Journal: Oncogene

Article Title: Cyclin E1 overexpression sensitizes ovarian cancer cells to WEE1 and PLK1 inhibition.

doi: 10.1038/s41388-025-03312-4

Figure Lengend Snippet: Fig. 2 TP53 inactivation is not a universal predictive biomarker of the response to PLK1 and WEE1 inhibitors. Validation of TP53 knockdown efficiency with two types of siRNA specific to TP53 based on TP53 protein levels (A, B) and mRNA expression (C) in OVTOKO and TOV21G cell lines. Analysis of cell viability in response to volasertib (D) or adavosertib (E) under TP53 knockdown. Reported p values were determined via one-way ANOVA based on IC50 values obtained from multiple repeated experiments. Statistical significance is denoted as **p < 0.01 and ***p < 0.001.

Article Snippet: Cells were treated with volasertib (PLK1 inhibitor; Selleckchem, Japan; S2235) or adavosertib (WEE1 inhibitor; Selleckchem, Japan; S1525) for 72 h. Alternatively, after siCCNE1 treatment for 48 h, cells were exposed to volasertib or adavosertib for an additional 72 h and cultured until further experimentation.

Techniques: Biomarker Discovery, Knockdown, Expressing

Journal: Oncogene

Article Title: Cyclin E1 overexpression sensitizes ovarian cancer cells to WEE1 and PLK1 inhibition.

doi: 10.1038/s41388-025-03312-4

Figure Lengend Snippet: Fig. 1 The sensitivity of PLK1 or WEE1 inhibitors correlates with cyclin E1 expression, rather than CCNE1 amplification in ovarian cancer cell lines. A Protein expression of Cyclin E1 and β-actin, as determined via western blotting. B mRNA expression levels of CCNE1 and its expression relative to β-actin, as determined via RT-qPCR. CCNE1 copy numbers were obtained from the DepMap portal and Cell Model Passports databases. C Heatmap showing drug sensitivity of ovarian cancer cell lines to volasertib and adavosertib, assessed via cell viability assays. The status of CCNE1 and TP53 is indicated. Drug sensitivity is measured based on adjusted IC50 values. D IC50 values of the corresponding cell lines, as presented in (C), with a comparison of IC50 values between the CCNE1 high and low/normal expression groups. HGSOC high-grade serous ovarian carcinoma, CCOC clear cell ovarian carcinoma, EOC endometrioid ovarian carcinoma.

Article Snippet: Cells were treated with volasertib (PLK1 inhibitor; Selleckchem, Japan; S2235) or adavosertib (WEE1 inhibitor; Selleckchem, Japan; S1525) for 72 h. Alternatively, after siCCNE1 treatment for 48 h, cells were exposed to volasertib or adavosertib for an additional 72 h and cultured until further experimentation.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Comparison

Journal: Oncogene

Article Title: Cyclin E1 overexpression sensitizes ovarian cancer cells to WEE1 and PLK1 inhibition.

doi: 10.1038/s41388-025-03312-4

Figure Lengend Snippet: Fig. 3 Cyclin E1 expression is a predictive biomarker for the efficacy of PLK1 and WEE1 inhibitors. Knockdown efficiency of CCNE1 using siRNA (siCCNE1) was assessed based on protein (A) and mRNA (B) levels. Cell viability assays following CCNE1 knockdown in OVCAR3, OVISE, and KURAMOCHI cells treated with PLK1 inhibitor volasertib (C) or WEE1 inhibitor adavosertib (D). The p values were determined via one-way ANOVA based on IC50 values obtained from multiple repeated experiments. Statistical significance is denoted as ***p < 0.001.

Article Snippet: Cells were treated with volasertib (PLK1 inhibitor; Selleckchem, Japan; S2235) or adavosertib (WEE1 inhibitor; Selleckchem, Japan; S1525) for 72 h. Alternatively, after siCCNE1 treatment for 48 h, cells were exposed to volasertib or adavosertib for an additional 72 h and cultured until further experimentation.

Techniques: Expressing, Biomarker Discovery, Knockdown

Journal: Oncogene

Article Title: Cyclin E1 overexpression sensitizes ovarian cancer cells to WEE1 and PLK1 inhibition.

doi: 10.1038/s41388-025-03312-4

Figure Lengend Snippet: Fig. 4 Cyclin E1 overexpression sensitizes ovarian cancer cells to PLK1 or WEE1 inhibitors by deregulating the cell cycle. A–E Cells were exposed to DMSO (control) or two concentrations of volasertib (0.02 and 0.1 μM) for 24 to 72 h in four ovarian cancer cell lines: cyclin E1-high (OVCAR3, OVISE) and cyclin E1-low (OVTOKO, JHOC-5). A, B Flow cytometry analysis and corresponding cell cycle phase distribution after 24 h of volasertib treatment, with statistical analysis (One-Way ANOVA). C Cyclin A2 and 7-AAD dual staining showing the proportion of cells in G1, S, M, and G2 phases (excluding sub-G1 and others). D, E Cells were treated with volasertib for 72 h. F, G Cells were exposed to DMSO (control) or three concentrations of adavosertib (0.1, 0.5, and 1 μM) for 24 h in the four cell lines. All comparisons were made between DMSO- and volasertib- or adavosertib-treated groups. Statistical significance was assessed by One-Way ANOVA (*p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: Cells were treated with volasertib (PLK1 inhibitor; Selleckchem, Japan; S2235) or adavosertib (WEE1 inhibitor; Selleckchem, Japan; S1525) for 72 h. Alternatively, after siCCNE1 treatment for 48 h, cells were exposed to volasertib or adavosertib for an additional 72 h and cultured until further experimentation.

Techniques: Over Expression, Control, Flow Cytometry, Staining

Journal: Oncogene

Article Title: Cyclin E1 overexpression sensitizes ovarian cancer cells to WEE1 and PLK1 inhibition.

doi: 10.1038/s41388-025-03312-4

Figure Lengend Snippet: Fig. 5 PLK1 and WEE1 inhibitors induce apoptosis in cyclin E1-overexpressing ovarian cancer cells. A Protein levels of apoptosis-related proteins (cleaved caspase-3 and cleaved PARP) and γH2AX (indicative of DNA double-strand breaks) at 72 h after treatment of cyclin E1-high (OVCAR3, OVISE) and cyclin E1-low cells (OVTOKO, JHOC-5) with 0.1 and 1 µM volasertib evaluated using western blotting. B Protein levels of phospho-CDK1 (Tyr15), apoptosis-related proteins, and γH2AX at 72 h after treatment with 0.5 and 1 µM adavosertib in the four cell lines described in (A). V-0.1, volasertib 0.1 µM; V-1, volasertib 1 µM; A-0.5, adavosertib 0.5 µM; A-1, adavosertib 1 µM. C–E Time-lapse microscopy analysis of apoptosis in Cyclin E1-high and Cyclin E1-low expressing cells. C Representative images of Caspase-3/7 Green fluorescence, indicating apoptotic cells, captured at 0, 18, and 24 h. D Normalized Caspase-3/7 positive cell counts expressed relative to 0 h. E Normalized cell confluence, expressed relative to 0 h. Data are shown as mean ± SEM. Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ns (not significant).

Article Snippet: Cells were treated with volasertib (PLK1 inhibitor; Selleckchem, Japan; S2235) or adavosertib (WEE1 inhibitor; Selleckchem, Japan; S1525) for 72 h. Alternatively, after siCCNE1 treatment for 48 h, cells were exposed to volasertib or adavosertib for an additional 72 h and cultured until further experimentation.

Techniques: Western Blot, Time-lapse Microscopy, Expressing

Journal: Oncogene

Article Title: Cyclin E1 overexpression sensitizes ovarian cancer cells to WEE1 and PLK1 inhibition.

doi: 10.1038/s41388-025-03312-4

Figure Lengend Snippet: Fig. 6 Cyclin E1 expression is a predictive biomarker of the response to PLK1 or WEE1 inhibitors in vivo. A–D Mouse xenograft experiments in 6-week-old female BALB/cSlc-nu/nu mice inoculated with four ovarian cancer cells. Mice were treated with vehicle, volasertib BIW (twice weekly) at 25 mg/kg, or adavosertib OD (once daily) at 60 mg/kg via oral administration for 12–22 days (n = 8 per group). The figure includes photos of resected tumors and graphs showing tumor volume. A two-way ANOVA with Tukey’s multiple comparisons test was used to compare various experimental arms with the vehicle arm. E–H Analyses of cleaved PARP levels via western blotting. One-way ANOVA or the Kruskal–Wallis test was used for analysis. I–L Representative H&E staining and detection of cyclin E1 and γH2AX (Scale bar, 100 μm) by immunohistochemistry. Cyclin E1-high cells: A A2780, B OVISE, and cyclin E1-low cells: C TOV21G and D OVTOKO. Statistical significance is denoted as ns, non-significant; **p < 0.01; and ***p < 0.001.

Article Snippet: Cells were treated with volasertib (PLK1 inhibitor; Selleckchem, Japan; S2235) or adavosertib (WEE1 inhibitor; Selleckchem, Japan; S1525) for 72 h. Alternatively, after siCCNE1 treatment for 48 h, cells were exposed to volasertib or adavosertib for an additional 72 h and cultured until further experimentation.

Techniques: Expressing, Biomarker Discovery, In Vivo, Western Blot, Staining, Immunohistochemistry

Journal: Oncogene

Article Title: Cyclin E1 overexpression sensitizes ovarian cancer cells to WEE1 and PLK1 inhibition.

doi: 10.1038/s41388-025-03312-4

Figure Lengend Snippet: Fig. 7 PLK1 and WEE1 inhibitors induce synthetic lethality in cyclin E1-overexpressing cells through different mechanisms of action. The diagram illustrates the mechanisms and differential responses to treatment with volasertib and adavosertib of high cyclin E1-expressing cells versus low cyclin E1-expressing cells. The figure highlights how these drugs affect cell cycle progression and apoptosis in cells with varying levels of cyclin E1. This model proposes that synthetic lethality with a PLK1 inhibitor takes advantage of the arrested mitotic (spindle) assembly checkpoint and mitotic catastrophe (during the M phase), while lethality induced by a WEE1 inhibitor depends on the persistence of unrepaired DNA damage and replication catastrophe (during the S phase).

Article Snippet: Cells were treated with volasertib (PLK1 inhibitor; Selleckchem, Japan; S2235) or adavosertib (WEE1 inhibitor; Selleckchem, Japan; S1525) for 72 h. Alternatively, after siCCNE1 treatment for 48 h, cells were exposed to volasertib or adavosertib for an additional 72 h and cultured until further experimentation.

Techniques: Expressing

Journal: Science Advances

Article Title: Nucleolar integrity during interphase supports faithful Cdk1 activation and mitotic entry

doi: 10.1126/sciadv.aap7777

Figure Lengend Snippet: ( A ) Increased Wee1 levels caused by NOL11 depletion. HeLa cells were treated with the siRNAs for NOL11 and synchronized at the G 2 /M border. Whole-cell extracts were immunoblotted with the indicated antibodies. ( B ) Reduced Wee1 turnover by NOL11 depletion. NOL11-depleted cells were synchronized by single thymidine block. Six hours after release, cells were treated with 10 μM RO-3306 and collected at the indicated times for immunoblotting. The number in the upper immunoblot panels shows hours after the treatment of RO-3306.

Article Snippet: Mouse monoclonal antibodies against α-β-actin (sc47778, Santa Cruz Biotechnology): cyclin B1 (sc245, Santa Cruz Biotechnology), UBF (sc13125, Santa Cruz Biotechnology), Cdk1 (sc45, Santa Cruz Biotechnology), Wee1 (sc5285, Santa Cruz Biotechnology), tubulin (DM1A, Millipore), nucleolin (M019-3, MBL), H3-pS10 (MABI0312, MBL), MPM2 (05-368, Millipore), NPM (FC61991, Thermo Fisher Scientific), and RPL11 (3A4A7, Thermo Fisher Scientific).

Techniques: Blocking Assay, Western Blot

Journal: Science Advances

Article Title: Nucleolar integrity during interphase supports faithful Cdk1 activation and mitotic entry

doi: 10.1126/sciadv.aap7777

Figure Lengend Snippet: ( A ) Increased Wee1 levels caused by nucleolar disruption. HeLa cells were treated with the indicated siRNAs and synchronized at the G 2 /M border. Whole-cell extracts were immunoblotted with the indicated antibodies. ( B ) Reduced Wee1 turnover by nucleolar disruption. Cells were treated as and examined by immunoblotting with the indicated antibodies. The number in the upper immunoblot panels shows hours after the treatment of RO-3306. ( C ) Reduction of enhanced Cdk1-pY15 by MK1775 treatment. Cells treated with indicated siRNAs were synchronized at the G 2 /M border. Subsequently, the cells were treated with 100 nM MK1775 for 3 hours and collected for immunoblotting using the indicated antibodies. ( D ) Restoration of mitotic entry by the Wee1 inhibitor. HeLa cells were treated with or without 100 nM MK1775 at 6 hours after release from the double thymidine block. The cells were collected at 9 hours after release from the double thymidine block, and mitotic index was examined using FACS. Values are shown as mean ± SD, n = 3.

Article Snippet: Mouse monoclonal antibodies against α-β-actin (sc47778, Santa Cruz Biotechnology): cyclin B1 (sc245, Santa Cruz Biotechnology), UBF (sc13125, Santa Cruz Biotechnology), Cdk1 (sc45, Santa Cruz Biotechnology), Wee1 (sc5285, Santa Cruz Biotechnology), tubulin (DM1A, Millipore), nucleolin (M019-3, MBL), H3-pS10 (MABI0312, MBL), MPM2 (05-368, Millipore), NPM (FC61991, Thermo Fisher Scientific), and RPL11 (3A4A7, Thermo Fisher Scientific).

Techniques: Disruption, Western Blot, Blocking Assay

Journal: Science Advances

Article Title: Nucleolar integrity during interphase supports faithful Cdk1 activation and mitotic entry

doi: 10.1126/sciadv.aap7777

Figure Lengend Snippet: ( A ) Knockdown efficiency of siRNA for rRNA transcription factor or processing factors. HeLa cells transfected with the indicated siRNAs were cultured for 48 hours and immunoblotted by the indicated antibodies. ( B ) The alteration of nucleolar structure by depletion of rRNA transcription factor, not by processing factors. HeLa cells were transfected with the indicated siRNAs and cultured for 48 hours. The cells were stained with anti-nucleolin (green) and anti-MYBBP1A (red) antibodies. DNA was counterstained by DAPI (blue). Scale bars, 5 μm. ( C ) Increased Wee1 and Cdk1-pY15 in cells with the disrupted nucleolus. HeLa cells were synchronized at the G 2 /M border after siRNA transfection and immunoblotted using the indicated antibodies. ( D ) Delayed mitotic entry in cells with the disrupted nucleolus. HeLa cells were treated with the indicated siRNAs and synchronized by double thymidine as the same protocol shown in . Nine hours after the release from G 1 /S synchronization, the cells were collected for analysis of mitotic index using flow cytometry. ( E ) Correlation between nuclear disruption and delayed mitotic entry. HeLa cells were transfected with the indicated siRNAs and cultured for 48 hours. Thereafter, cells were incubated in the presence of HPG for 2 hours and collected for the analysis by flow cytometry. Values are shown as means ± SD, n = 3. In parallel, the nucleolar disruption with reference to (B) and and the delayed mitotic entry with reference to (D) and and are shown under the graph [+, nucleolar disruption induced (top); −, nucleolar disruption not induced (top); +, delayed mitotic entry induced (bottom); −, delayed mitotic entry not induced (bottom)]. a.u., arbitrary units; CHX, cycloheximide.

Article Snippet: Mouse monoclonal antibodies against α-β-actin (sc47778, Santa Cruz Biotechnology): cyclin B1 (sc245, Santa Cruz Biotechnology), UBF (sc13125, Santa Cruz Biotechnology), Cdk1 (sc45, Santa Cruz Biotechnology), Wee1 (sc5285, Santa Cruz Biotechnology), tubulin (DM1A, Millipore), nucleolin (M019-3, MBL), H3-pS10 (MABI0312, MBL), MPM2 (05-368, Millipore), NPM (FC61991, Thermo Fisher Scientific), and RPL11 (3A4A7, Thermo Fisher Scientific).

Techniques: Knockdown, Transfection, Cell Culture, Staining, Flow Cytometry, Disruption, Incubation

Journal: Cell Reports Medicine

Article Title: CCNE1 copy number is a biomarker for response to combination WEE1-ATR inhibition in ovarian and endometrial cancer models

doi: 10.1016/j.xcrm.2021.100394

Figure Lengend Snippet: Combination WEE1i-ATRi decreases viability and colony formation in CCNE1 Amp compared with CCNE1 Low cells (A) Copy number analysis in CCNE1 Amp (blue), CCNE1 Gain (orange), and CCNE1 Low (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 CCNE1 Amp 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 CCNE1 Amp (blue), CCNE1 Gain (orange), and CCNE1 Low (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 log 2 (CDI) plotted against CCNE1 CN, displayed as log 2 (CN/2) for standard low doses (right lower). The trend line with correlation coefficient R 2 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). Log 2 (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 CCNE1 Amp OVCAR3 cells and CCNE1 Low 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.

Article Snippet: Rabbit anti- WEE1 , Cell Signaling Technology , cat. # 13084, Lot# 1, RRID: AB_2713924.

Techniques: Western Blot, Molecular Weight, Control, Comparison, Knockdown, Transfection

Journal: Cell Reports Medicine

Article Title: CCNE1 copy number is a biomarker for response to combination WEE1-ATR inhibition in ovarian and endometrial cancer models

doi: 10.1016/j.xcrm.2021.100394

Figure Lengend Snippet:

Article Snippet: Rabbit anti- WEE1 , Cell Signaling Technology , cat. # 13084, Lot# 1, RRID: AB_2713924.

Techniques: Virus, Recombinant, Infection, Transfection, BrdU Staining, Flow Cytometry, Imaging, Derivative Assay, Plasmid Preparation, Software, Enzyme-linked Immunosorbent Assay, Microscopy

Journal: International Journal of Cancer

Article Title: Therapeutic potential of targeting the FLNA‐regulated Wee1 kinase in adrenocortical carcinomas

doi: 10.1002/ijc.35239

Figure Lengend Snippet: Differential FLNA and Wee1 protein expression in ACC, ACA, and NAG. (A) Representative immunoblots of FLNA, Wee1 and GAPDH expression in 6 patient‐derived ACC, 8 ACA, and 8 NAG. (B) The graphs show densitometric analysis of FLNA and Wee1 expression normalized to GAPDH and expressed as fold over NCI‐H295R. NCI‐H295R cell lysates were included to normalize all blots to the same control sample. Horizontal bars represent median. * p < 0.05; *** p < 0.001. Kruskal–Wallis multiple comparison test with Dunn's post‐hoc test.

Article Snippet: Wee1 antibody (Santa Cruz Biotechnology, Dallas, TX) and phospho‐Wee1(Ser123) (Bioss Antibodies, Woburn, MA) were diluted 1:100 and 1:200.

Techniques: Expressing, Western Blot, Derivative Assay, Control, Comparison

Journal: International Journal of Cancer

Article Title: Therapeutic potential of targeting the FLNA‐regulated Wee1 kinase in adrenocortical carcinomas

doi: 10.1002/ijc.35239

Figure Lengend Snippet: Expression of FLNA and Wee1 in human ACC cell lines, and effects of FLNA silencing and overexpression on Wee1 protein levels. (A) Protein expression levels of FLNA and Wee1 in NCI‐H295R, MUC‐1, and TVBF‐7 cell lines. Representative immunoblots of FLNA and Wee1 expression normalized to GAPDH are shown. (B) NCI‐H295R, MUC‐1, and TVBF‐7 cell lines were transfected with FLNA siRNA or negative control (C‐) siRNA for 6 days. The graphs show densitometric analysis of Wee1 normalized to GAPDH (median and IQR of at least 3 independent experiments, respectively). Representative immunoblots are shown. * p < 0.05 vs C‐ siRNA. Mann–Whitney test. (C) MUC‐1 cells were transfected with FLNA siRNA or negative control (C‐) siRNA for 6 days. The graphs show densitometric analysis of phosphorylated CDK1 (Tyr15) normalized to total CDK1 (median and IQR of at least 3 independent experiments), and of cyclin B1 normalized to GAPDH (median and IQR of at least 3 independent experiments). Representative immunoblots are shown. * p < 0.05 vs C‐ siRNA. Mann–Whitney test. (D) Patient‐derived primary cultured ACC cells (ACC #1) were transfected with FLNA siRNA or negative control (C‐) siRNA for 6 days. The values above immunoblot images indicate densitometric analysis of Wee1 normalized to GAPDH. (E) MUC‐1 cells were transiently transfected with myc‐tagged FLNA plasmid for 72 h. Mock‐transfected cells were used as negative control. The graphs show densitometric analysis of Wee1 and cyclin B1 normalized to GAPDH (median and IQR of at least 3 independent experiments, respectively), and phosphorylated CDK1 (Tyr15) normalized to total CDK1 (median and IQR of at least 3 independent experiments). Representative immunoblots are shown. *** p < 0.001 vs mock. Mann–Whitney test.

Article Snippet: Wee1 antibody (Santa Cruz Biotechnology, Dallas, TX) and phospho‐Wee1(Ser123) (Bioss Antibodies, Woburn, MA) were diluted 1:100 and 1:200.

Techniques: Expressing, Over Expression, Western Blot, Transfection, Negative Control, MANN-WHITNEY, Derivative Assay, Cell Culture, Plasmid Preparation

Journal: International Journal of Cancer

Article Title: Therapeutic potential of targeting the FLNA‐regulated Wee1 kinase in adrenocortical carcinomas

doi: 10.1002/ijc.35239

Figure Lengend Snippet: Lactacystin treatment reverted Wee1 depletion in FLNA‐transfected in MUC‐1 cell line. (A) MUC‐1 cells were transfected with FLNA siRNA or C‐ siRNA for 6 days, and with myc‐tagged FLNA plasmid for 72 h, and then RNA was extracted. The graphs show expression levels of Wee1 transcript measured by qRT‐PCR after FLNA genetic silencing and FLNA overexpression normalized to GAPDH (median and IQR of at least 3 independent experiments). Mann–Whitney test. (B) MUC‐1 cells were transiently transfected with myc‐tagged FLNA plasmid for 72 h, and then incubated with lactacystin 10 μM for 20 h. Mock‐transfected cells were used as negative controls. The graph shows densitometric analysis of Wee1 normalized to GAPDH (median and IQR of at least 3 independent experiments), expressed as fold vs mock (untreated). Representative immunoblots are shown. * p < 0.05 of untreated myc‐FLNA vs untreated mock, and of lactacystin‐treated myc‐FLNA vs untreated myc‐FLNA. Kruskal–Wallis test with Dunn's post‐hoc test. (C) MUC‐1 cells were transiently transfected with myc‐tagged FLNA plasmid for 72 h. Mock‐transfected cells were used as negative control. Representative immunoblots are shown. Due to the poor quality of p‐Wee1 (Ser123) antibody, densitometrical analysis could not be performed. Three independent experiments were carried out.

Article Snippet: Wee1 antibody (Santa Cruz Biotechnology, Dallas, TX) and phospho‐Wee1(Ser123) (Bioss Antibodies, Woburn, MA) were diluted 1:100 and 1:200.

Techniques: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Over Expression, MANN-WHITNEY, Incubation, Western Blot, Negative Control

Journal: International Journal of Cancer

Article Title: Therapeutic potential of targeting the FLNA‐regulated Wee1 kinase in adrenocortical carcinomas

doi: 10.1002/ijc.35239

Figure Lengend Snippet: Effects of Wee1 knockdown and Wee1 kinase inhibitor AZD1775 on ACC cells. (A) Proliferation assay in Wee1‐silenced MUC‐1 cells. Cells were transfected with Wee1 siRNA or negative control (C‐) siRNA for 72 h. Cells were incubated with BrdU for 2 h, and its incorporation into newly synthetized DNA was measured (median and IQR of at least 3 independent experiments). Representative immunoblots of Wee1 silencing are shown. *** p < 0.001 vs C‐ siRNA. Mann–Whitney test. (B) Detection of MUC‐1 cell apoptosis by Pacific Blue Annexin V/SYTOX AAdvanced Apoptosis kit. Unlabelled cells were used as negative control. After 72 h of transfection, the percentage of Annexin V+ and SYTOX+ cell fractions were measured. Representative flow cytometric plots are shown. Early apoptotic, late apoptotic, and necrotic cells are plotted graphically and are expressed as fold vs C‐ siRNA (median and IQR of at least 3 independent experiments). ** p < 0.01 vs C‐ siRNA. Mann–Whitney test. (C) Cell proliferation assay in MUC‐1, NCI‐H295R, and TVBF‐7 cell lines. Subconfluent cells were stimulated with increasing concentrations of AZD1775 for 72 h. After 2 h of incubation with BrdU, its incorporation into newly synthetized DNA was measured (median and IQR of at least 3 independent experiments, respectively). * p < 0.05; ** p < 0.01; *** p < 0.001 vs basal condition. Friedman test with Dunn's post‐hoc test. (D) Cell viability assay in MUC‐1, NCI‐H295R, and TVBF‐7 cell lines. Subconfluent cells were stimulated with increasing concentrations of AZD1775 for 72 h. MTT assay was used to verify cell viability in response to AZD1775 treatment (median and IQR of at least 3 independent experiments, respectively). * p < 0.05; ** p < 0.01; *** p < 0.001 vs basal condition. Friedman test with Dunn's post‐hoc test. (E) Cell proliferation assay in two different patient‐derived primary cultures of ACC (ACC #1 and ACC #2). Subconfluent cells were stimulated with increasing concentrations of AZD1775 for 72 h. After 24 h of incubation with BrdU, its incorporation into newly synthetized DNA was measured (median and IQR of 5 replicates for each condition). * p < 0.05; ** p < 0.01; *** p < 0.001 vs basal absorbance value. Kruskal–Wallis test with Dunn's post‐hoc test. (F) Detection of MUC‐1 cell apoptosis by Pacific Blue Annexin V/SYTOX AAdvanced Apoptosis kit. Unlabelled cells were used as negative control. After 72 h of treatment with AZD1775, the percentage of Annexin V+ and SYTOX+ cell fractions were measured. Representative flow cytometric plots are shown. Early apoptotic, late apoptotic, and necrotic cells are plotted graphically and are expressed as fold vs basal condition (median and IQR of at least 3 independent experiments). * p < 0.05; ** p < 0.01 vs basal condition. Friedman test with Dunn's post‐hoc test.

Article Snippet: Wee1 antibody (Santa Cruz Biotechnology, Dallas, TX) and phospho‐Wee1(Ser123) (Bioss Antibodies, Woburn, MA) were diluted 1:100 and 1:200.

Techniques: Knockdown, Proliferation Assay, Transfection, Negative Control, Incubation, Western Blot, MANN-WHITNEY, Viability Assay, MTT Assay, Derivative Assay

Journal: Leukemia

Article Title: SETD2 mutations confer chemoresistance in acute myeloid leukemia partly through altered cell cycle checkpoints

doi: 10.1038/s41375-019-0456-2

Figure Lengend Snippet: Checkpoint inhibition increases mitotic catastrophe in chemotreated cells. a Flow cytometry analysis of the phospho-Histone H3 (PHH3)-positive (PHH3+) mitotic population (indicated by boxes) in Mll-Af9 and Mll-Af9/Setd2F2478L/WT primary cells after exposure to culture medium alone or Ara-C treatment for 24 h. b The total number of PHH3+ cells in Mll-Af9 and Mll-Af9/Setd2 F2478L/WT primary cells in (a) were calculated and illustrated. c After Mll-Af9 and Mll-Af9/ Setd2F2478L/WT primary cells were treated with Ara-C for 24 h, the WEE1 inhibitor MK-1775 was added and cells were collected at 1, 5, and 12 h from the same culture for pHH3+ analysis. d Total number of PHH3+ cells in Mll-Af9 and Mll-Af9/Setd2F2478L/WT primary populations in (c) were illustrated and calculated. e After Mll-Af9 and Mll- Af9/Setd2F2478L/WT primary cells were treated with Ara-C for 24 h, MK-1775 was added and the cells were collected at 5 h for confocal analysis. Mitotic catastrophe is visualized by micronucleation detected with an antibody against Lamin B (green). Nuclei or condensed chromosomes are shown by counterstaining with DAPI (blue). Scale bar indicates 10 μm. Yellow triangles are used to show abnormal interphase cells; white arrows are used to show pro-/meta-/anaphase cells. f Representative data of three independent experiments in (e) are shown. In total, 200 cells per slide were counted. Three biological replicates of each genotype are performed in triplicate and the data are presented as the mean ± SD values. *P < 0.05; ***P < 0.001; ****P < 0.0001

Article Snippet: WEE1 inhibitor MK-1775 and CHK1 inhibitor MK-8776 were obtained from Selleckchem.

Techniques: Inhibition, Flow Cytometry

Journal: Leukemia

Article Title: SETD2 mutations confer chemoresistance in acute myeloid leukemia partly through altered cell cycle checkpoints

doi: 10.1038/s41375-019-0456-2

Figure Lengend Snippet: Checkpoint inhibition resensitizes resistant Mll-Af9/Setd2 double-mutant AML cells to chemotherapy. a Mll-Af9 and Mll-Af9/ Setd2F2478L/WT primary bone marrow leukemic cells were treated with variable concentrations of the WEE1 inhibitor MK-1775 for 48 h. b Mll-Af9 and Mll-Af9/Setd2F2478L/WT primary bone marrow leukemic cells were treated with variable concentrations of the combination of Ara-C and MK-1775 for 48 h. c Mll-Af9 and Mll-Af9/Setd2F2478L/WT primary cells were treated with variable concentrations of the CHK1 inhibitor MK-8776 for 48 h. d Mll-Af9 and Mll-Af9/Setd2F2478L/WT primary cells were treated with variable concentrations of the combination of Ara-C and MK-8776 for 48 h. e–g shRNA-mediated Setd2 knockdown was performed in MLL-AF9 human CD34+ cells (e, f) and the MV4–11 cell line (g). After puromycin selection, the stable cell lines were treated with variable concentrations of the WEE1 inhibitor MK-1775 (e, g) or CHK1 inhibitor MK-8776 (f). h MLL-AF9 human CD34+ Scrambled and shSETD2 cells tested in the combination of Ara-C and MK-1775 for 48 h. i MLL-AF9 human CD34+ Scrambled and shSETD2 cells tested with the combination of Ara-C and MK-8776 for 48 h. j MV4–11 Scrambled and shSETD2 cells tested with the combination of Ara-C and MK-1775 for 48 h. The data in (a, c, e, f, g) are shown as three biological replicates of each genotype performed in triplicate and presented as the mean ± SD values. The data in (b, d, h, i, j) are shown as the mean ± SD values (n = 3 technical replicates) of one independent clone

Article Snippet: WEE1 inhibitor MK-1775 and CHK1 inhibitor MK-8776 were obtained from Selleckchem.

Techniques: Inhibition, Mutagenesis, shRNA, Knockdown, Selection, Stable Transfection

Journal: EMBO Molecular Medicine

Article Title: Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis

doi: 10.15252/emmm.201506059

Figure Lengend Snippet: Cell proliferation for PC3 cells transfected with siSCR or siCLU monitored in real time for 60 h with SP Dynamic xCELLigence system (Voltage 2.5 K). Error bars represent mean ± SEM, n = 4. Slopes compared by ANCOVA test, P < 0.0001. Left panel: Cell cycle profile of PC3 cells transfected with siSCR or siCLU. Right panel: Percentage quantification of variation in G1, S, and G2/M phases. Error bars represent mean ± SEM, n = 3, ** P < 0.01 by paired Student's t ‐test (G2/M population P = 0.0058; G1 P = 0.008). Inset: Western blot with CLU antibody in PC3 cells after CLU silencing. Vinculin was used as loading control. Left panel: RNA microarray analysis comparing PC3 cells transfected with siCLU versus siSCR, and Kinexus phosphokinome microarray analysis comparing LNCaP cells transfected with siCLU versus siSCR. Target genes expression is shown as fold change relative to siSCR, P ≤ 0.05 by an unpaired t ‐test for all the genes listed. Central panel: Venn diagram to graphically illustrate the overlap of mRNA and proteins involved in the regulation of mitosis in both transcriptome and phosphokinome analysis. The comparison of these two experiments shows modification of 4 mitosis regulators. Right panel: Western blot validating the expression levels of target proteins.

Article Snippet: PCR primers and probes for the following genes were purchased from Life Technology and used according to the manufacturer's recommendations (clusterin: Hs00156548_m1; Cdc25C: Hs00156411_m1; GAPDH: Hs02758991_g1; Wee1: Hs01119384_g1), using the ABI ViiA TM 7 Real‐Time PCR detection system (Applied Biosystems) with a TaqMan Gene Expression Master Mix (Applied Biosystems).

Techniques: Transfection, Western Blot, Control, Microarray, Expressing, Comparison, Modification

Journal: EMBO Molecular Medicine

Article Title: Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis

doi: 10.15252/emmm.201506059

Figure Lengend Snippet: Gene clusters identified by differential expression profiling of RNA microarray analysis comparing PC3 cells transfected with siSCR versus siCLU. Data were analyzed through the use of QIAGEN's Ingenuity Pathway Analysis (IPA; QIAGEN, Redwood City) ( www.quiagen.com/ingenuity ). ( P < 0.05; right‐tailed Fisher's exact test). In silico correlation analysis of Cdc25C and CLU mRNA levels in 460 prostate cancer patients using GeneSapiens data set http://ist.medisapiens.com/ (Spearman correlation: r = −0.23; P < 0.001). Co‐immunoprecipitation from parental PC3 cells. Proteins were immunoprecipitated with specific anti‐Cdc25C and CLU antibodies and analyzed by immunoblotting. Actin and IgG were used as negative controls. Duolink proximity ligation assay between Cdc25C and CLU in PC3 cells. Confocal microscopy was used to detect the interaction (red dots). DNA was counterstained with DAPI (blue). Actin and eIF4G were used as a negative control. Scale bar represents 10 μm. FACS analysis showing percentage of the PC3 cells positive for phosphohistone H3 after treatment with siCLU compared to siSCR. Error bar represents mean ± SEM, n = 3, *** P < 0.0001 by the Mann–Whitney test. Western blot analysis for CLU, Cdc25C, Cdc25C‐T48, and pERK antibodies in PC3 cells after transfection with siSCR or siCLU, followed by treatment with or without MAPK inhibitor UO126, synchronization and nocodazole release at the indicated time. Vinculin was used as loading control.

Article Snippet: PCR primers and probes for the following genes were purchased from Life Technology and used according to the manufacturer's recommendations (clusterin: Hs00156548_m1; Cdc25C: Hs00156411_m1; GAPDH: Hs02758991_g1; Wee1: Hs01119384_g1), using the ABI ViiA TM 7 Real‐Time PCR detection system (Applied Biosystems) with a TaqMan Gene Expression Master Mix (Applied Biosystems).

Techniques: Quantitative Proteomics, Microarray, Transfection, In Silico, Immunoprecipitation, Western Blot, Proximity Ligation Assay, Confocal Microscopy, Negative Control, MANN-WHITNEY, Control

Journal: EMBO Molecular Medicine

Article Title: Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis

doi: 10.15252/emmm.201506059

Figure Lengend Snippet: Real‐time PCR for CLU and Cdc25C in three different prostate cancer cell lines. Target genes expression was calculated relative to GAPDH and normalized to siSCR. Error bars represent mean ± SD, n = 3. Western blot for CLU and Cdc25C after siSCR or siCLU transfection in the same prostate cancer cell lines as in A. Vinculin was used as loading control. Western blot for CLU and Cdc25C in PC3 cells after transfection with siSCR or siCLU followed by transfection with CLU or empty expression plasmid for an additional 48 h. Vinculin was used as loading control.

Article Snippet: PCR primers and probes for the following genes were purchased from Life Technology and used according to the manufacturer's recommendations (clusterin: Hs00156548_m1; Cdc25C: Hs00156411_m1; GAPDH: Hs02758991_g1; Wee1: Hs01119384_g1), using the ABI ViiA TM 7 Real‐Time PCR detection system (Applied Biosystems) with a TaqMan Gene Expression Master Mix (Applied Biosystems).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Control, Plasmid Preparation

Journal: EMBO Molecular Medicine

Article Title: Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis

doi: 10.15252/emmm.201506059

Figure Lengend Snippet: Upper panel: Western blot for CLU and Cdc25C from LNCaP xenografts harvested from mice treated with OGX‐011 ( n = 8) or SCR ASO control ( n = 6). Vinculin was used as loading control. Lower panel: Quantification of the protein levels, relative to the loading control, reported as dot plots. * P < 0.05 by t ‐test followed by Welch's correction (CLU P = 0.049, Cdc25C P = 0.024). Western blot with the correspondent quantification of protein levels (left) and real‐time PCR (right) from untreated LNCaP xenografts harvested at endpoint. The negative correlation between CLU‐RNA and Cdc25C‐RNA ΔΔ C T values was calculated using a spearman test ( ρ = −0.86; P = 0.0107). Immunohistochemical staining for CLU and Cdc25C of radical prostatectomy samples from historical control specimens with no neoadjuvant therapy ( n = 5) or neoadjuvant hormone therapy (NHT) ( n = 5) or the combination of NHT and OGX‐011 ( n = 9) for 3 months prior to surgery. Average score staining from the three groups (left) and representative sections for CLU and Cdc25C (right). Error bars represent mean ± SEM. CLU: * P < 0.05; untreated versus 3mNHT ( P = 0.0213); 3mNHT versus 3mNHT + OGX‐011 ( P = 0.012) by ANOVA followed by Bonferroni's post hoc analysis. All cases were normalized by clinical stage, Gleason score, and serum PSA. For each patient, the pathologist selected the area with the highest Gleason score. Scale bar represents 100 μm.

Article Snippet: PCR primers and probes for the following genes were purchased from Life Technology and used according to the manufacturer's recommendations (clusterin: Hs00156548_m1; Cdc25C: Hs00156411_m1; GAPDH: Hs02758991_g1; Wee1: Hs01119384_g1), using the ABI ViiA TM 7 Real‐Time PCR detection system (Applied Biosystems) with a TaqMan Gene Expression Master Mix (Applied Biosystems).

Techniques: Western Blot, Control, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Staining

Journal: EMBO Molecular Medicine

Article Title: Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis

doi: 10.15252/emmm.201506059

Figure Lengend Snippet: Co‐immunoprecipitation from parental PC3 cells transfected with CLU and Cdc25C plasmid expression vectors. Proteins were immunoprecipitated with specific anti‐Cdc25C (left) and CLU (right) antibodies and analyzed by immunoblotting. Dual immunofluorescence staining with Cdc25C (green) and CLU (red) antibodies in PC3 cells. Confocal microscopy was used to identify the interaction (white arrows). DNA was counterstained with (DAPI) (blue). Scale bar represents 10 μm. Duolink proximity ligation assay between Cdc25C and CLU in PC3 cells. Confocal microscopy was used to detect the interaction (red dots). DNA was counterstained with DAPI (blue). PC3 cells transfected with siCLU were used as a negative control. Scale bar represents 10 μm.

Article Snippet: PCR primers and probes for the following genes were purchased from Life Technology and used according to the manufacturer's recommendations (clusterin: Hs00156548_m1; Cdc25C: Hs00156411_m1; GAPDH: Hs02758991_g1; Wee1: Hs01119384_g1), using the ABI ViiA TM 7 Real‐Time PCR detection system (Applied Biosystems) with a TaqMan Gene Expression Master Mix (Applied Biosystems).

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Expressing, Western Blot, Immunofluorescence, Staining, Confocal Microscopy, Proximity Ligation Assay, Negative Control

Journal: EMBO Molecular Medicine

Article Title: Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis

doi: 10.15252/emmm.201506059

Figure Lengend Snippet: Flow cytometry for PC3 cells after transfection with siSCR or siCLU and synchronization to the M phase with thymidine/nocodazole block. DNA content was analyzed at the indicated time point after nocodazole release. Histograms show % of cells in G2/M, over 5 h period. Error bars represent mean ± SEM, n = 3, * P < 0.05, **** P < 0.0001 by ANOVA followed by Bonferroni's post hoc correction. (2 h P = 0.0142). Immunofluorescence in PC3 cells after siSCR or siCLU transfection, synchronization and nocodazole release at indicated time points. Mitotic spindle formation was assessed by confocal microscopy, using pericentrin (centrioles; red) and β‐tubulin (microtubules; green) antibodies. DNA was counterstained with (DAPI) (blue). White arrows represent segregation abnormalities. Scale bar represents 10 μm. Flow cytometry to assess phosphohistone H3 levels in the same setting as in (A). Error bar represents mean ± SEM, n = 3, **** P < 0.0001 by ANOVA followed by Bonferroni's post hoc correction. Western blot for CLU, Cdc25C, Cdk1, and cyclin B1 antibodies in PC3 cells after transfection with siSCR or siCLU, synchronization and nocodazole release at the indicated time. Vinculin was used as loading control.

Article Snippet: PCR primers and probes for the following genes were purchased from Life Technology and used according to the manufacturer's recommendations (clusterin: Hs00156548_m1; Cdc25C: Hs00156411_m1; GAPDH: Hs02758991_g1; Wee1: Hs01119384_g1), using the ABI ViiA TM 7 Real‐Time PCR detection system (Applied Biosystems) with a TaqMan Gene Expression Master Mix (Applied Biosystems).

Techniques: Flow Cytometry, Transfection, Blocking Assay, Immunofluorescence, Confocal Microscopy, Western Blot, Control

Journal: EMBO Molecular Medicine

Article Title: Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis

doi: 10.15252/emmm.201506059

Figure Lengend Snippet: Left panel: Western blot for CLU, Cdc25C, Cdc25C‐T48, Cdc25C S216, and Cdc25C S198 in PC3 cells after siSCR or siCLU transfection, synchronization and nocodazole release at indicated time points. Vinculin was used as loading control. Right panel: Immunofluorescence for Cdc25C‐T48 in PC3 cells after siSCR or siCLU transfection. DNA was counterstained with (DAPI) (blue). Scale bar represents 10 μm. Western blot for CLU, Cdc25C‐T48, Cdk1, and PP2A in PC3 cells after siSCR or siCLU transfection and after synchronization followed by treatment with or without okadaic acid (OA) (100 nM) for 12 h. Proteins were extracted 15 min after nocodazole release. Vinculin was used as loading control. Western blot for PP2A, CLU, Cdc25C‐T48, Cdc25C, Wee1, and Cdk1 in PC3 cells transfected with siSCR or siPP2A synchronized and nocodazole released at indicated time points. Vinculin was used as loading control. PP2A phosphatase activity measurement in PC3 cells after siSCR or siCLU transfection, synchronization and nocodazole release at indicated time points. Error bar represents mean ± SEM, n = 3, ** P < 0.01 ( P = 0.0025) by ANOVA followed by Bonferroni's post hoc comparisons. Cells treated with no peptide were used as a negative control.

Article Snippet: PCR primers and probes for the following genes were purchased from Life Technology and used according to the manufacturer's recommendations (clusterin: Hs00156548_m1; Cdc25C: Hs00156411_m1; GAPDH: Hs02758991_g1; Wee1: Hs01119384_g1), using the ABI ViiA TM 7 Real‐Time PCR detection system (Applied Biosystems) with a TaqMan Gene Expression Master Mix (Applied Biosystems).

Techniques: Western Blot, Transfection, Control, Immunofluorescence, Activity Assay, Negative Control

Journal: EMBO Molecular Medicine

Article Title: Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis

doi: 10.15252/emmm.201506059

Figure Lengend Snippet: Western blot for CLU, Cdk1‐Y15, Cdk1, and Wee1 in PC3 cells after siSCR or siCLU transfection followed by synchronization and nocodazole release at indicated time points. Vinculin was used as loading control. Wee1 expression level was evaluated in PC3 cells after CLU silencing by real‐time PCR. Error bar represents mean ± SEM, n = 3, *** P < 0.001 ( P = 0.0001) by unpaired t ‐test followed by Welch's correction. FACS analysis showing percentage of the PC3 cells positive for phosphohistone H3 after transfection with siSCR or siCLU in the presence and absence of Wee1 inhibitor, MK‐1775. Error bar represents mean ± SEM, n = 3, **** P < 0.0001 by the Mann–Whitney test. Western blot for CLU, Wee1, Cdk1‐Y15, Cdk1, and cyclin B1 in PC3 cells after transfection with siSCR or siCLU followed by synchronization and treatment with or without MK‐1775, a specific Wee1 inhibitor, for 12 h. Cells were harvested at different time points after nocodazole release. Vinculin was used as loading control. Source data are available online for this figure.

Article Snippet: PCR primers and probes for the following genes were purchased from Life Technology and used according to the manufacturer's recommendations (clusterin: Hs00156548_m1; Cdc25C: Hs00156411_m1; GAPDH: Hs02758991_g1; Wee1: Hs01119384_g1), using the ABI ViiA TM 7 Real‐Time PCR detection system (Applied Biosystems) with a TaqMan Gene Expression Master Mix (Applied Biosystems).

Techniques: Western Blot, Transfection, Control, Expressing, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis

doi: 10.15252/emmm.201506059

Figure Lengend Snippet: Cabazitaxel IC50 calculation using WST1 assay in PC3 cells after transfection with siSCR or siCLU (left), and SCR or OGX‐011 (right) followed by 72 h of cabazitaxel treatment. Percentage of surviving cells was calculated relative to control. Error bar represents mean ± SEM, n = 3. Inset: Western blot was performed to verify CLU knockdown in cells. Mitotic cells and cells in mitotic catastrophe were counted using DAPI in PC3 cells after SCR or OGX‐011 transfection followed by different concentration of cabazitaxel as indicated (> 1,000 counts). Error bar represents mean ± SEM., * P < 0.05, *** P < 0.001, and **** P < 0.0001 by Student's t ‐test (caba 1.5 nM P = 0.00115; 3 nM P = 0.0105; 4.5 nM P = 1.93 × 10 −7 ; 6.25 nM P = 8.9 × 10 −9 ; 12.5 nM P = 6.8 × 10 −9 ; 25 nM P = 1.2 × 10 −9 ). Clonogenic assay in PC3 cells after SCR or OGX‐011 transfection followed by treatment with different concentrations of cabazitaxel as indicated. The number of survival clones was assessed after 15 days using crystal violet assay. Error bars represent mean ± SEM n = 3, * P < 0.05, ** P < 0.01 by the Mann–Whitney test (0 nM caba P = 0.039; 0.5 nM P = 0.0079; 2 nM P = 0.0079).

Article Snippet: PCR primers and probes for the following genes were purchased from Life Technology and used according to the manufacturer's recommendations (clusterin: Hs00156548_m1; Cdc25C: Hs00156411_m1; GAPDH: Hs02758991_g1; Wee1: Hs01119384_g1), using the ABI ViiA TM 7 Real‐Time PCR detection system (Applied Biosystems) with a TaqMan Gene Expression Master Mix (Applied Biosystems).

Techniques: Transfection, Control, Western Blot, Knockdown, Concentration Assay, Clonogenic Assay, Clone Assay, Crystal Violet Assay, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis

doi: 10.15252/emmm.201506059

Figure Lengend Snippet: Cell cycle profile of IGR‐CaP1, IGR‐CaP1‐R‐caba; and PC3, PC3‐R‐caba cells in the presence or absence of 10 nM cabazitaxel. Schema of molecular interaction between CLU and Cdc25C that regulate Cdk1/cyclin B1 activity.

Article Snippet: PCR primers and probes for the following genes were purchased from Life Technology and used according to the manufacturer's recommendations (clusterin: Hs00156548_m1; Cdc25C: Hs00156411_m1; GAPDH: Hs02758991_g1; Wee1: Hs01119384_g1), using the ABI ViiA TM 7 Real‐Time PCR detection system (Applied Biosystems) with a TaqMan Gene Expression Master Mix (Applied Biosystems).

Techniques: Activity Assay

Journal: EMBO Molecular Medicine

Article Title: Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis

doi: 10.15252/emmm.201506059

Figure Lengend Snippet: Cell cycle distribution of synchronized PC3 and PC3‐R‐caba cells 60 min after nocodazole release at indicated time points. Western blot for CLU, Cdc25C, Cdc25C‐T48, Cdk1, Cdk1‐Y15, and Wee1 in IGR‐CaP1 and PC3 parental (WT) or cabazitaxel‐resistant (R‐caba) cells as well as for PC3‐R‐caba cells transfected with siSCR or siCLU. Vinculin was used as loading control. Cell survival measured using WST1 in PC3‐R‐caba cells after SCR or OGX‐011 transfection and treatment with 10 nM cabazitaxel for 48 h. Error bar represents mean ± SEM, n = 3, *** P < 0.001 by one‐way ANOVA followed by Bonferroni's post hoc correction. Cell survival measured using WST1 in PC3 resistant cells after transfection with siSCR or siCLU followed by treatment with cabazitaxel (10 nM; C10), MK‐1775 (10 nM; MK10), or both for 72 h. Error bar represents the mean ± SEM, n = 3, *** P < 0.001 by one‐way ANOVA followed by Bonferroni's post hoc correction. PARP cleavage was assessed by Western blot in cells treated as in (D) and its relative expression was quantified by densitometry using ImageJ. Vinculin was used as loading control. Left panel: Quantification of tumor volume over 52 days in nude male mice xenotransplanted with PC3‐docetaxel‐resistant cells and treated with either MK‐1775 (M, black line), MK‐1775 and micellar taxane (MT, blue line), micellar taxane and OGX‐011 (TO, red line), or MK‐1775, micellar taxane and OGX‐011 (MTO, green line). The error bars indicate mean ± SEM, n = 8. Right panel: Slopes of tumor growth between groups were compared by ANOVA followed by Dunnett's post hoc analysis. * P < 0.05, *** P < 0.001. P = 0.0488 (M to TO); P = 0.0007 (M to MTO).

Article Snippet: PCR primers and probes for the following genes were purchased from Life Technology and used according to the manufacturer's recommendations (clusterin: Hs00156548_m1; Cdc25C: Hs00156411_m1; GAPDH: Hs02758991_g1; Wee1: Hs01119384_g1), using the ABI ViiA TM 7 Real‐Time PCR detection system (Applied Biosystems) with a TaqMan Gene Expression Master Mix (Applied Biosystems).

Techniques: Western Blot, Transfection, Control, Expressing