Fig 2: Schematic diagram of KDM5B-mediated regulation for Ewing sarcoma proliferation by the FBXW7/CCNE1 axis.(Left) In Ewing sarcoma cells, KDM5B overexpression inhibits FBXW7 transcription by demethylation of H3K4me3 at the promoter region, blocking the degradation of CCNE1 and accumulating CCNE1 protein, leading to acceleration of cell cycle process and cell proliferation of Ewing sarcoma. (Right) Epigenetic drug AS-8351 suppresses the function of KDM5B and elevates H3K4me3 level at the promoter region of FBXW7 gene, then up-regulates FBXW7 and enhances CCNE1 degradation, resulting in cell cycle arrest and tumor growth inhibition.

Fig 3: Effects of theaflavin-3,3′-digallate (TF3) on PDGF-BB–induced proliferation in rat aortic smooth cells (RASMCs). Serum-starved RASMCs were pretreated with 1-, 10-, or 20-μM TF3 for 1 h and then stimulated with 20 ng/ml PDGF-BB for 48 h. (A) An EdU assay was performed to evaluate the proliferation of RASMCs. (B) Statistical data of the EdU assay are shown. (C) The effect of TF3 on PDGF-BB–induced proliferation of RASMCs was also measured using CCK-8 assay. (D) The effect of TF3 on PDGF-BB–induced apoptosis of RASMCs was measured using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. (E) Statistical data from the TUNEL assay are shown. (F) The levels of the cell proliferation-associated proteins PCNA, CDK1, CDK2, CDK4, and CCNE1 were detected by western blotting. The western blot data are shown. Each experiment was performed in triplicate. Scale bar, 100 μm, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: Differential effects of WEE1i and ATRi upon CCNE1 induction results in fork collapse in early S phase(A) Representation of cell-cycle distribution for FT282 CCNE1induc cells ± doxycycline. Cells were synchronized by 24 h FBS deprivation (0.1%), released with 10% FBS, and collected at the indicated time points (± doxycycline). Cell-cycle distribution was measured with EdU and 7-AAD. Representative images (left) and quantification (right) are shown (n = 3; mean ± SD; p < 0.0001 for S phase ± doxycycline at 12 and 16 h, shown by an asterisk).(B) Detection of the indicated proteins after FT282 cells were arrested, released as in (A), and treated with DMSO (control) or 1 µM ATRi, 200 nM WEE1i, or both for 8 h (± doxycycline).(C and D) Cell-cycle distribution after drug treatment as in (B). Representative images (C, left), quantification of early, mid-, and late S phase (C, right) are shown (n = 3; mean ± SD; control versus WEE1i, statistics shown for early S phase, ****p < 0.0001). Nucleotide incorporation by mean EdU intensity (S/G0-G1) (D) is shown (control versus WEE1i, p < 0.0001).(E) Detection of S-phase entry and nucleotide incorporation by PCNA and EdU immunofluorescences after cells were treated as in (B) and fixed at 8 h. Percentages of PCNA+, PCNA+EdU+, and PCNA+EdU- cells were quantified by ImageJ. Representative images of cells with CCNE1 induction (bottom) are shown (scale bar, 20 µM; n = 3; mean ± SD; control versus WEE1i, p = 0.012).(F) Measurement of ?H2AX and EdU after treatment as in (B). Representative images (top) and quantification of ?H2AX+EdU-, ?H2AX+EdU+, and ?H2AX+EdU+/EdU+ (bottom) ± doxycycline are presented in CCNE1induc cells. With CCNE1induc, addition of ATRi to WEE1i affected ?H2AX+EdU- (WEE1i versus both, p < 0.0001; ?H2AX+EdU-, p < 0.0001; ?H2AX+EdU+/EdU+, p < 0.0001) at 16 h.Data were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test (C–F). Representative data are shown for one of 3 biologically independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.

Fig 5: 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.