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

Extended Data Fig. 4. RP-6306 activates cyclin B-CDK1.

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 TP53R175H 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 TP53R175H 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 TP53R175H 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 TP53R175H 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 TP53R175H (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 TP53R175H (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