Fig 2: AZD1775 accelerated S phase progression and mitotic entry. (a) AZD1775 induced S phase accumulation in the MDA-MB-231 and BT-549 cell lines. TNBC cells were treated with AZD1775 at 350 nmol/L for 5 d, and cellular DNA contents were measured by flow cytometry after PI staining. The proportions of cells in each stage of the cell cycle are shown in the bar graph. Bars represent ±SDs (n = 3). **P < 0.01, ***P < 0.001. (b) AZD1775 accelerated S phase progression in MDA-MB-231 cells. Following treatment with 350 nmol/L AZD1775 for 24 h, S phase progression was examined by EdU-BrdU dual pulse labeling. EdU/BrdU(−/+) indicates the early S phase, and EdU/BrdU(+/+) indicates the mid/late S phase. At least 10000 cells were counted per group in an experiment. Data were collected from 3 independent experiments. Bars represent ±SDs. *P < 0.05. (c) Early mitotic entry induced by WEE1 inhibition in the MDA-MB-231 cell line. After treatment under the above conditions, p-HH3 (orange colored) antibody was applied to TNBC cells that had incorporated EdU (green colored) to investigate mitotic phase cells in S phase cells. At least 10000 cells were counted per group in an experiment. Data were collected from 3 independent experiments. Bars represent ±SDs. ***P < 0.001. (d) AZD1775 increased the proportion of cells with a DNA content of more than 4n. TNBC cells were treated with 350 nmol/L AZD1775 for 5 d and PI-stained. Cells with high DNA contents were identified by flow cytometry. Bars represent ±SDs (n = 3). *P < 0.05, **P < 0.01. (e) The proportion of multinucleated MDA-MB-231 cells increased in response to AZD1775. The nuclei of MDA-MB-231 and MDA-MB-468 cells were stained with DAPI, and the proportions of multinucleated cells were determined by confocal microscopy. White arrows indicate multinucleated nuclei. MDA-MB-231 and MDA-MB-468 cells were treated with 200 nmol/L AZD1775 for 5 d. A total of 100 cells per group were analyzed. Bars represent ±SDs (n = 3). **P < 0.01.

Fig 3: CompC increased the phosphorylation of Cdk1 on Tyr15 and Wee1 on Ser642. (a) Renca cells were cultured for one day and then serum-starved by incubation with SFM overnight. The cells were treated with SFM (C) or 10% FBS for the indicated times (8–24 h) in the absence (−) or presence (+) of 10 μM CompC. Cell lysates were analyzed by western blot using antibodies against phosphorylated Cdk1 (P-Tyr15-Cdk1), Cdk1, phosphorylated Wee1 (P-Ser642-Wee1), Wee1, Myt1, phosphorylated histone H3 (P-Ser10-H3), histone H3 (H3), phosphorylated p53 (P-Ser15-p53), p53, p21, and β-actin. (b) Renca cells treated with vehicle or 25–50 nM Adavo in the absence (−) or presence (+) of 10 μM CompC for 16 h. Cell lysates were analyzed by western blot using antibodies against P-Tyr15-Cdk1, Cdk1, P-Ser10-H3, H3, P-Ser15-p53, p53, and β-actin. The band densities were normalized against β-actin and the fold changes compared to that of control (vehicle/−CompC/−Adavo) are written under each band in (a,b). (c) The cell viability of 5–10 μM CompC-, 50–100 nM Adavo-, and CompC/Adavo-cotreated cells was measured by MTT assay, and the percentage of cell viability was plotted as the mean ± SD with n = 8. The p-values were determined as *** p < 0.001, compared with vehicle-treated control cells (−CompC/−Adavo) and ### p < 0.001, compared to vehicle- or CompC-treated cells in the absence of Adavo. (d) Renca cells were cultured for one day and treated with the vehicle or 5–500 nM Adavo for two days. The cell viability was measured by the MTT assay and the percentage was plotted as the mean ± SD with n = 8. *** p < 0.001, compared with vehicle-treated control cells.

Fig 4: Combination WEE1i-ATRi decreases viability and colony formation in CCNE1Amp compared with CCNE1Low cells(A) Copy number analysis in CCNE1Amp (blue), CCNE1Gain (orange), and CCNE1Low (black) cells based on the Cancer Cell Line Encyclopedia (CCLE) data (https://sites.broadinstitute.org/ccle).(B) Immunoblot of the indicated proteins, including full-length and low molecular weight (LMW) cyclin E1, in cell lines normalized by cell number. Histone H3 was nuclear loading control.(C) Immunoblot for indicated proteins in CCNE1Amp OVCAR3 and KLE cells after treatment (200 nM WEE1i, 1 µM ATRi, or both) at the indicated times.(D) Cell viability of the indicated cell lines after 5 days of treatment at the indicated doses (left). Selected dose is defined as the "standard low dose" for comparison, highlighted in red (n = 3–5; mean ± SD). CDI relative to the Fa plot comparing CCNE1Amp (blue), CCNE1Gain (orange), and CCNE1Low (black) cell lines (right upper). The standard low dose is the bold dot, the higher doses are open dots, and lower doses are faded dots. Synergy is represented by log2(CDI) plotted against CCNE1 CN, displayed as log2(CN/2) for standard low doses (right lower). The trend line with correlation coefficient R2 is shown.(E) Colony formation (CF) after all lines were treated with 0.25 µM ATRi, 0.05 µM WEE1i, or both for 10 days (n = 3; representative image shown). CF quantified with ImageJ (Figure S1) and mean CF were used to calculate CDI. CDI relative to the Fa plot as in (D) (middle). Log2(CDI) versus CCNE1 CN as in (D) (right).(F) Knockdown efficiency of WEE1 siRNA (2 nM) and ATR siRNA (10 nM) in OVCAR3 cells measured by immunoblot 48 h post-transfection (top). Viability for combination WEE1 and ATR siRNA treatment in CCNE1Amp OVCAR3 cells and CCNE1Low WO-20 cells at 48 h (n = 5; mean ± SD; CDI = 0.9).Significance determined by one-way ANOVA followed by Tukey’s multiple comparison test for (D) and (F). Representative data are shown for one of 3 biologically independent experiments. ****p < 0.0001; ns, not significant.

Fig 5: miR-506-3p dependent regulatory axis is synthetically lethal with cell-cycle checkpoint inhibitors. (A) Abrogation of G2/M cell cycle checkpoint activation in RAD17 silenced SKOV3 cells. Western blot analysis on total cell lysates from SKOV3 cells silenced for RAD17 (siRAD17) or with scrambled (sictrl) siRNA and treated or not with 1 and 3 µM Pt. Immunoblottings were performed with Abs against the proteins indicated. Vinculin was used as loading control. Representative western blot images (left panel) and quantification (right panel) of relative phosphorylation levels calculated as the ratio between phosphorylated and total protein normalized on loading control. Bars in the graph represent the phosphorylation ratio between Pt-treated and untreated cells. Comparison assessed by Student’s t test (* p<0.05; ** p<0.01). (B) Forced expression of miR-506-3p or RAD17 silencing induces sensitivity to Chk1 and Wee1 inhibitors in SKOV3 cells. SKOV3 cells were transfected with miR-506-3p mimic or control scrambled miR (miR-506 and scr, left panels), or silenced with siRNA against RAD17 or with a control siRNA (siRAD17 and sictrl, right panels) and tested by clonogenic assays for sensitivity to Chk1 inhibitor (LY603618) or Wee1 inhibitors (MK1775) at the indicated doses. (C) Wee1 or Chk1 inhibition in miR-506-3p reconstituted cells enhanced effect of Pt treatment. Both scr (black lines) and miR-506 (blue lines) SKOV3 cells were treated with Pt at the indicated doses, alone (solid line) or in combination with 100 nM Chk1 inhibitor (Chk1i; LY2603618, dotted lines, upper panel) or 50 nM Wee1 inhibitor (Wee1i; MK1775, dotted lines, lower panel). For each panel, percentages of relative colony formation rate are reported. To compare miR-506 or siRAD17 cells versus their relative control cells (panel B) or to compare miR-506 cells treated with Pt alone versus combinations with cell cycle checkpoint inhibitors (panel C), two way ANOVA and Bonferroni’s post test was used (*p<0.05; ** p<0.01;***p<0.001).