Extended Data Fig. 1: Characterization of CCNE1-high isogenic cell lines and validation of PKMYT1 synthetic lethality.
From: CCNE1 amplification is synthetic lethal with PKMYT1 kinase inhibition
a,d, Whole cell lysates of RPE1-hTERT TP53-/- Cas9 CCNE1-2A-GFP (a) and FT282-hTERT TP53R175H (d) CCNE1-high and parental (WT) cells were immunoblotted with cyclin E, CHK1-pS345 and actinin specific antibodies. b, FACS analysis of RPE1-hTERT TP53-/- Cas9 CCNE1-high and parental (WT) cells for EdU incorporation and DNA content (DAPI). Percentage of each population gated for EdU+ (S) and EdU- 2C (G2/M) are indicated. c. QIBC analysis of RPE1-hTERT TP53-/- Cas9 CCNE1-high and parental cells for chromatin-bound MCM2 nuclear intensity, EdU incorporation and DNA content (DAPI). e, FACS analysis FT282-hTERT TP53R175H CCNE1-high and parental (WT) cells for EdU incorporation and DNA content (DAPI). Percentage of each population gated for EdU- 1C (G1), EdU+ (S) and EdU- 2C (G2/M) are indicated. f, Left, QIBC analysis of FT282-hTERT TP53R175H CCNE1-high and parental cells for chromatin-bound MCM4 nuclear intensity, EdU incorporation and DNA content (DAPI). Right, quantitation of EdU- 1C (G1) and EdU+ (S) nuclei with chromatin-bound MCM4 (A.U. > 120). Data are shown as mean ± s.d. (n = 3). g–j, Clonogenic survival assays of (g,h) the indicated RPE1-hTERTTP53-/- Cas9 cell lines transduced with lentivirus expressing the indicated sgRNAs and (i,j) the indicated FT282-hTERT TP53R175H cell lines nucleofected with Cas9 ribonucleoproteins assembled with the indicated sgRNAs. Shown in (g,i) are representative plates with colonies stained with crystal violet. Quantitation is shown in (h, j). Data are shown as mean ± s.d. (n = 3). k, Whole cell lysates of RPE1-hTERT TP53-/- Cas9 CCNE1-high and parental cells expressing doxycycline-inducible sgRNA-resistant Flag alone (-), Flag-PKMYT1 or Flag-PKMYT1N238A were immunoblotted with Flag and actinin antibodies. l, m, Whole cell lysates of RPE1-hTERT TP53−/− Cas9 parental and two independent PKMYT1-/- clones either untreated or treated for 24 h with 3 mM hydroxyurea (HU) were immunoblotted for PKMYT1 (l) and either total CDK1, CDK1-pT14 or CDK-pY15 (m). Actinin was used as loading control. For gel source data, see Supplementary Fig. 1.
