Figure 3. Cyclin E overexpression depends on CDK2 to sensitize cells to Wee1 kinase inhibition.
(A) Kaplan-Meier survival plots of cyclin EhighpCDK2high and cyclin ElowpCDK2low tumors from the TNBC patient cohorts from fig. 1D. (B) Percentages of γH2AX- and RAD51-positive cells of 76NE6 cyclin E-inducible cells cultured with (+) or without (−)10 ng/mL doxycycline (DOX) for 24 hours by IHC staining. (C) 76NE6 cyclin ER130A inducible cells cultured with (DOX) or without (CNL) 10 ng/mL doxycycline for 24 hours were treated with AZD1775 for 48 hours and subjected to HTSA as in fig. S2C. Inset shows immunoblot of cyclin E expression. (D) Representative images of RPA, γH2AX, RAD51, and 53BP1 foci in MDA231 cyclin E-inducible cells as in (A). Scale bar, 10 μm. (F) Quantification of foci-positive cell (>5 foci per cell) population in the samples as in (D), n=3. (F) Representative ongoing replication forks of DNA tracts pulse-labeled with IdU and CIdU from the parental cells and CDK2 knockout (KO) cells (via CRISPR/CAS9) of MDA231 with or without cyclin E induction for 48 hours by 10 ng/mL doxycycline (DOX). Scale bar, 10 μm. The data in the blue box are presented in fig 2A. (G) Analysis of replication fork progression of the ongoing replication forks in the samples as in (F). More than 89 DNA fibers were analyzed for each sample. The data in the blue box were presented in fig 2B. (H) 76NE6 cyclin E–inducible cells with scrambled shRNA (Scr.) or cyclin E shRNA knockdown (shCyclin E) (left) with (+) or without (−) 10 ng/mL doxycycline (DOX) for 24 hours (left) and MDA231 cyclin E-inducible cells as in (F) (right) were subjected to immunoblot with the indicated antibodies. Actin is the loading control. (I) 76NE6 cyclin E–inducible cells with CDK2 or scramble shRNA were subjected to HTSA as in fig. S2E. (J) Parental and CDK2 knockout cells of MDA231 cyclin E–inducible cells were subjected to HTSA as in fig. S10F. (K) MDA231 cyclin E–inducible cells were concomitantly treated with 6 nM dinaciclib and different concentrations of AZD1775 with or without 10 ng/mL doxycycline for 48 hours as in fig. S10H. The bar graph shows the changes in cell survival between the cells without and with 6nM dinaciclib at absence (CNL) or presence (DOX) of 10 ng/mL doxycycline at the different AZD1775 doses.