Journal: Cancer Research Communications

Article Title: Histone Deacetylase Inhibitors Target DNA Replication Regulators and Replication Stress in Ewing Sarcoma Cells

doi: 10.1158/2767-9764.CRC-25-0058

Figure Lengend Snippet: HDAC inhibitors decrease RRM1, RRM2, CHK1, and WEE1 protein levels. A, EW8 and TC71 cells were treated with fimepinostat (25 or 100 nmol/L) for 24 hours, and then cellular lysates were collected for immunoblotting. B, EW8 and TC71 cells were treated with LY3023414 (1,000 nmol/L) for 24 hours, and then cellular lysates were collected for immunoblotting. C, EW8 and TC71 cells were treated with panobinostat (10 nmol/L) or romidepsin (5 nmol/L) for 24 hours, and then cellular lysates were collected for immunoblotting. D and E, EW8 cells were treated with different doses of panobinostat ( D ) or romidepsin ( E ) for 24 hours, and then cellular lysates were collected for immunoblotting. F, Cell lines representing additional sarcoma subtypes were treated with romidepsin (5 nmol/L) for 24 hours, and then cellular lysates were collected for immunoblotting.

Article Snippet: Antibodies to the following proteins were used in the immunoblots: FLI1 (Abcam, #ab133485, 1:1,000, RRID:AB_2722650), actin (Cell Signaling Technology, #4970, 1:5,000; RRID:AB_2223172), α-tubulin (Proteintech, # 66031-1-Ig, 1:5,000, RRID:AB_11042766), histone H3 (Cell Signaling Technology, #4499, 1:1,000, RRID:AB_10544537), acetyl-histone H3-Lys 9 (Cell Signaling Technology, #9649, 1:1,000, RRID:AB_823528), acetyl-histone H3-Lys 14 (Cell Signaling Technology, #7627, 1:1,000, RRID:AB_10839410), acetyl-histone H3-Lys 18 (Cell Signaling Technology, #13998, 1:1,000, RRID:AB_2783723), acetyl-histone H3-Lys 27 (Cell Signaling Technology, #8173, 1:1,000, RRID:AB_10949503), acetyl-histone H3-Lys 56 (Cell Signaling Technology, #4243, 1:1,000, RRID:AB_10548193), c-myc (Cell Signaling Technology, #18583, 1:1,000, RRID:AB_2895543), BRD4 (Cell Signaling Technology, #13440, 1:1,000, RRID:AB_2687578), replication protein A (RPA2; Bethyl Laboratories, A300-244A, 1:2,000, RRID:AB_185548), MCM2 (Cell Signaling Technology, #3619, 1:1,000, RRID:AB_2142137), MCM3 (Proteintech, #15597-1-AP, 1:500, RRID:AB_2141973), MCM5 (Proteintech, #11703-1-AP, 1:1,000, RRID:AB_2235162), CDT1 (Cell Signaling Technology, #8064, 1:1,000, RRID:AB_10896851), RRM1 (Cell Signaling Technology, #8637, 1:1,000, RRID:AB_10896851), RRM2 (Proteintech, #11661-1-AP, 1:500, RRID:AB_2180392), CHK1 (Cell Signaling Technology, #2360, 1:1,000, RRID:AB_11217623), WEE1 (Cell Signaling Technology, #13084, 1:1,000, RRID:AB_2713924), and survivin (Cell Signaling Technology, #2808, 1:1,000, RRID:AB_2063948).

Techniques: Western Blot

Journal: Cancer Research Communications

Article Title: Histone Deacetylase Inhibitors Target DNA Replication Regulators and Replication Stress in Ewing Sarcoma Cells

doi: 10.1158/2767-9764.CRC-25-0058

Figure Lengend Snippet: HDAC inhibitors downregulate the expression of genes related to DNA replication and the cellular response to DNA replication stress. A, EW8 cells were treated with panobinostat or romidepsin for 24 hours, and then cellular lysates and mRNA were collected for immunoblotting and RNA-seq analysis. B and C, Volcano plots of differentially expressed genes (fold change >2, adjusted P value < 0.05) in the EW8 cells treated with romidepsin (5 nmol/L; B ) or panobinostat (10 nmol/L; C ). D–E, RNA-seq data, fold change and adjusted P value, for the RRM1, RRM2, CHK1, and WEE1 genes in EW8 cells treated with romidepsin (5 nmol/L) and panobinostat (10 nmol/L). F and G, Gene sets (Kyoto Encyclopedia of Genes and Genomes pathway) enriched in the genes that are downregulated in EW8 cells treated with romidepsin ( F ) and panobinostat ( G ). H, RNA-seq data, fold change and adjusted P value, for the MCM2–7 genes in EW8 cells treated with romidepsin and panobinostat. I, EW8 and TC71 cells were treated with romidepsin (5 nmol/L) for 24 hours, and then cellular lysates were collected for immunoblotting.

Article Snippet: Antibodies to the following proteins were used in the immunoblots: FLI1 (Abcam, #ab133485, 1:1,000, RRID:AB_2722650), actin (Cell Signaling Technology, #4970, 1:5,000; RRID:AB_2223172), α-tubulin (Proteintech, # 66031-1-Ig, 1:5,000, RRID:AB_11042766), histone H3 (Cell Signaling Technology, #4499, 1:1,000, RRID:AB_10544537), acetyl-histone H3-Lys 9 (Cell Signaling Technology, #9649, 1:1,000, RRID:AB_823528), acetyl-histone H3-Lys 14 (Cell Signaling Technology, #7627, 1:1,000, RRID:AB_10839410), acetyl-histone H3-Lys 18 (Cell Signaling Technology, #13998, 1:1,000, RRID:AB_2783723), acetyl-histone H3-Lys 27 (Cell Signaling Technology, #8173, 1:1,000, RRID:AB_10949503), acetyl-histone H3-Lys 56 (Cell Signaling Technology, #4243, 1:1,000, RRID:AB_10548193), c-myc (Cell Signaling Technology, #18583, 1:1,000, RRID:AB_2895543), BRD4 (Cell Signaling Technology, #13440, 1:1,000, RRID:AB_2687578), replication protein A (RPA2; Bethyl Laboratories, A300-244A, 1:2,000, RRID:AB_185548), MCM2 (Cell Signaling Technology, #3619, 1:1,000, RRID:AB_2142137), MCM3 (Proteintech, #15597-1-AP, 1:500, RRID:AB_2141973), MCM5 (Proteintech, #11703-1-AP, 1:1,000, RRID:AB_2235162), CDT1 (Cell Signaling Technology, #8064, 1:1,000, RRID:AB_10896851), RRM1 (Cell Signaling Technology, #8637, 1:1,000, RRID:AB_10896851), RRM2 (Proteintech, #11661-1-AP, 1:500, RRID:AB_2180392), CHK1 (Cell Signaling Technology, #2360, 1:1,000, RRID:AB_11217623), WEE1 (Cell Signaling Technology, #13084, 1:1,000, RRID:AB_2713924), and survivin (Cell Signaling Technology, #2808, 1:1,000, RRID:AB_2063948).

Techniques: Expressing, Western Blot, RNA Sequencing

Journal: Cancer Research Communications

Article Title: Histone Deacetylase Inhibitors Target DNA Replication Regulators and Replication Stress in Ewing Sarcoma Cells

doi: 10.1158/2767-9764.CRC-25-0058

Figure Lengend Snippet: Integrated model for the regulation of DNA replication and replication stress response pathways by HDAC inhibitors. HDAC inhibitors downregulate the expression of multiple components of the pre-RC, including the MCM2–7 proteins and CDT1, as well as the RRM1 and RRM2 subunits of RNR, CHK1, and WEE1. HDAC inhibitors also downregulate the levels of the BRD4 and survivin proteins.

Article Snippet: Antibodies to the following proteins were used in the immunoblots: FLI1 (Abcam, #ab133485, 1:1,000, RRID:AB_2722650), actin (Cell Signaling Technology, #4970, 1:5,000; RRID:AB_2223172), α-tubulin (Proteintech, # 66031-1-Ig, 1:5,000, RRID:AB_11042766), histone H3 (Cell Signaling Technology, #4499, 1:1,000, RRID:AB_10544537), acetyl-histone H3-Lys 9 (Cell Signaling Technology, #9649, 1:1,000, RRID:AB_823528), acetyl-histone H3-Lys 14 (Cell Signaling Technology, #7627, 1:1,000, RRID:AB_10839410), acetyl-histone H3-Lys 18 (Cell Signaling Technology, #13998, 1:1,000, RRID:AB_2783723), acetyl-histone H3-Lys 27 (Cell Signaling Technology, #8173, 1:1,000, RRID:AB_10949503), acetyl-histone H3-Lys 56 (Cell Signaling Technology, #4243, 1:1,000, RRID:AB_10548193), c-myc (Cell Signaling Technology, #18583, 1:1,000, RRID:AB_2895543), BRD4 (Cell Signaling Technology, #13440, 1:1,000, RRID:AB_2687578), replication protein A (RPA2; Bethyl Laboratories, A300-244A, 1:2,000, RRID:AB_185548), MCM2 (Cell Signaling Technology, #3619, 1:1,000, RRID:AB_2142137), MCM3 (Proteintech, #15597-1-AP, 1:500, RRID:AB_2141973), MCM5 (Proteintech, #11703-1-AP, 1:1,000, RRID:AB_2235162), CDT1 (Cell Signaling Technology, #8064, 1:1,000, RRID:AB_10896851), RRM1 (Cell Signaling Technology, #8637, 1:1,000, RRID:AB_10896851), RRM2 (Proteintech, #11661-1-AP, 1:500, RRID:AB_2180392), CHK1 (Cell Signaling Technology, #2360, 1:1,000, RRID:AB_11217623), WEE1 (Cell Signaling Technology, #13084, 1:1,000, RRID:AB_2713924), and survivin (Cell Signaling Technology, #2808, 1:1,000, RRID:AB_2063948).

Techniques: Expressing

Journal: Nature Cancer

Article Title: First-in-class ultralong-target-residence-time p38α inhibitors as a mitosis-targeted therapy for colorectal cancer

doi: 10.1038/s43018-024-00899-7

Figure Lengend Snippet: a , Representative western blot analysis in KAP 2D cells upon 1 day of treatment with 1 µM SKL, BIRB-796, 1639, 2015 or DMSO (cropped blot images; n = 3 biologically independent experiments). b , Representative western blot analysis for p38α–Wee1 kinase assay (cropped blot images; n = 3 independent kinase assays). c , Representative western blot analysis in KAP 2D cells upon 1 day of treatment with 1 µM SKL, BIRB-796, 1639, 2015 or DMSO (cropped blot images; n = 3 biologically independent experiments). d – f , Quantification ( d ), representative pictures ( e ) and determination of mitotic phases ( f ) of KAP 2D cells stained for P-H3 S10 and α-tubulin after 1 day of treatment with 1 µM, SKL, 1639, 2015 or DMSO ( n = 3 cultures per condition; data are presented as the mean ± s.d.). Statistical significance was calculated using an ANOVA and Tukey’s multiple-comparisons test ( P < 0.001). Scale bars, 100 µm. g , h , Representative confocal microscopy pictures of stained KAP 2D ( g ) and PDO5 ( h ) after 1 day of treatment with 1 µM 2015 or DMSO ( n = 3 cultures per condition). Scale bars, 15 µm. i , Quantification of viable KAP 2D upon treatment with 1 µM SKL, 1639, 2015 or DMSO ( n = 3 cultures per condition; data are presented as the mean ± s.d.). Statistical significance was calculated using an ANOVA and Tukey’s multiple-comparisons test ( P < 0.0001). j , k , Quantification ( j ) and representative pictures ( k ) of EdU-labeled KAP 2D after 2 days of treatment with 1 µM SKL, 1639, 2015 or DMSO ( n = 4 cultures per condition; values represent the mean ± s.d.). Statistical significance was calculated using an ANOVA and Tukey’s multiple-comparisons test ( P < 0.0001). Scale bars, 50 µm. l , Representative size analysis of KAP 2D upon 2 days of treatment with 1 µM SKL, 1639, 2015 or DMSO (measurements without gating; n = 3 cultures per condition). m , Representative DNA content analysis in KAP 2D upon 1 day of treatment with 1 µM SKL, 1639, 2015 or DMSO (gating strategy in Extended Data Fig. ; n = 3 cultures per condition). The experiments in a – c were independently performed three times and the experiments in d – m were independently performed twice, all with similar results.

Article Snippet: The Wee1 kinase assay was performed with recombinant human active p38α protein (Abcam, ab271647), mostly inactive MK2 protein (Abcam, ab79910) and mostly inactive Wee1 protein (Novus Biologicals, H00007465-P01).

Techniques: Western Blot, Kinase Assay, Staining, Confocal Microscopy, Labeling

Journal: Cells

Article Title: Role of Alternative Splicing and Polyadenylation in Regulation of Spleen Development

doi: 10.3390/cells15060496

Figure Lengend Snippet: Confirmation of the regulatory roles of transcription factors E2F1 and MYBL2 in WEE1 gene expression. ( A ) Schematic structure of the WEE1 promoter and the putative binding sites of E2F1 and MYBL2. Binding sites and the position are given in the schematic structure, while sequences for each motif in the wild-type and mutant-type (Mut) promoters are given below. Sequences shown with strikethrough were deleted, and those highlighted in yellow were mutated in the Mut reporter constructs. ( B ) Log2Fold-change in E2F1, MYBL2 and WEE1 in 90- compared to 30-day-old pigs as revealed with RNA-seq. ( C ) Characterization of the promoter activity of WEE1 gene with dual-luciferase reporter analysis. WT, wild-type reporter gene. ( D ) Deletion of each putative binding site significantly decreased promoter activity. ( E ) Expression efficiency of plasmids overexpressing MYBL2 or E2F1 as revealed with Western blotting. ( F ) MYBL2 or E2F1 regulated reporter gene expression via the putative binding sites. ( G ) Overexpression of MYBL2 or E2F1 upregulated the mRNA level of WEE1 in PK-15 cells. ( H ) Overexpression of MYBL2 or E2F1 upregulated the protein level of WEE1 in PK-15 cells. ( I ) Direct binding of MYBL2 or E2F1 to the motifs as revealed by EMSA. EV, empty vector. * p < 0.05, ** p < 0.01, compared with WT reporters in Panels ( D , F ).

Article Snippet: The transferred PVDF membranes underwent protein blocking and were incubated with primary antibodies against WEE1 (1:5000 dilution; Catalog Number: 29474-1-AP; Proteintech, Wuhan, China) at 4 °C overnight, followed by incubation with secondary antibodies (1:20,000 dilution; LI-COR) at 37 °C for 1 h. Finally, the PVDF membranes were detected using a LI-COR near-infrared fluorescence imaging system (LI-COR, Lincoln, NE, USA).

Techniques: Gene Expression, Binding Assay, Mutagenesis, Construct, RNA Sequencing, Activity Assay, Luciferase, Expressing, Western Blot, Over Expression, Plasmid Preparation

Journal: Neuro-Oncology

Article Title: Transcriptomics-guided high-throughput drug screening identifies potent therapies for P53 pathway altered DIPG/DMG

doi: 10.1093/neuonc/noaf216

Figure Lengend Snippet: Transcriptomic Screening Identifies ATR Pathway Inhibitor AZ20 as a Promising Candidate to Overcome SN-38 Resistance in TP53 -Mutant DIPG. ( A ) KEGG analysis of 190313 cells treated with SN-38 (10 nM, 72 h) revealed upregulation of cell cycle and DNA replication pathways (* P < .05). (B) HALLMARK pathway analysis identified enrichment of E2F target genes (* P < .05). (C) Correlation analysis showed strong associations between E2F (E2F1, E2F2, E2F7, E2F8) and DNA damage repair genes (ATR, CHK1, PARP1, etc.) (* P < .05). (D) Western blot analysis showed significant upregulation of ATR, CHK1, PARP1, and WEE1 protein expression in TP53 -mutant DIPG cell lines (190313, 190326, 150728) treated with 10 nM SN-38 for 72 h, compared to untreated controls. (E-G) PARP1 inhibitor Olaparib had minimal effect on TP53-mutant DIPG (IC50 > 10 μM) and showed no synergy with SN-38 (NS). (H) The CHK1 inhibitor (SCH900776) exhibited potent cytotoxic effects on TP53 -mutant DIPG cells (190326), with an IC50 of ∼100 nM and minimal toxicity to PPCs. (I-J) Co-treatment with SCH900776 (100 nM) and SN-38 (10 nM) demonstrated significant synergy, reducing cell viability (* P < .05). (K) Screening of 23 ATR pathway inhibitors identified AZ20 as the most potent (>70% viability reduction at 1 μM). Heatmap includes TP53-KD and PPM1D-KD isogenic lines. (L) AZ20 exhibited strong activity in TP53-mutant DIPG (IC50 ∼200 nM) and limited toxicity to PPCs (IC50 > 1 μM). Viability assessed by CellTiter-Glo (mean ± SD, n = 3).

Article Snippet: P53 (DO-1, Cat# 18032S), WIP1 (E2X1I, Cat# 94886S), and GAPDH (D4C6R, Cat# 97166S) antibodies were obtained from Cell Signaling Technology (CST); BCL2 (Cat# 68103-1-Ig), BAX (Cat# 60267-1-Ig), Vinculin (Cat# 66305-1-Ig), and PARP1 (Cat# 66520-1-Ig) antibodies were purchased from ProteinTech; CHK1 (Cat# BM3968), WEE1 (Cat# A01319-2), and ATR (Cat# A00262-3) antibodies were sourced from Boster.

Techniques: Mutagenesis, Western Blot, Expressing, Activity Assay

Journal: Neuro-Oncology

Article Title: Transcriptomics-guided high-throughput drug screening identifies potent therapies for P53 pathway altered DIPG/DMG

doi: 10.1093/neuonc/noaf216

Figure Lengend Snippet: Synergistic anti-tumor effects of AZ20 and SN-38 in TP53 -mutant DIPG cells through inhibition of ATR pathway signaling and induction of apoptosis. (A) Twenty-one ATR pathway inhibitors were screened in combination with SN-38 (1 μM each) in TP53-mutant DIPG cells. Viability was measured by CellTiter-Glo ( n = 3) analyzed by a two-tailed unpaired t -test. (B-D) Synergy analysis using the BLISS model confirmed a robust synergistic interaction between SN-38 and AZ20 in 190326 cells (D). In contrast, this synergistic effect was not observed in TP53 wild-type DIPG cells (150714, DIPG17) (B and C). (E-G) Cell viability was measured after 24, 48, and 72 h of treatment with DMSO, SN-38 (10 nM), AZ20 (10 nM), or both in 190326, 150714, and DIPG17 cells. Combination significantly reduced viability in 190326 (**** P < .0001). (H) Western blot analysis of protein expression in 190326 cells following 72 h of treatment with DMSO (vehicle control), SN-38 (10 nM), AZ20 (10 nM), or their combination. SN-38 monotherapy activated ATR and its downstream targets, CHK1 and WEE1, while combination treatment with SN-38 and AZ20 suppressed ATR activation and downregulated CHK1 and WEE1 expression. The combination treatment also induced apoptosis, as evidenced by increased levels of cleaved PARP1. (I) Chou-Talalay-based combination index (CI) heatmap for SN-38 and AZ20 in TP53-mutant DIPG cell line 190326. Combination index values were calculated from a 72-h viability assay using fixed-ratio matrix combinations of SN-38 and AZ20. CI < 1 indicates synergy, CI = 1 indicates additivity, and CI > 1 indicates antagonism. (J) 190326 cells transfected with siATR and treated with SN-38 or AZ20 showed reduced viability in SN-38 + siATR and AZ20 + SN-38 + siATR groups (**** P < .0001).

Article Snippet: P53 (DO-1, Cat# 18032S), WIP1 (E2X1I, Cat# 94886S), and GAPDH (D4C6R, Cat# 97166S) antibodies were obtained from Cell Signaling Technology (CST); BCL2 (Cat# 68103-1-Ig), BAX (Cat# 60267-1-Ig), Vinculin (Cat# 66305-1-Ig), and PARP1 (Cat# 66520-1-Ig) antibodies were purchased from ProteinTech; CHK1 (Cat# BM3968), WEE1 (Cat# A01319-2), and ATR (Cat# A00262-3) antibodies were sourced from Boster.

Techniques: Mutagenesis, Inhibition, Two Tailed Test, Western Blot, Expressing, Control, Activation Assay, Viability Assay, Transfection

Journal: Science advances

Article Title: Rac1 promotes kidney collecting duct repair by mechanically coupling cell morphology to mitotic entry.

doi: 10.1126/sciadv.adi7840

Figure Lengend Snippet: Fig. 9. Rac1 via actomyosin regulates the G2-M checkpoint kinase Wee1 to prevent premature mitotic entry. (A and B) CD cells were G2-synchronized using RO- 3306, and lysates were collected at the indicated time points after RO-3306 washout (“G2 release”). Lysates were immunoblotted for pH3 to monitor mitotic entry. Three repeat experiments were quantified in (B). Fold change values ± SD. Arrows highlight the first pH3 peak of the respective groups indicating mitotic entry. (C and D) G2- synchronized CD cells were immunoblotted for cleaved caspase 3 (cl-Casp3) after G2 release and quantified in (D) as fold change values ± SD (n = 3). Asterisk (*) denotes between-group significance at the corresponding time point. (E and F) CD cells were G2-synchronized, and lysates were obtained immediately upon G2 washout (G2, T0) and immunoblotted in biological duplicates. p-Cdk1 Y15: phosphorylated Cdk1 tyrosine-15. Three repeat experiments were quantified in (F). Fold change values ± SD. (G and H) Asynchronous Rac1f/f (+DMSO) and Rac1−/− [+DMSO or blebbistatin (5 μM)] CD cells were treated with cycloheximide (CHX; 100 μg/ml), and lysates were obtained at the indicated time points and immunoblotted for Wee1. Three repeat experiments are quantified in (H) as fold change ± SD. Asterisk (*) denotes significance between Rac1−/− and Rac1f/f or blebbistatin-treated Rac1−/− at the corresponding time point. (I and J) Rac1f/f and Rac1−/− CD cells were G2-synchronized and treated with vehicle (DMSO) or 5 μM blebbistatin upon G2 release. Lysates were collected at the indicated time points and immunoblotted for pH3 to monitor mitotic entry. Three independent repeat experiments are quantified (for Rac1f/f, only the vehicle control is shown) in (J) as fold change values ± SD. Asterisk (*) denotes significance between Rac1−/− and Rac1f/f or blebbistatin-treated Rac1−/− at the corresponding time point. *P < 0.05.

Article Snippet: Primary antibodies used are pH3 (Cell Signaling Technology, #9701), cleaved caspase 3 (Cell Signaling Technology, #9664), Wee1 (Novus Biologicals, #NBP1- 33506), actin (Cell Signaling Technology, #4967), cyclin B1 (Cell Signaling Technology, #4138), Rac1 (Millipore, #05- 389), α- tubulin (Cell Signaling Technology, #2144), Cdk1 (Cell Signaling Technology, #77055), and phospho–(Y15) Cdk1 (Cell Signaling Technology, #4539).

Techniques: Control

Journal: Science advances

Article Title: Rac1 promotes kidney collecting duct repair by mechanically coupling cell morphology to mitotic entry.

doi: 10.1126/sciadv.adi7840

Figure Lengend Snippet: Fig. 10. Wee1 inhibition phenocopies Rac1 deficiency in mitosis. (A to D) Rac1f/f and Rac1−/− CD cells were G2-synchronized using RO-3306 and treated with the Wee1- specific inhibitor MK-1775 (1 μM) upon G2 release. Lysates were collected at the indicated time points and immunoblotted for pH3 to monitor mitotic entry or cleaved caspase 3 to monitor cell death. Densitometry was used to quantify fold changes ± SD of three repeat experiments in (B) and (D). Arrows in (B) highlight the first pH3 peak indicating mitotic entry. (E) F-actin (white)– and DNA (blue)–labeled Rac1f/f and Rac1−/− (+MK-1775; 1 μM) CD cell monolayers analyzed by confocal microscopy with a mitotic metaphase cell shown in the center (scale bars, 10 μm). The top row column depicts metaphase F-actin (scale bars, 5 μm) as outlined by a red continuous box in the bottom row. Images are representative of at least three experiments. (F) Circularity quantification of mitotic metaphase F-actin as shown in (E) with a minimum of 10 measurements shown per group. Bars are means ± SD. (G) Live confocal mitosis imaging of SPY650-DNA (blue)– and SPY555-actin (white)–labeled vehicle (DMSO)– or MK-1775 (1 μM)–treated Rac1f/f CD cells in vitro. The mitotic cell was manually segmented and colored in green. Scale bars, 10 μm. Sequences are representative of three repeat experiments with at least three to four mitoses analyzed per experiment. (H) Relative distribution of mitotic defects in vitro during live imaging cell division se- quences with at least 10 mitoses analyzed per group. *P < 0.05.

Article Snippet: Primary antibodies used are pH3 (Cell Signaling Technology, #9701), cleaved caspase 3 (Cell Signaling Technology, #9664), Wee1 (Novus Biologicals, #NBP1- 33506), actin (Cell Signaling Technology, #4967), cyclin B1 (Cell Signaling Technology, #4138), Rac1 (Millipore, #05- 389), α- tubulin (Cell Signaling Technology, #2144), Cdk1 (Cell Signaling Technology, #77055), and phospho–(Y15) Cdk1 (Cell Signaling Technology, #4539).

Techniques: Inhibition, Labeling, Confocal Microscopy, Imaging, In Vitro