE1-high (CCNE1) cells treated with RP-6306 (500 nM) and stained with DAPI (cyan), EdU (yellow) and a cyclin B-pS126 antibody, (magenta). The channels are merged and image is representative of three replicates. For gel source data, see Supplementary Fig. 1.

Source data