e2f4  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: Simultaneous Targeting of KRAS and CDK4 Synergistically Suppresses Pancreatic Cancer Cells

doi: 10.1101/2025.01.11.632518

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 seven 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, G, 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 Fig. 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, B, C, D, E, F, G, H, I, J unpaired t-test (A, B, C, D, E, F, G, H, I of AUC); ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Complete statistics in .

Article Snippet: Transient transfections were carried out using Lipofectamine 3000 (L3000008, Thermo Fisher Scientific). siRNAs were transfected at a final concentration of 10 nM as smart pool targeting CDKN1B (s2837, s2838, s2839, Thermo Fisher Scientific), E2F4 (s4414, s4416, s223455, Thermo Fisher Scientific), FOS (s5339, s5340, s5341), RB1 (s552, s523, s524, Thermo Fischer Scientific), RBL1 (s11852, s11853, s11854, Thermo Fischer Scientific), RBL2 (s11855, s11856, s11857, Thermo Fisher Scientific); scrambled siRNA was used as control (s4390844, s4390847, Thermo Fisher Scientific).

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

Journal: Frontiers in Cell and Developmental Biology

Article Title: Spatial transcriptomics reveals Inhba/Smad2/E2f4 axis in Lrp2 high thecal cell proliferation in androgen-induced PCOS mice

doi: 10.3389/fcell.2025.1633254

Figure Lengend Snippet: Proliferative and androgenic features of Lrp2 high TC in PCOS ovaries. (A) Expression analysis of androgen synthesis genes Cyp11a1, Cyp17a1, and Hsd3b1 across clusters. (B) KEGG pathway enrichment analysis for upregulated genes in Lrp2 high TC, emphasizing cell cycle regulation. (C,D) Intersection and STRING network analysis predict an Inhba/Smad2/E2f4 signaling axis involved in cell cycle regulation within Lrp2 high TC. (E) AUC analysis reveals Lrp2 high TC as having the highest co-expression of Inhba, Smad2, and E2f4. (F) Quantification of co-localized regions for Inhba, Smad2, E2f4 and Lrp2, showing a significant increase in PCOS ovaries. (G) Spatial co-localization of Inhba, Smad2, E2f4 and Lrp2 in control and PCOS ovaries. Co-expressed spots are marked as “TRUE.” Data are represented as mean ± SD, *P < 0.05 by t-test.

Article Snippet: For functional assays, thecal cells were transfected with small interfering RNAs (siRNAs) targeting Inhba (Santa Cruz, Cat. No. sc-39783), Smad2 (Santa Cruz, Cat. No. sc-44338), or E2f4 (Santa Cruz, Catalog No. sc-35248) using Lipofectamine 3,000 (Invitrogen, Cat. No. L3000015) according to the manufacturer’s instructions.

Techniques: Expressing, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Spatial transcriptomics reveals Inhba/Smad2/E2f4 axis in Lrp2 high thecal cell proliferation in androgen-induced PCOS mice

doi: 10.3389/fcell.2025.1633254

Figure Lengend Snippet: Inhba/Smad2/E2f4 Signaling Promotes Thecal Cell Proliferation. (A) Immunofluorescence staining showing the subcellular localization of Inhba (cytoplasmic), Smad2, and E2f4 (nuclear) in primary thecal cells under control and DHEA-treated conditions. DAPI (blue) marks nuclei. Bar = 20 µm. (B) Representative EdU staining images of proliferating thecal cells following DHEA treatment and siRNA-mediated knockdown of Inhba, Smad2, or E2f4. Bar = 200 µm. (C) Quantification of EdU-positive nuclei across treatment groups. Data are presented as mean ± SD. Different lowercase letters denote statistically significant differences (one-way ANOVA followed by Tukey’s post hoc test, P < 0.05).

Article Snippet: For functional assays, thecal cells were transfected with small interfering RNAs (siRNAs) targeting Inhba (Santa Cruz, Cat. No. sc-39783), Smad2 (Santa Cruz, Cat. No. sc-44338), or E2f4 (Santa Cruz, Catalog No. sc-35248) using Lipofectamine 3,000 (Invitrogen, Cat. No. L3000015) according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Staining, Control, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: Spatial transcriptomics reveals Inhba/Smad2/E2f4 axis in Lrp2 high thecal cell proliferation in androgen-induced PCOS mice

doi: 10.3389/fcell.2025.1633254

Figure Lengend Snippet: Inhba/Smad2/E2f4 Axis Promotes Cell Cycle Progression in TCs. (A) Flow cytometry analysis of cell cycle phases. (B) Distribution of cells in G1, S, and G2/M phases (%). (C–E) qRT-PCR analysis of Inhba, Smad2, and E2f4 mRNA levels. (F) Western blot analysis of protein expression. (G–I) Quantification of protein levels for Inhba, Smad2, and E2f4. Data are represented as mean ± SD. Different lowercase letters at the top of each bar denote significant differences among groups (one-way ANOVA followed by Tukey’s post hoc test, P < 0.05).

Article Snippet: For functional assays, thecal cells were transfected with small interfering RNAs (siRNAs) targeting Inhba (Santa Cruz, Cat. No. sc-39783), Smad2 (Santa Cruz, Cat. No. sc-44338), or E2f4 (Santa Cruz, Catalog No. sc-35248) using Lipofectamine 3,000 (Invitrogen, Cat. No. L3000015) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Quantitative RT-PCR, Western Blot, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Spatial transcriptomics reveals Inhba/Smad2/E2f4 axis in Lrp2 high thecal cell proliferation in androgen-induced PCOS mice

doi: 10.3389/fcell.2025.1633254

Figure Lengend Snippet: Schematic overview of the study design and proposed mechanism. Spatial transcriptomic profiling of ovaries from DHEA-induced PCOS mice revealed expansion of Lrp2 high TC with elevated proliferative and steroidogenic activity. Mechanistically, DHEA stimulates Inhba expression, activating Smad2 and E2f4 signaling to drive thecal cell proliferation, contributing to androgen excess and ovarian dysfunction in PCOS.

Article Snippet: For functional assays, thecal cells were transfected with small interfering RNAs (siRNAs) targeting Inhba (Santa Cruz, Cat. No. sc-39783), Smad2 (Santa Cruz, Cat. No. sc-44338), or E2f4 (Santa Cruz, Catalog No. sc-35248) using Lipofectamine 3,000 (Invitrogen, Cat. No. L3000015) according to the manufacturer’s instructions.

Techniques: Activity Assay, Expressing

Journal: Advanced Science

Article Title: TTK Inhibition Alleviates Postinjury Neointimal Formation and Atherosclerosis

doi: 10.1002/advs.202409250

Figure Lengend Snippet: TTK is upregulated during neointima formation in vascular injury and atherosclerosis. A) Venn diagram of the differentially expressed genes (DEGs) in four Gene Expression Omnibus (GEO) datasets was used to identify novel molecules involved in the phenotypic switching of VSMCs under different pathological conditions. B) Quantitative real‐time polymerase chain reaction (qRT‐PCR) validation of the expression of novel VSMC phenotype‐related genes in sham‐operated or wire‐injured carotid arteries of C57 mice on day 28 post‐surgery ( n = 6). C) qRT‐PCR validation of the expression of novel VSMC phenotype‐related genes in sham‐operated or ligated carotid arteries of C57 mice on day 28 post‐surgery ( n = 6). D) qRT‐PCR analysis of the relative mRNA level of TTK, ACTA2, TAGLN, and CNN1 in VSMCs transfected with scrambled siRNA or TTK‐specific siRNA ( n = 6). E) Representative hematoxylin and eosin (HE) staining (left) and immunofluorescence (right) staining of TTK (green) and tdTomato (red) in the sham‐operated or wire‐injured carotid sections of Myh11‐CreER T2 / Rosa26 tdTomato mice on days 14 and 28 post‐surgery. IgG was used as a negative control. Nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue). Scale bar = 50 µm (left) or 10 µm (right). F) The percentage of TTK‐positive, tdTomato‐positive VSMCs in the neointima ( n = 14). G) Analysis of the correlation between the neointima area and the percentage of TTK‐positive, tdTomato‐positive VSMCs in neointima ( n = 28). H) Representative immunofluorescence staining of TTK (green) and tdTomato (red) in the aortic root sections of Myh11‐CreER T2 / Rosa26 tdTomato / ApoE −/− mice fed on a high‐fat diet (HFD) for 0, 8, and 16 weeks. Nuclei were stained with DAPI (blue). Scale bar = 50 µm. I) The percentage of TTK‐positive, tdTomato‐positive VSMCs in atherosclerotic plaques ( n = 14). J) Analysis of the correlation between plaque area and the percentage of TTK‐positive, tdTomato‐positive VSMCs in atherosclerotic plaques ( n = 28). Data are presented as the mean ± SEM; unpaired t ‐test, one‐way ANOVA.

Article Snippet: Small interfering RNAs (siRNAs) against TTK, CEP55, NCAPH, IRF1, E2F4, C/EBPβ, and a scrambled siRNA were designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Biomarker Discovery, Expressing, Transfection, Staining, Immunofluorescence, Negative Control

Journal: Advanced Science

Article Title: TTK Inhibition Alleviates Postinjury Neointimal Formation and Atherosclerosis

doi: 10.1002/advs.202409250

Figure Lengend Snippet: IRF1 upregulates TTK transcription in VSMCs upon pathological stimulation. A,B) Relative levels of TTK mRNA A) and protein B) in VSMCs stimulated with platelet‐derived growth factor‐BB (PDGF‐BB) (0, 5, 10, 20, and 40 ng mL −1 ) for 24 h ( n = 6). C,D) Relative levels of TTK mRNA C) and protein D) in VSMCs stimulated with oxidized low‐density lipoprotein (ox‐LDL) (0, 5, 10, 25, 50, and 100 µg mL −1 ) for 24 h ( n = 6). E) TTK promoter activity in MOVAS cells treated with PDGF‐BB (0, 5, 10, 20, and 40 ng mL −1 ) for 24 h ( n = 6). F) TTK promoter activity in MOVAS cells treated with ox‐LDL (0, 5, 10, 25, 50, and 100 µg mL −1 ) for 24 h ( n = 6). G) qRT‐PCR analysis of the relative mRNA level of TTK in VSMCs transfected with scrambled, IRF1, E2F4, or C/EBPβ‐specific siRNAs in the presence or absence of 20 ng mL −1 PDGF‐BB ( n = 6). H) qRT‐PCR analysis of the relative mRNA level of TTK in VSMCs transfected with scrambled, IRF1, E2F4, or C/EBPβ‐specific siRNAs in the presence or absence of 20 ng mL −1 ox‐LDL ( n = 6). I) Schematic illustration of putative IRF1 binding sequences in the TTK promoter region. J) Luciferase reporters of TTK promoter with native (p‐TTK), mutated IRF1binding site 1 (mut‐p‐TTK 1), mutated IRF1binding site 2 (mut‐p‐TTK 2), or mutated IRF1binding site 1 and 2 (mut‐p‐TTK 1&2) were cloned and co‐transfected with IRF1overexpressing vector into MOVAS cells for 48 h ( n = 10). K,L) Chromatin immunoprecipitation (ChIP) assay K) and quantification L) demonstrated that PDGF‐BB and ox‐LDL promoted the binding of IRF1 to the TTK promoter ( n = 6). IgG lane: negative control. Data are presented as the mean ± SEM; unpaired t ‐test, one‐way ANOVA, two‐way ANOVA.

Article Snippet: Small interfering RNAs (siRNAs) against TTK, CEP55, NCAPH, IRF1, E2F4, C/EBPβ, and a scrambled siRNA were designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Derivative Assay, Activity Assay, Quantitative RT-PCR, Transfection, Binding Assay, Luciferase, Clone Assay, Plasmid Preparation, Chromatin Immunoprecipitation, Negative Control

Journal: Advanced Science

Article Title: TTK Inhibition Alleviates Postinjury Neointimal Formation and Atherosclerosis

doi: 10.1002/advs.202409250

Figure Lengend Snippet: TTK promotes VSMCs phenotypic switching by phosphorylating p120‐catenin at T310. A) Overlapping analysis of the upregulating phosphorylation proteins in hemagglutinin (HA)‐tagged TTK construct‐transfected group in phosphorylated proteomics and interacting proteins of TTK. B) Lysates of VSMCs transfected with the hemagglutinin (HA)‐tagged TTK lentivirus were immunoprecipitated with anti‐HA antibodies, and the precipitates were analyzed using immunoblotting with anti‐p120 antibodies. C) Lysates of VSMCs transfected with the HA‐tagged TTK lentivirus were immunoprecipitated with anti‐p120 antibodies, and the precipitates were analyzed using immunoblotting with anti‐HA antibodies. D) Representative western blotting and quantification of p120 phosphorylated at T310 in VSMCs transfected with empty vector or LV‐TTK ( n = 6). E) Representative western blotting and quantification of p120 phosphorylated at T310 in VSMCs transfected with scramble small interfering RNA (siRNA) or TTK‐specific siRNAs ( n = 6). F) Representative western blotting and quantification of p120 phosphorylated at T310, α‐SMA, SM22α, and calponin1 in VSMCs co‐transfected with vector or LV‐TTK and wild‐type p120 (p120‐wt) or mutant p120 (p120‐T310A) ( n = 6). G,H) Quantification of Ki67 immunofluorescence staining G) and transwell assay results H) of VSMCs co‐transfected with empty vector or LV‐TTK and p120‐wt or p120‐T310A vector ( n = 6). Data are presented as the mean ± SEM; unpaired t ‐test, one‐way ANOVA.

Article Snippet: Small interfering RNAs (siRNAs) against TTK, CEP55, NCAPH, IRF1, E2F4, C/EBPβ, and a scrambled siRNA were designed and synthesized by RiboBio (Guangzhou, China).

Techniques: Phospho-proteomics, Construct, Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Small Interfering RNA, Mutagenesis, Immunofluorescence, Staining, Transwell Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Knockdown of E2F4 suppresses the growth of ovarian cancer cells through the cell cycle pathway

doi:

Figure Lengend Snippet: E2F4 is elevated in ovarian cancer cell lines. (A) E2F4 expression level in ovarian cancer cells was demonstrated by qRT-PCR. (B) E2F4 protein expression in a series of human OC cell lines was analyzed by western blot. Immunoblot analysis was performed on whole cell lysate for E2F4 expression and the relative protein level of E2F4 represented the protein level normalized to β-actin. (C) Quantitation of the relative gray scale of the expression of E2F4 in HaCaT, SKOV3, A2780, OVCA433, and OVCA429 cells. Data are presented as the mean ± SD (one-way ANOVA with Bonferroni post hoc multiple comparison test, ***P<0.001, ****P<0.0001; D), (E) The expression of E2F4 in A2780 and OVCA433 cells after transfection with E2F4-siRNA or negative control was analyzed by qRT-PCR. GAPDH was used for normalization.

Article Snippet: E2F4 knockdown of the A2780 and OVCA433 cell line was performed using the E2F4 small interfering RNA (siRNA) duplex, purchased from Sigma-Aldrich (Cat. {"type":"entrez-nucleotide","attrs":{"text":"NM_001950","term_id":"1519244736","term_text":"NM_001950"}} NM_001950 ).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Quantitation Assay, Transfection, Negative Control

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Knockdown of E2F4 suppresses the growth of ovarian cancer cells through the cell cycle pathway

doi:

Figure Lengend Snippet: Knockdown of E2F4 in ovarian cancer cells inhibits cell proliferation. A. Determination of cell viability in ovarian cancer cell lines by CCK-8 kit. B. Colony formation assay showed that knockdown of E2F4 decreased cell proliferation in A2780 and OVCA433. Negative control or E2F4 knockdown cells were subjected to clonogenic assay. The colonies, observed by bright-field microscopy at day 12 of growth are also shown. C. Representative images for colony growth are shown. All values are expressed as mean ± SD. **P<0.01, ***P<0.001.

Article Snippet: E2F4 knockdown of the A2780 and OVCA433 cell line was performed using the E2F4 small interfering RNA (siRNA) duplex, purchased from Sigma-Aldrich (Cat. {"type":"entrez-nucleotide","attrs":{"text":"NM_001950","term_id":"1519244736","term_text":"NM_001950"}} NM_001950 ).

Techniques: CCK-8 Assay, Colony Assay, Negative Control, Clonogenic Assay, Microscopy

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Knockdown of E2F4 suppresses the growth of ovarian cancer cells through the cell cycle pathway

doi:

Figure Lengend Snippet: E2F4 knockdown suppresses cell migration ability of ovarian cancer cells. A. Wound-healing assay was performed in control siRNA or siE2F4 cells. Images were captured to display the process of gap closure at 0, 24 h, and 48 h. The area was measured by ImageJ software to evaluate the scratch by quantification of the areas in arbitrary units for three independent experiments performed in triplicate. Scale bar, 200 μm. B. Quantification of wound healing assay showed the cell migration ability of A2780 and OVCA433 cells. The results are from three independent experiments. Bars represent the mean ± SD (n=3). *P<0.05, **P<0.01.

Article Snippet: E2F4 knockdown of the A2780 and OVCA433 cell line was performed using the E2F4 small interfering RNA (siRNA) duplex, purchased from Sigma-Aldrich (Cat. {"type":"entrez-nucleotide","attrs":{"text":"NM_001950","term_id":"1519244736","term_text":"NM_001950"}} NM_001950 ).

Techniques: Migration, Wound Healing Assay, Software

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Knockdown of E2F4 suppresses the growth of ovarian cancer cells through the cell cycle pathway

doi:

Figure Lengend Snippet: E2F4 knockdown induced expression of cell cycle markers in ovarian cancer cells. A. CDK2, CDK6, Cyclin A1, Cyclin D1, and Cyclin E1 mRNA expression pattern in A2780 and OVCA433 cells assessed by quantitative RT-PCR. B. Western blot analysis of cell cycle regulators to evaluate the E2F4 knockdown effect of CDK2, CDK6, and Cyclin D1 in A2780 and OVCA433 cells treated with negative control or siRNA. β-actin levels are shown as loading controls. C. Gray-scale analysis of the level of CDK2, CDK6, and Cyclin D1. All data are presented as mean ± SD. **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: E2F4 knockdown of the A2780 and OVCA433 cell line was performed using the E2F4 small interfering RNA (siRNA) duplex, purchased from Sigma-Aldrich (Cat. {"type":"entrez-nucleotide","attrs":{"text":"NM_001950","term_id":"1519244736","term_text":"NM_001950"}} NM_001950 ).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Negative Control