Journal: Protein & Cell

Article Title: Unveiling E2F4, TEAD1 and AP-1 as regulatory transcription factors of the replicative senescence program by multi-omics analysis

doi: 10.1007/s13238-021-00894-z

Figure Lengend Snippet: Transcriptional signatures and regulators of replicative senescence . (A) Schematic outlining the design of multi-omics analysis of cellular replicative senescence. Skin fibroblasts from 2-month-old mice were used, and samples were collected at P1, P3, P5 and P7 for RNA-seq, ATAC-seq and RRBS. (B) PCA of the transcriptome of skin fibroblast during RS. P1 (blue), P3 (dark red), P5 (dark green) and P7 (purple), ( n = 4). (C) Heatmap showing 6 major modules of differentially expressed genes during RS. Genes that differentially expressed in any group when compared with P1 are shown (adjusted P -value < 0.05 and fold change > 2). The number of genes within each cluster is given in parentheses, ( n = 4). (D) Transcription factor enrichment analysis based on the genes from each cluster. Only the top significantly enriched TFs are shown. AP-1 family TFs and E2F4 are highlighted. The analysis was performed with RcisTarget. (E) Selected biological process GO terms enriched in each gene cluster, with the top 5 most significantly enriched terms shown. The size of each point represents the gene count in the GO term, and the color of each point represents the significance of the enrichment. (F) GSEA showing cell cycle-related terms based on the transcriptome of P1 versus P7, ( n = 4). (G) GSEA showing inflammatory response-related terms based on the transcriptome of P5 versus P7, ( n = 4)

Article Snippet: For E2f4 overexpression vector construction, E2f4 cDNA (NM_148952.1) was cloned into lentiviral vector FU-tet-on-P2A-EGFP (Addgene) through homologous recombination using ClonExpress II One Step Cloning Kit (Vazyme, C112).

Techniques: Biomarker Discovery, RNA Sequencing

Journal: Protein & Cell

Article Title: Unveiling E2F4, TEAD1 and AP-1 as regulatory transcription factors of the replicative senescence program by multi-omics analysis

doi: 10.1007/s13238-021-00894-z

Figure Lengend Snippet: E2F4 affects replicative senescence-associated phenotypes as a regulatory TF . (A) Structures of plasmids used for EGFP-labeled E2F4 expression under doxycycline induction and corresponding control. (B) RT-qPCR showing the change in the E2f4 expression level after overexpression with lentivirus, ( n = 3). (C) Immunoblot showing the change in E2F4 protein level after overexpression. (D) Change in SA-β-Gal activity after E2f4 overexpression. SA-β-Gal positive percentage was estimated based on 3 different views, and the points indicated the average value, ( n = 3). Scale bar = 200 μm. (E) Scatter plot showing the nuclear size distribution after E2f4 overexpression based on nuclear staining. Nuclear size was calculated using ImageJ, ( n = 3). (F) EdU staining showing the change in proliferating capacity after E2f4 overexpression. Red dots indicate EdU positive cells (white arrows), and the EdU-positive percentage was calculated with ImageJ. The positive percentage was estimated based on 5 different views, and the points indicated the average value, ( n = 3). Scale bar = 200 μm. (G) GSEA showing changes in cell cycle-related terms after E2f4 overexpression based on RNA-seq. (H) Heatmap showing changes in the expression levels of cell cycle-related genes, including positive regulators (up) and negative regulators (down). P3 samples were used as proliferating cell control, ( n = 2). (I) RT-qPCR detecting the change in E2f4 expression level after shRNA-mediated knockdown, ( n = 3). (J) Immunoblot showing the change in E2F4 protein level after knockdown. (K) EdU staining showing the change in proliferating capacity after E2f4 knockdown. Green dots represent EdU-positive cells (white arrows), ( n = 3). Scale bar = 200 μm. (L) Scatter plot showing the nuclear size distribution after E2f4 knockdown based on nuclear staining. Nuclear size was calculated using ImageJ, ( n = 3). (M) Bar plot showing the changes of RS-associated pathways after E2f4 knockdown through GSEA analysis. Blue bar indicates down-regulated pathways, and red bar indicates up-regulated pathways after E2f4 knockdown, ( n = 3). Data were analyzed by one-way ANOVA (I, K and L) and t -test (B, D, E and F). Error bars denote for the SD. **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05

Article Snippet: For E2f4 overexpression vector construction, E2f4 cDNA (NM_148952.1) was cloned into lentiviral vector FU-tet-on-P2A-EGFP (Addgene) through homologous recombination using ClonExpress II One Step Cloning Kit (Vazyme, C112).

Techniques: Labeling, Expressing, Control, Quantitative RT-PCR, Over Expression, Western Blot, Activity Assay, Staining, RNA Sequencing, shRNA, Knockdown

Journal: Nature communications

Article Title: Trim33 masks a non-transcriptional function of E2f4 in replication fork progression.

doi: 10.1038/s41467-023-40847-0

Figure Lengend Snippet: Fig. 3 | Deletion of Trim33 promotes E2f4 interactions with chromatin and with the Recql helicase. a Genome browser tracks of E2f4 ChIP-seq in p19/Nras Trim33- WT and Trim33-KO cells; n = 2. b Quantification of ChIP-seq tag coverage at E2f4 peaks in p19/Nras Trim33-WT and Trim33-KO cells. c Relationship between changes (log2FC) in E2f4 binding (normalized E2f4 ChIP-seq tags) and mRNA expression (log2FC) of the encoded gene for p19/Nras Trim33-KO vs Trim33-WT cells. Each dot is a bin of 5 genes sorted by the change in mRNA levels. Trendlines are derived from linear regression analysis. d LC-MS/MS analysis of E2f4 interactome in p19/Nras Trim33-WT and Trim33-KO cells. Known interaction partners are highlighted in blue. e PLA analysis of Recql-E2f4 association in p19/Nras Trim33-WT and Trim33- KO cells stably expressing HA-tagged E2f4 or vector Ctrl; from left, n = 49,49,50,50

Article Snippet: E2f4-GST fusion proteins were generated by cloning the PCR-amplified E2f4 cDNA fragments (1–413, 1–105, 90–200, 300–413) into the pGEX-4T3 vector using the HiFi assembly kit (Cell Signaling).

Techniques: ChIP-sequencing, Binding Assay, Expressing, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Stable Transfection, Plasmid Preparation

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: After blocking with 5% skim milk or 5% BSA in TBST, the membranes were incubated at 4 °C overnight with primary antibodies against TTK (AF6028, R&D Systems), TTK (A2500, Abclonal), IRF1 (#8478, CST), E2F4 (10923‐1‐AP, Proteintech), C/EBP β (sc‐7962, Santa Cruz), α‐SMA (ab5694, Abcam), SM22α (ab10135, Abcam), calponin (ab46794, Abcam), p120‐catenin (12180‐1‐AP, Proteintech), p‐p120 (Thr310) (bs‐12990R, Bioss), HA (ab9110, Abcam), β‐catenin (51067‐2‐AP, Proteintech), N‐cadherin (ab18203, Abcam), SRF (16821‐1‐AP, Proteintech), MYOCD (SAB4200539, Sigma‐Aldrich), Cre Recombinase (#15036, CST), H3 (AF0009, Beyotime), β‐tubulin (AC015, Abclonal) and β‐actin (AC026, Abclonal).

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