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: 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: Nucleic Acids Research

Article Title: The p21CIP1-CDK4-DREAM axis is a master regulator of genotoxic stress-induced cellular senescence

doi: 10.1093/nar/gkae426

Figure Lengend Snippet: MCF7 cells were exposed to 1 μM B[a]P ( A, C ) or 5 Gy IR ( B, D ). Expression of E2F1, E2F4, E2F5, CCNA1, CCNA2, CCNB1, CCNB2, CCND1 and CCND2 was analysed by qPCR (A, B). ACTB and GAPDH were used as internal loading control, differences between treatment and control were statistically analysed using Student's t test (non labelled = non significant, * P < 0.1 ** P < 0.01, *** P < 0.001). Expression of E2F4, pRB, RB, pp130 and p130 was analysed by immunodetection (C, D). β-Actin or HSP90 were used as internal loading control, quantification of band intensities from two blots is shown.

Article Snippet: For gene silencing, predesigned siRNAs specific for p16 INK4A (sc-37622, Santa Cruz Biotechnology), p21 CIP1 (sc-29427, Santa Cruz Biotechnology), E2F4 (sc-29300, Santa Cruz Biotechnology) and E2F5 (sc-35250, Santa Cruz Biotechnology) were used; control human non-silencing siRNA (Silencer Select Predesigned siRNA Negative Control #1 siRNA; Ambion) was used as negative control.

Techniques: Expressing, Control, Immunodetection

Journal: Nucleic Acids Research

Article Title: The p21CIP1-CDK4-DREAM axis is a master regulator of genotoxic stress-induced cellular senescence

doi: 10.1093/nar/gkae426

Figure Lengend Snippet: ( A ) Expression of E2F4 and E2F5 was detected 48 and 72 h after siRNA-mediated knockdown by immunodetection; β-Actin was used as internal loading control; quantification of band intensities indicates knock-down efficiency. ( B, C ) MCF7 cells were treated with E2F4-, E2F5- or non-specific siRNA for 24 h and thereafter exposed to B[a]P or IR for 120 h. ( B ) Senescence was measured microscopically by β-Gal staining. ( C ) Expression of CDK1, FOXM1, MYBL2, HMGB2, CCNB1, LMNB1, PLK1, CENPF, TK1 and ASPM was measured by qPCR; ACTB and GAPDH were used as internal loading control. (B, C) Experiments were performed in triplicates, differences between treatment in absence or presence of siRNA were statistically analysed using Student's t test (non labelled = non significant, * P < 0.1 ** P < 0.01, *** P < 0.001).

Article Snippet: For gene silencing, predesigned siRNAs specific for p16 INK4A (sc-37622, Santa Cruz Biotechnology), p21 CIP1 (sc-29427, Santa Cruz Biotechnology), E2F4 (sc-29300, Santa Cruz Biotechnology) and E2F5 (sc-35250, Santa Cruz Biotechnology) were used; control human non-silencing siRNA (Silencer Select Predesigned siRNA Negative Control #1 siRNA; Ambion) was used as negative control.

Techniques: Expressing, Knockdown, Immunodetection, Control, Staining

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