Journal: Cancer cell

Article Title: Long-term breast cancer response to CDK4/6 inhibition defined by TP53-mediated geroconversion

doi: 10.1016/j.ccell.2024.09.009

Figure Lengend Snippet: (A) GESA plot with normalized enrichment score (NES) of REACTOME gene sets. NES was analyzed between MCF7 and p53KO treated with 50 nM abemaciclib at day21. REACTOME-E2F-mediated-regulation-of-DNA-replication (top), REACTOME-Transcription-of-E2F-targets-under-negative-control-by-DREAM-complex (middle) and REACTOME-Transcription-of-E2F-targets-under-negative-control-byp107 RBL1-and-p130 RBL2-in-complex-with-HDAC1 (bottom). (B) Immunoblotting of DREAM complex components with 50 nM abemaciclib. (C) Cells were treated with 100 nM abemaciclib for 48 h. Lysates were immunoprecipitated with p130 and IgG antibodies. (D) Cells were treated with 7 days of abamacilib and tested by ChIP followed by real-time PCR. Bar showed technical duplicate. Unpaired, Student’s t test. (E) Western blot results of MCF7 cells and p53KO transduced with doxycycline (dox)-inducible HA-tagged p21. The cells were treated with 50 nM abemaciclib or DMSO +/− dox for 48 h. (F) Dox-inducible HA-p21 MCF7 and p53KO cells were treated with 100 nM abemaciclib and +/− dox for 72 h. Lysates were immunoprecipitated with E2F4 and IgG antibodies. (G) Time course change of the proportion of G1 phase after drug withdrawal. FUCCI labeled MCF7 were treated with 50 nM abemaciclib and FUCCI labeled p53KO cells were treated with or without dox and with 50 nM abemaciclib for 4 days. The cells were cultured without abemaciclib for 62 h under time-lapse imaging. Two-way ANOVA, Tukey’s. (H) Cell viability treated with 50 nM abemaciclib or DMSO +/− dox. Data are means ± SEM of four replicates. Two-way ANOVA, Tukey’s. (I) Flow cytometry analysis showing the percentage of SA-β-Gal positive cells. Data are means ± SD of three replicates. Two-way ANOVA, Sidak’s. (J) Colony formation assay. Cells were treated with 50 nM abemaciclib ± dox for 11 days and reseeded without drug. (K) Cell viability of p130KO in dox inducible HA-p21 cells. The cells were treated with 50 nM abemaciclib or DMSO +/− dox. Data are means ± SEM of six replicates. Two-way ANOVA, Tukey’s. See also and .

Article Snippet: E2F4 (IP) , Santa Cruz Biotechnology , sc-398543.

Techniques: Negative Control, Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction, Transduction, Labeling, Cell Culture, Imaging, Flow Cytometry, Colony Assay

Journal: Nature communications

Article Title: ZC3H18 specifically binds and activates the BRCA1 promoter to facilitate homologous recombination in ovarian cancer.

doi: 10.1038/s41467-019-12610-x

Figure Lengend Snippet: Fig. 6 ZC3H18 binds the BRCA1 promoter and inhibits E2F1 binding. a Schematic of the BRCA1 proximal promoter with E2FA and E2FB sites indicated. Nucleotide sequences of the DNA probes used in the electrophoretic mobility shift assays (EMSA). E2FA and E2FB mutation sites are indicated in open rectangles. b EMSA with purified recombinant SFB-ZC3H18 using BRCA1 promoter probe with wild-type sequence (E2FA/BWT) or mutations in the E2FA site (E2FΔA), the E2FB site (E2FΔB), or both E2F sites (E2FΔA/B). A probe with randomly shuffled sequences was used as negative control. For supershift assays, an anti-S-Tag monoclonal antibody, which binds the SFB tag in SFB-ZC3H18, was used. c, d ZC3H18 and E2F4 co-occupy the endogenous BRCA1 promoter. Sequential ChiP (ChIP-Re-ChIP) assays in OVCAR-8 cells using anti-ZC3H18 antibody for primary ChIP and anti-E2F4 antibody for secondary ChIP (c) and using anti-E2F4 antibody for primary ChIP and anti-ZC3H18 antibody for secondary ChIP (d). e EMSA with purified recombinant SFB-ZC3H18 and SFB-E2F1 using BRCA1 promoter probe with mutated E2FB site (E2FΔB). f EMSA with purified SFB-E2F4 and SFB-E2F1 using BRCA1 promoter probe with mutated E2FA site (E2FΔA). The images of EMSA in b, e, and f are representative of three independent experiments that gave similar results. Data in c and d are means ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t-test

Article Snippet: Immunoblotting was done using the following primary antibodies: rabbit polyclonal ZC3H18 (1:1000, A304-682A, Bethyl Laboratories Inc.); mouse monoclonal BRCA1 (1:2000, sc-6954, Santa Cruz Biotechnology); mouse monoclonal E2F1 (1:500, ab4070, Abcam); rabbit polyclonal E2F4 (1:1000, NBP1-21374, Novus Biologicals); mouse monoclonal DNMT1 (1:5000, ab13537, Abcam); mouse monoclonal HSP90 (D. Toft, Mayo Clinic, H9010), and rabbit monoclonal HA-tag (1:1000, CST-3724S, Cell Signaling Technology).

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Mutagenesis, Recombinant, Sequencing, Negative Control, ChIP-chip

Journal: Nature communications

Article Title: ZC3H18 specifically binds and activates the BRCA1 promoter to facilitate homologous recombination in ovarian cancer.

doi: 10.1038/s41467-019-12610-x

Figure Lengend Snippet: Fig. 7 ZC3H18 and E2F4 expression correlates with BRCA1 levels in HGSOC patient and PDX tumors. a Scatter plots of BRCA1 mRNA expression as a function of either ZC3H18 or E2F4 mRNA expression in HGSOC tumors from patients and PDX models. mRNA expression is in RPKM units. b Model for the role of ZC3H18 in BRCA1 transcription. Left panel: in ZC3H18-proficient cells, ZC3H18 directly binds to the E2FA site on the BRCA1 promoter, where it promotes E2F4 occupancy at the E2FB site, thereby preventing E2F1-dependent DNMT1 occupancy and promoter methylation and inducing BRCA1 transcription. Right panel: in ZC3H18-deficient cells, E2F1 occupies both E2FA and E2FB sites and causes DNMT1 loading onto the promoter, leading to methylation of the promoter, reduced expression of BRCA1, and disruption of HR. Spearman correlations are shown in the images

Article Snippet: Immunoblotting was done using the following primary antibodies: rabbit polyclonal ZC3H18 (1:1000, A304-682A, Bethyl Laboratories Inc.); mouse monoclonal BRCA1 (1:2000, sc-6954, Santa Cruz Biotechnology); mouse monoclonal E2F1 (1:500, ab4070, Abcam); rabbit polyclonal E2F4 (1:1000, NBP1-21374, Novus Biologicals); mouse monoclonal DNMT1 (1:5000, ab13537, Abcam); mouse monoclonal HSP90 (D. Toft, Mayo Clinic, H9010), and rabbit monoclonal HA-tag (1:1000, CST-3724S, Cell Signaling Technology).

Techniques: Expressing, Methylation, Disruption

Journal: Cell Death & Disease

Article Title: Simultaneous targeting of KRAS and CDK4 synergistically induces durable growth arrest in pancreatic cancer cells

doi: 10.1038/s41419-025-08362-w

Figure Lengend Snippet: A Proliferation of MIA PaCa-2 cells measured by automated transmission microscopy (Celigo®). Cells were reverse transfected by siRNAs to RB1 ( A ), RBL1 ( B ), RBL2 ( C ), E2F4 ( D ), or FOS ( E ); (scrb = ctrl siRNA). On day 1 after transfection, the cells were treated with DMSO, 10 µM (5 µM for ( E )) Palbociclib, 5 µM Sotorasib or the combination, for 48 h, followed by 7 days of recovery in normal medium. Means of three technical replicates ±SD. F MIA PaCa-2 cells were transfected by siRNAs to deplete CDKN1B ( F – H ) or control siRNA (scrb) during seeding. On day 1, the cells were treated with 1, 2.5, or 5 µM Palbociclib, with or without 5 µM Sotorasib, 48 h, followed by seven days of recovery without drugs. Three technical replicates, means ± SD. I Proliferation of 8661 cells (murine PDAC) wild type (WT) or CDKN1B knock-out (KO) lines (n = 3 clones, 3 technical replicates each). All cells were treated and observed as in ( A ), with the following specifics: 1 µM Palbociclib, 0.1 µM MRTX1133 or the combination. J Cell viability of 8661 WT and CDKN1B KO cells evaluated at D3 and D7 corresponding to ( I ). Statistical analyses: A – J unpaired t-test ( A – I of AUC); ns not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Complete statistics in Supplementary Fig. .

Article Snippet: E2F4 , Cell Signaling , Cat# 40291; RRID: AB_2799174.

Techniques: Transmission Assay, Microscopy, Transfection, Control, Knock-Out, Clone Assay

Journal: Cells

Article Title: Weighted Correlation Network Analysis Reveals CDK2 as a Regulator of a Ubiquitous Environmental Toxin-Induced Cell-Cycle Arrest

doi: 10.3390/cells9010143

Figure Lengend Snippet: E2F1 and E2F4 were downregulated by OTA exposure. ( A ) E2F1 and E2F4 were significantly downregulated following 100 nM OTA exposure according to RNA-sequencing data in both cell lines, while E2F7 was found to be upregulated only in HEK293-T cells. Due to the consistent results between the two cell lines, the expression of E2F1 and E2F4 only was verified by RT-qPCR ( B ) and Western blot ( C ) (mean ± SD, * p < 0.05, N = 3).

Article Snippet: CDKN1A/p21 (Cell Signaling Technology, cat. no. 2946, Frankfurt, Germany), CDK2 (Cell Signaling Technology, cat. no. 2546), and E2F4 (R&D, cat. no. AF5139) were detected using IRDye-coupled fluorescent secondary antibodies (LI-COR Biosciences, Bad Homburg, Germany) and an Odyssey imaging system from LI-COR Biosciences.

Techniques: RNA Sequencing, Expressing, Quantitative RT-PCR, Western Blot