Fig 2: Inhibition of WEE1 with AZD1775 increased G2/M cell cycle arrest in LTED cells. (A–D) MCF7 and T47D and their LTED derivatives were treated with vehicle, E2 deprivation (E2 dep), 500nM ribociclib (ribo), 500nM AZD1775, combination of E2 dep and ribo or the combination of E2 dep, ribo and AZD1775 for 6 days. Cell cycle profiles under different treatment conditions were analyzed by fluorescence-activated cell sorting (FACS) and compared to vehicle treated cells. Results are reported as mean percent cell cycle distribution + SE **p < 0.01 and ***p < 0.001 (ANOVA) for % cell in G2/M phase for indicated treatment conditions compared with vehicle control in respective cell lines. Bar graphs represent data from three independent experiments (n=3).

Fig 3: Inhibition of WEE1 kinase induces growth arrest and apoptosis in PDAC cells. A, immunoblot analysis of PDAC cell lines following treatment with nonspecific (NS) or WEE1-targeting (W1-1 and W1-2) siRNA for 24 or 48 h (n = 3). B, immunoblot analysis of Pa02C cells following treatment with WEE1i for 48 h (n = 4). C, proliferation of PDAC cells following treatment for 5 days with WEE1i (adavosertib) at various concentrations. Cell numbers at endpoint were normalized to vehicle-treated control (100% growth) for each cell line. Curves were fit using a 4-parameter log-logistic function and the drm package in R (n = 3). D, PDAC cells were plated at low density to allow for clonogenic growth, and various concentrations of WEE1i were added. After 10 days, plates were stained with crystal violet, and colonies were imaged (n = 4). E, cells were treated with vehicle or adavosertib (100 nM for Pa02C and 200 nM for Pa16C) for 24 h, then fixed, and stained with propidium iodide. Flow cytometry quantification of cell cycle populations is shown for two representative cell lines (n = 3). F, annexin V–FITC staining followed by flow cytometry in PDAC cell lines after 72 h treatment with 500 nM WEE1i (n = 4). Molecular weight markers are noted next to all immunoblots. PDAC, pancreatic ductal adenocarcinoma.

Fig 4: MUS81 overexpression links with poor prognosis and WEE1 deficiency in gastric cancer. a Heatmap of differentially expressed genes (DEGs) between gastric cancer and paracancerous tissues in the GSE62254 data set. The top 10 upregulated genes and top 10 downregulated genes are shown. b Volcano map of DEGs related to DNA damage and repair. The DEGs with ∣log (FC)∣ > 0.05 and -log (P-value) >1 are indicated; red, green, and yellow dots represent upregulated DEGs, down-regulated DEGs, and DEGs with non-significant changes, respectively. c High expression of MUS81 is associated with short overall survival time in patients with gastric cancer (GSE62254 data set). d Pearson correlation analysis of MUS81 and WEE1 in the GSE62254 data set. e Representative immunohistochemical staining of MUS81 and WEE1 in patients with gastric cancer. Scale bar: 100 μm. f Pearson correlation analysis was performed to validate the correlation of MUS81 and WEE1 protein expression in 26 patients with gastric cancer from Wuhan Union Hospital

Fig 5: WEE1 inhibition increases DNA damage and mitotic entry in CIC-DUX4 sarcoma cells.(A) γH2AX IF staining of NCC_CDS1_X1_C1 and NCC_CDS2_C1 cells treated with adavosertib (0.5 μM) or DMSO (48 hours). Representative figure; performed in triplicate. Scale bars: 10 μm. (B) Quantitative analysis of γH2AX IF in A demonstrating the percentage of NCC_CDS1_X1_C1 or NCC_CDS2_C1 cells with more than 5 nuclear γH2AX foci following treatment with adavosertib or control. Mean percentage over 10 HPFs. ***P < 0.001, 1-way ANOVA. Representative cell cycle histograms (PI staining) of NCC_CDS1_X1_C1 (C and D) and NCC_CDS2_C1 (I and J) cells treated with adavosertib or DMSO (48 hours). Percentage of adavosertib- (0.5 μM) and DMSO-treated (48 hours) NCC_CDS1_X1_C1 (E) and NCC_CDS2_C1 (K) cells in sub-G1, G1, S, and G2/M phases as identified in C, D, I, and J. **P < 0.05, 1-way ANOVA. Performed in triplicate. Error bars represent SEM. Values for each fraction of cells in sub-G1, G1, S, and G2/M are provided in Supplemental Figure 4, A and B. γH2AX expression in NCC_CDS1_X1_C1 (F and G) and NCC_CDS2_C1 (L and M) cells treated with adavosertib or DMSO. Percentage of γH2AX-positive cells among NCC_CDS1_X1_C1 (H) and NCC_CDS2_C1 (N) cells analyzed in F, G, L, and M. Student’s t test. Error bars represent SEM.