Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The G Protein Estrogen Receptor (GPER) is involved in the resistance to the CDK4/6 inhibitor palbociclib in breast cancer

doi: 10.1186/s13046-024-03096-7

Figure Lengend Snippet: EGFR or GPER silencing restore palbociclib sensitivity in MCF7/PalbR cells. A Representative pictures of spheroids (a single spheroid/well) from MCF7/PalbR/shRNA and MCF7/PalbR/shEGFR spheroid cultures grown for 6 days on agar-coated plates. B Quantification of spheroid growth; values of MCF7/PalbR/shRNA cells were set as 100% upon which the number of MCF7/PalbR/shEGFR cells was determined. C Efficacy of EGFR silencing in MCF7/PalbR/shEGFR cells. D Representative pictures of spheroids (a single spheroid/well) from the MCF7/PalbR/shRNA and MCF7/PalbR/shGPER spheroid cultures grown for 6 days on agar-coated plates. Scale bar 500 μm. E Quantification of spheroid growth; values of MCF7/PalbR/shRNA cells were set as 100% upon which the number of MCF7/PalbR/shGPER cells was determined. F Efficacy of GPER silencing in MCF7/PalbR/shGPER cells. G Colony formation assay in in MCF7/PalbR/shRNA and MCF7/PalbR/shGPER cells. Plates were stained with Crystal Violet and colonies were counted following 10 days of incubation. ( H ). I Protein levels of cyclin D1, cyclin E1 and GPER in MCF7/PalbR/shRNA and MCF7/PalbR/shGPER cells. Side panels show densitometric analyses of the blots normalized to β-actin, which served as loading control. Values represent the mean ± SD of three independent experiments performed in triplicate. (*) indicates p < 0.05. Kaplan-Meier survival curves representing the overall survival ( J ) and relapse-free survival ( K ) in ER-positive BC patients of the METABRIC database, based on low vs high EGFR and GPER mRNA levels (median values were used as threshold)

Article Snippet: Equal amounts of whole-protein extract were resolved on an 8% or 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes (Merck, Milan, Italy), which were probed with primary antibodies against ERα (F-10), EGFR (A-10), c-Fos (E-8), EGR1 (S-25), Cyr61 (A-10), p21 (H164), phosphorylated ERK1/2 (E-4), ERK2 (C-14), and β-actin (AC-15) (Santa Cruz Biotechnology, DBA, Milan, Italy), GPER (AB137479; Abcam, DBA, Milan, Italy), pAKT (4060) and AKT (9272) (Cell Signaling, Euroclone, Milan, Italy), cyclin D1 (TA801655) and cyclin E1 (AP06082PU-N) (purchased from OriGene Technologies, DBA, Milan, Italy) , and then revealed using the chemiluminescent substrate for western blotting ClarityTM Western ECL Substrate (Bio-Rad, Milan, Italy).

Techniques: shRNA, Colony Assay, Staining, Incubation, Control

Journal: The Journal of Pathology

Article Title: DNA methylation of microRNA‐coding genes in non‐small‐cell lung cancer patients

doi: 10.1002/path.5079

Figure Lengend Snippet: Predicted miR‐1179 targets in NSCLC cells and in NSCLC patients. (A) Expression of the predicted miR‐1179 targets CCNE1 , NUAK2 and SPOCK1 was measured in miR‐1179 mimic‐transfected NCI‐H1993 and NCI‐H2073 cells. Fold changes relative to control cells are shown. * p < 0.05. (B) Stably transfected A549 pCMV‐miR‐1179 cells were treated with an miR‐1179 inhibitor, and RT‐PCR showed upregulated expression of CCNE1 , NUAK2 and SPOCK1 . * p < 0.05. (C) Western blot analysis of cyclin E1 in NCI‐H1993 and NCI‐H2073 cells transfected with control RNA (–) or miR‐1179 (+). (D) Luciferase activity in cells that were cotransfected with a luciferase reporter construct containing either the wild‐type (WT) or the mutated (MUT) 3′‐UTR of CCNE1 and with miR‐1179 mimics. RLU, relative luminescence units.

Article Snippet: Cells were cotransfected with a luciferase reporter construct containing the 3′‐UTR of CCNE1 (SC207262; Origene, Rockville, MD, USA) and with miRNA‐1179 mimics (4464066_MC13164; Ambion) or random sequence miRNA mimic controls.

Techniques: Expressing, Transfection, Control, Stable Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Luciferase, Activity Assay, Construct

Journal: Nature Communications

Article Title: G1/S restriction point coordinates phasic gene expression and cell differentiation

doi: 10.1038/s41467-022-31101-0

Figure Lengend Snippet: A DNA-content analysis of cells from whole embryos ranging from embryonic day 6.5 (E6.5) to E13.5 (representative litters, n = 2 biologically independent samples for each stage, embryos from each litter were pooled). B Schematic of the data collection and analysis. E7.5, E8.5, and E9.5 embryos were dissociated to single cells, sorted by cell cycle phase based on DNA-content, and subjected to RNA-sequencing for differential expression analysis between the phases at each timepoint. C – E The expression of Actb ( C ), Prc1 (adjusted P value (adj. P ), * P = 0.029 [E7.5]; *** P = 0.00017 [E8.5]; *** P = 1.02e−9 [E9.5]) ( D ) and Slbp (* adj. P = 0.05) ( E ) at each developmental stage. n = 2 biologically independent litters for each stage and phase. Litters of 6 and 4 embryos at E7.5, 8 and 6 embryos at E8.5, and 3 and 5 embryos at E9.5 were pooled. F The number of genes showing differential expression between G1 and G2/M (adj. P < 0.1) increases between E7.5 and E9.5 (*** P < 0.001, Chi-square test). G The distribution of adjusted P values at each developmental stage for union of all genes identified in F (*** P < 0.001, Wilcoxon Rank Test, two-sided). H Heat map of P values for all genes shown in G (*** P < 0.001, Wilcoxon Rank Test, two-sided). I The distribution of fold-change values for all genes shown in G (*** P < 0.001, Tukey’s post hoc, one-way ANOVA). J The distribution of log2 counts per million for all genes shown in ( G ) (no significant change, P > 0.5, one-way ANOVA, n = 714 genes). White dot, median. Box edges, 25th and 75th quartiles. Whiskers, 1.5× the IQR of the box edge. K The relative distribution of Cyclin E1 and Cyclin B1 protein in E7.5, E8.5, and E9.5 embryos. The fraction of cells expressing both Cyclin E1 and Cyclin B1 declines between E7.5 and E9.5 (** P < 0.01, one-way ANOVA; n = 521 (E7.5), n = 680 (E8.5), n = 1516 (E9.5) biologically independent cells). C – H Differential expression was evaluated with a Wald Chi-squared test and adjusted for multiple tests using the Benjamini-Hochberg (BH) approach, n = 2 biologically independent samples for each stage and phase (each dot represents an individual sample in C – E ). The Source Data for ( C – E , K ) are provided in “ ”.

Article Snippet: Embryos were fixed in 4% PFA overnight at 4 °C, and targets detected using the following primary antibodies: GFP at 1:400 (Aves Labs, Aves-1020), Cyclin E1 at 1:200 (R&D Systems, AF6810), Cyclin B1 at 1:200 (Cell Systems Technology, 4138).

Techniques: RNA Sequencing, Quantitative Proteomics, Expressing

Journal: Nature Communications

Article Title: G1/S restriction point coordinates phasic gene expression and cell differentiation

doi: 10.1038/s41467-022-31101-0

Figure Lengend Snippet: A Targeting Ccne1 with homology directed repair gave rise to N-terminal truncation and segment duplication (SD) mutants. Cyclin E1 western blot of control, Ccne1 Trunc and Ccne1 SD mutant ES cell lines. The upper band seen in wt and absent in both mutants is of unknown origin. B Rb remains constitutively phosphorylated at site 807/811 of Ccne1 SD mutants during assay of neural differentiation. (left, middle panels) The fraction of pRb 807/811+ cells is higher in Ccne1 SD than controls after 7 days differentiation (two-way ANOVA, P = 0.0031, Sidak’s multiple comparison test *** P = 0.0005 (day 7), n = 2 biologically independent samples across two independent experiments). (right panel) Schematic of data from “B” illustrating the presence of pRb throughout G1 in neural differentiation assay of Ccne1 SD mutants. C Neural differentiation of Ccne1 SD mutants is impaired (** P < 0.01, *** P < 0.001, Sidak’s multiple comparison test, two-way ANOVA, P < 0.0001). D Consistent with deficient neural differentiation, Ccne1 SD mutants rarely generate βIII-tubulin+ neurons (scale bar = 200 μm). E Mesodermal differentiation of Ccne1 SD mutants is impaired (** P = 0.0037, Sidak’s multiple comparison test, two-way ANOVA, P = 0.0011). F , G Rather than neural expression, the expression of pluripotency markers NANOG ( F ) and OCT4 ( G ) persist during neuronal culture of Ccne1 SD mutants (scale bar = 200 μm). The Source Data for ( B , C , E ) are provided in “ ”.

Article Snippet: Embryos were fixed in 4% PFA overnight at 4 °C, and targets detected using the following primary antibodies: GFP at 1:400 (Aves Labs, Aves-1020), Cyclin E1 at 1:200 (R&D Systems, AF6810), Cyclin B1 at 1:200 (Cell Systems Technology, 4138).

Techniques: Western Blot, Control, Mutagenesis, Comparison, Differentiation Assay, Expressing

Journal: Molecular Oncology

Article Title: Integrative miRNOMe profiling reveals the miR ‐195‐5p– CHEK1 axis and its impact on luminal breast cancer outcomes

doi: 10.1002/1878-0261.70077

Figure Lengend Snippet: Relapse‐free survival probabilities for breast carcinoma patients stratified by subtype, therapy, and expression of studied genes. Relapse‐free survival (RFS) of adjuvant chemotherapy‐treated patients with luminal subtypes ( N = 171) (A) or solely the patients with the luminal A subtype ( N = 54) (B) stratified by the CHEK1 gene expression. RFS of adjuvant chemotherapy‐treated patients with luminal subtypes ( N = 171) (C) and solely the patients with the luminal B subtype ( N = 113) (D) stratified by the CCNE1 gene expression. Neoadjuvant chemotherapy‐treated patients with luminal subtypes ( N = 38) stratified by miR‐195‐5p (E) and CDC25A (F) gene expression levels. The optimal cut‐off for dividing patients with low vs. high expression was the 50th percentile (Q1‐Q2 vs Q3‐Q4) unless otherwise specified.

Article Snippet: We identified CHK1 (1:1000, mouse monoclonal antibody, 2G1D5, Novus Biologicals, Centennial, CO, USA), CCNE1 (1:200, mouse monoclonal antibody, CCNE1/2460, Novus Biologicals), CDC25A (1:500, mouse monoclonal antibody, #336445, R&D Systems, Minneapolis, MN, USA), ERα (1:000, rabbit monoclonal antibody, #8644, Cell Signaling, Danvers, Massachusetts, USA), cleaved caspase‐3 (1:500, rabbit monoclonal antibody, #9664, Cell Signaling), and cleaved PARP (1:750, rabbit polyclonal antibody, #9542, Cell Signaling).

Techniques: Expressing, Adjuvant, Gene Expression

Journal: Molecular Oncology

Article Title: Integrative miRNOMe profiling reveals the miR ‐195‐5p– CHEK1 axis and its impact on luminal breast cancer outcomes

doi: 10.1002/1878-0261.70077

Figure Lengend Snippet: Relative changes of the CHEK1, CDC25A, and CCNE1 gene expression in breast cancer cell lines transfected with miR‐195‐5p miRNA mimics compared to control in vitro . Baseline protein expression of the CHEK1, CDC25A, CCNE1, and ERα across BT‐20, MCF‐7, and T‐47D breast cancer cell lines is shown in (A). Relative changes in protein (B, C) and mRNA levels (D) in the same cell lines transfected with miR‐195‐5p miRNA mimics or negative controls for 24 h are presented. Cell lines treated with Lipofectamine RNAiMAX alone or with scrambled miRNAs were used as negative controls. The genes were considered significantly differentially expressed at the transcript level if the absolute value of fold change compared to control was ≥1.5 and P ‐value ≤0.05. The upper and lower lines of the boxplots represent the upper (75%) and lower quartile (25%), the line across the box represents the median, and the solid circles indicate outliers. Results of transcript expression measured by qPCR are presented as the mean ± SD of at least three independent experiments. Densitometric data of protein expression measured by western blot are presented as a percentage of the control (mean ± SD of three independent experiments), except for CDC25A in T‐47D cells and CCNE1 in BT‐20 cells (based on two replicates due to lack of expression in the third experiment). Western blot images show a representative experiment. Data were analyzed with an unpaired Student's t ‐test; * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: We identified CHK1 (1:1000, mouse monoclonal antibody, 2G1D5, Novus Biologicals, Centennial, CO, USA), CCNE1 (1:200, mouse monoclonal antibody, CCNE1/2460, Novus Biologicals), CDC25A (1:500, mouse monoclonal antibody, #336445, R&D Systems, Minneapolis, MN, USA), ERα (1:000, rabbit monoclonal antibody, #8644, Cell Signaling, Danvers, Massachusetts, USA), cleaved caspase‐3 (1:500, rabbit monoclonal antibody, #9664, Cell Signaling), and cleaved PARP (1:750, rabbit polyclonal antibody, #9542, Cell Signaling).

Techniques: Gene Expression, Transfection, Control, In Vitro, Expressing, Western Blot

Journal: Scientific Reports

Article Title: The role of long non-coding RNA LINC00839 in oral squamous cell carcinoma based on bioinformatics and experimental research

doi: 10.1038/s41598-024-82922-6

Figure Lengend Snippet: Correlation and expression analysis of the LINC00839/miR-195-5p/CCNE1 axis according to TCGA database data. ( A ) A Pearson correlation analysis was conducted to determine the relationship between the expression levels of LINC00839 and CCNE1. ( B ) The expression of LINC00839, classified as an oncogenic lncRNA, was compared between OSCC tumor tissues ( n = 293) and normal tissues ( n = 32). ( C ) The expression of miR-195-5p, a tumor suppressor miRNA, was analyzed in tumor samples ( n = 303) versus normal tissues ( n = 32), and ( D ) the expression of CCNE1, recognized as an oncogene, was assessed in both OSCC tumor and normal tissues. All the results yielded significant p-values and false discovery rates.

Article Snippet: Subsequently, the slides were rinsed with TBS containing Triton X-100 and incubated with the Cyclin E1 Polyclonal Antibody (Elabscience, E-AB-31074) overnight at 4 oC.

Techniques: Expressing

Journal: Scientific Reports

Article Title: The role of long non-coding RNA LINC00839 in oral squamous cell carcinoma based on bioinformatics and experimental research

doi: 10.1038/s41598-024-82922-6

Figure Lengend Snippet: Protein-protein interaction (PPI) network of CCNE1. ( A ) The CCNE1 PPI network is constituted of 21 nodes and 259 edges. The node size and edge thickness are associated with the degree value and strong data sources, respectively. Additionally, the unique colors assigned to the edges represent different types of interactions. ( B ) The percentages of different interactions that created the network: physical interactions, co-expression, predicted, co-localization, genetic interactions, pathway, and shared protein domains.

Article Snippet: Subsequently, the slides were rinsed with TBS containing Triton X-100 and incubated with the Cyclin E1 Polyclonal Antibody (Elabscience, E-AB-31074) overnight at 4 oC.

Techniques: Expressing

Journal: Scientific Reports

Article Title: The role of long non-coding RNA LINC00839 in oral squamous cell carcinoma based on bioinformatics and experimental research

doi: 10.1038/s41598-024-82922-6

Figure Lengend Snippet: Dot plot and cnetplot of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Each dot plot indicates significantly enriched terms in GO categories, including ( A ) biological process (BP), ( B ) cellular component (CC), and ( C ) molecular function (MF), accompanied by enriched terms in ( D ) KEGG. The terms at the top of the dot plot are more significant than those at the bottom. The cnetplot represents the connections between individual proteins of the CCNE1 PPI network and GO categories, including ( E ) BP, ( F ) CC, ( G ) MF, and ( H ) KEGG. CCNE1 is depicted in bold.

Article Snippet: Subsequently, the slides were rinsed with TBS containing Triton X-100 and incubated with the Cyclin E1 Polyclonal Antibody (Elabscience, E-AB-31074) overnight at 4 oC.

Techniques:

Journal: Scientific Reports

Article Title: The role of long non-coding RNA LINC00839 in oral squamous cell carcinoma based on bioinformatics and experimental research

doi: 10.1038/s41598-024-82922-6

Figure Lengend Snippet: Analysis of LINC00839/miR-195-5p/cyclin E1 (CCNE1) axis expression and their correlation in OSCC tissues. The relative expression of LINC00839 ( A ), miR-195-5p ( B ), and CCNE1 mRNA ( C ) in 30 OSCC tissues versus adjacent non-tumor tissues was evaluated using RT-qPCR. ( D ) The protein level of CCNE1 was determined through western blotting (corresponding graph is represented as mean ± SD following normalization against glyceraldehyde 3-phosphate dehydrogenase [GAPDH]). ( E ) Correlation analysis between LINC00839 and CCNE1 expression levels (Spearman’s nonparametric test). ( F ) Pearson’s test was conducted to analyze the correlation between LINC00839 and miR-195-5p expression levels. ( G ) The correlation between miR-195-5p and CCNE1 was determined based on the Pearson correlation coefficient. ** p < 0.01 and **** p < 0.0001.

Article Snippet: Subsequently, the slides were rinsed with TBS containing Triton X-100 and incubated with the Cyclin E1 Polyclonal Antibody (Elabscience, E-AB-31074) overnight at 4 oC.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: The role of long non-coding RNA LINC00839 in oral squamous cell carcinoma based on bioinformatics and experimental research

doi: 10.1038/s41598-024-82922-6

Figure Lengend Snippet: Immunofluorescence staining of oral squamous cell carcinoma tumor and margin tissues. The nuclei were stained using 4′,6-Diamidino‐2-phenylindole (DAPI), which emits a blue fluorescence, while the cyclin E1 protein was detected using a green fluorescent antibody. Scale bar = 50 μm. * p < 0.05. Original magnification, × 200.

Article Snippet: Subsequently, the slides were rinsed with TBS containing Triton X-100 and incubated with the Cyclin E1 Polyclonal Antibody (Elabscience, E-AB-31074) overnight at 4 oC.

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Scientific Reports

Article Title: The role of long non-coding RNA LINC00839 in oral squamous cell carcinoma based on bioinformatics and experimental research

doi: 10.1038/s41598-024-82922-6

Figure Lengend Snippet: Association of LINC00839, miR-195-5p, and cyclin E1 expression with clinico-pathological parameters in OSCC patients.

Article Snippet: Subsequently, the slides were rinsed with TBS containing Triton X-100 and incubated with the Cyclin E1 Polyclonal Antibody (Elabscience, E-AB-31074) overnight at 4 oC.

Techniques: Expressing

Journal: Scientific Reports

Article Title: The role of long non-coding RNA LINC00839 in oral squamous cell carcinoma based on bioinformatics and experimental research

doi: 10.1038/s41598-024-82922-6

Figure Lengend Snippet: ROC curve analysis of LINC00839, miR-195-5p, CCNE1, and their combination for OSCC diagnosis in tumor tissues. AUC: area under the curve, CCNE1: cyclin E1.

Article Snippet: Subsequently, the slides were rinsed with TBS containing Triton X-100 and incubated with the Cyclin E1 Polyclonal Antibody (Elabscience, E-AB-31074) overnight at 4 oC.

Techniques: Biomarker Discovery

Journal: Scientific Reports

Article Title: The role of long non-coding RNA LINC00839 in oral squamous cell carcinoma based on bioinformatics and experimental research

doi: 10.1038/s41598-024-82922-6

Figure Lengend Snippet: Schematic diagram illustrating our bioinformatics analysis and experimental validation of the LINC00839/miR-195-5p/CCNE1 axis in the progression of OSCC.

Article Snippet: Subsequently, the slides were rinsed with TBS containing Triton X-100 and incubated with the Cyclin E1 Polyclonal Antibody (Elabscience, E-AB-31074) overnight at 4 oC.

Techniques: Biomarker Discovery

Journal: EMBO Reports

Article Title: Oncogenic YAP sensitizes cells to CHK1 inhibition via CDK4/6 driven G1 acceleration

doi: 10.1038/s44319-025-00514-5

Figure Lengend Snippet: ( A ) Immunostaining of YAP in MCF10A cells expressing doxycycline-inducible YAP5SA. dox doxycycline. ( B ) Growth curve of parental MCF10A and MCF10A-YAP5SA cells treated with doxycycline as indicated. Mean +/− SEM. P values were calculated using the extra sum-of-squares F test ( n = 3 independent replicates). ( C ) Cell cycle distribution of cells 48 h after YAP5SA induction was analyzed by high-content microscopy. Mean +/− SD. Students t test ( n = 3 independent replicates). ( D ) A EdU pulse-chase experiment was performed to measure the S to M transition. Cells were pulse-treated with 10 µM EdU for 20 min and chased for the indicated times. Cells were then stained for the mitosis marker pH3 (S10). The fraction of EdU-positive entering mitosis (pH3-positive) was plotted against the time of the chase. Mean +/− SD. P values were calculated using the extra sum-of-squares F test ( n = 3 independent replicates). ( E ) Cell cycle distribution of CCNE1-expressing cells treated for 48 h with doxycycline. Mean +/− SD. Students t test ( n = 3 independent replicates). ( F ) Growth curve of MCF10A cells expressing doxycycline-inducible CCNE1. Mean +/− SEM. P values were calculated using the extra sum-of-squares F test ( n = 3 independent replicates). .

Article Snippet: pInducer20 Cyclin E1 , Addgene , 109348.

Techniques: Immunostaining, Expressing, Microscopy, Pulse Chase, Staining, Marker

Journal: EMBO Reports

Article Title: Oncogenic YAP sensitizes cells to CHK1 inhibition via CDK4/6 driven G1 acceleration

doi: 10.1038/s44319-025-00514-5

Figure Lengend Snippet: ( A ) High-content microscopy-based analysis of chromatin-bound MCM7. S-phase cells were labeled with EdU. We assessed for DNA content ( x axis), EdU incorporation ( y axis) and MCM7. Each dot represents a single cell and is color-coded according to mean MCM7 fluorescent intensity. For each sample, 3000 cells were randomly selected from a representative experiment ( n = 3 independent replicates). ( B ) Chromatin-bound MCM7 in early S-phase. Violin plots display single-cell measurements from a representative experiment, with ≥748 cells analyzed per condition ( n = 3 independent replicates). ( C ) EdU intensities of YAP5SA-expressing cells. Violin plots display single-cell measurements from a representative experiment, with ≥2196 cells analyzed per condition ( n = 3 independent replicates). ( D ) Chromatin-bound MCM7-positive cells in early S-phase after expression of CCNE1. Violin plots display single-cell measurements from a representative experiment, with ≥1466 cells analyzed per condition ( n = 3 independent replicates). ( E ) EdU intensities of CCNE1-expressing cells. Single-cell measurements from a representative experiment, with ≥2358 cells analyzed per condition ( n = 3 independent replicates). ( F – H ) DNA fiber assays of cells sequentially labeled with IdU and CldU. ( F ) Labeling scheme and representative DNA fiber images. ( G ) Quantification ((IdU+CldU)/2) track lengths) of unidirectional DNA replication structures of at least 145 fibers per condition ( n = 3 independent replicates). ( H ) Sister fork asymmetry (longer/shorter sister fork length). At least 39 bidirectional replication structures were measured per condition per experiment ( n = 3 independent replicates). ( B – E , G , H ) P values were calculated using ordinary one-way ANOVA. .

Article Snippet: pInducer20 Cyclin E1 , Addgene , 109348.

Techniques: Microscopy, Labeling, Expressing

Journal: EMBO Reports

Article Title: Oncogenic YAP sensitizes cells to CHK1 inhibition via CDK4/6 driven G1 acceleration

doi: 10.1038/s44319-025-00514-5

Figure Lengend Snippet: ( A ) Viability of cells exposed to the CHK1 inhibitor prexasertib was analyzed by MTT assay. Cells were treated for 3 days with increasing concentrations of CHK1i. ( B ) MTT viability assay of MCF10A cells expressing doxycycline-inducible Cyclin E1 after treatment with prexasertib. ( C , D ) MTT viability assay of cells exposed to the ATR inhibitor AZD6738 or the WEE1 inhibitor AZD1775. Mean +/− SEM. ( E ) Cells expressing YAP5SA,S94A were exposed to the CHK1 inhibitor prexasertib. Viability was analyzed by MTT assays. In Fig. 5A–E data are presented as mean ± SEM. P values were calculated using the extra sum-of-squares F test. ( A – D ) n = 3 independent replicates, ( E ) n = 4 independent replicates. ( F ) Immunoblot analysis of pKAP1 and YAP. YAP5SA and YAP5SA-S94A were expressed for 48 h and treated with 100 nM CHK1i for 8 h. Actin served as a loading control. ( G ) Quantification of nuclear EU intensities in S-phase after labeling for 30 min with 0.5 mM EU. The PIP-FUCCI cell cycle reporter (Fig. ) was used to identify S-phase cells. Violin plots display single-cell measurements from a representative experiment, with at least 3337 cells analyzed per condition. P values were calculated using ordinary one-way ANOVA ( n = 3 independent replicates). ( H ) Effect of treating YAP-expressing cells with DRB on γH2AX staining. Violin plots display single-cell measurements from a representative experiment, with at least 1767 cells analyzed per condition ( n = 2 independent replicates). ( I ) PLA with PCNA and RNAPII pSer2 antibodies. Quantification of nuclear PLA foci. Violin plots display single-cell measurements from a representative experiment, with at least 2323 nuclei analyzed per condition. P values were calculated using ordinary one-way ANOVA ( n = 3 independent replicates). ( J ) Immunoblot of YAP, pRB and pKAP1 in cells treated with 100 nM prexasertib (CHK1i, 100 nM, 8 h) and palbociclib (CDK4/6i, 500 nM, 24 h). Actin served as a loading control. ( K ) Cells were treated with 100 nM CHKi for 4 h and with 50 nM palbociclib for 24 h. γH2AX in S-phase in cells was determined by high-content microscopy. Violin plots display single-cell measurements from a representative experiment, with at least 2309 cells analyzed per condition. P values were calculated using ordinary one-way ANOVA. ( n = 3 independent replicates). ( L ) Model and summary. .

Article Snippet: pInducer20 Cyclin E1 , Addgene , 109348.

Techniques: MTT Assay, MTT Viability Assay, Expressing, Western Blot, Control, Labeling, Staining, Microscopy