Journal: Cell reports. Medicine

Article Title: KRAS G12D -driven pentose phosphate pathway remodeling imparts a targetable vulnerability synergizing with MRTX1133 for durable remissions in PDAC.

doi: 10.1016/j.xcrm.2025.101966

Figure Lengend Snippet: Figure 4. KRASG12D amplifies UBE2T transcription by Rb/E2F1/p53 axis-mediated positive feedback loops (A) Schematic diagram illustrating the identification of UBE2T transcription factors. (B) Volcano plot showing DEGs between KRASWT and KRASG12D PDO-1. (C) DNA pull-down assay showing the interaction of E2F1 with UBE2T promoter (top). Dual-luc assays detecting the transcriptional activity of the indicated UBE2T promoter with or without E2F1 overexpression (bottom) (n = 6). (D) Dual-luc assays detecting the transcriptional activity of UBE2T promoter (full length, 886 to 876 bp, and its mutant version) with or without E2F1 over- expression (n = 6). (E) CoIP assays showing the interaction between Rb and E2F1 in BxPC-3 cells. Green fluorescent protein (GFP) as control. (F and G) Immunoblotting (IB) analysis with the indicated antibodies in control or KRASG12D-overexpressed BxPC-3 cells with or without palbociclib treatment (F)/ E2F1 knockdown (G). (H and I) Ubiquitination assay showing the degree of p53 ubiquitination using BxPC-3 cells expressing the indicated plasmids. (J) IB analysis with the indicated antibodies in control or KRASG12D-overexpressed BxPC-3 cells with or without TP53 knockdown. (K) Dual-luc assays detect the transcriptional activities of the UBE2T promoter (886 to 876 bp) with or without E2F1 and/or p53 overexpression (n = 6). (L) IB analysis with the indicated antibodies in KRASG12D-overexpressed BxPC-3 cells with or without p53 overexpression and/or palbociclib treatment.

Article Snippet: The specific procedure of immunoblotting was as previously described.53 The primary antibodies used in the article were as follows: mouse anti-FLAG antibody (1:1000, Sigma, #F1804), rabbit anti-HA antibody (1:1000, Invitrogen, #71–5500), mouse anti-His antibody (1:1000, ABclonal, #AE003), rabbit anti-E2F1 antibody (1:1000, CST, #3742), rabbit anti-p21 (1:1000, Abcam, #ab109520), mouse anti-p53 antibody (1:1000, CST, #48818S), rabbit anti-phospho-Rb (1:1000, CST, #8516), rabbit anti-Rb antibody (1:1000, Abcam, #ab181616), rabbit anti-RING1 (1:1000, CST, #13069S), rabbit anti-UBE2T (1:1000, Proteintech, #10105-2-AP), rabbit anti-RASG12D antibody (1:1000, Abcam, #ab221163), rabbit anti-GAPDH (1:1000, Proteintech, #10494-1-AP), rabbit anti-RPLP0 (1:1000, Abcam, #ab192866), rabbit anti-phosphor-ERK1/2 antibody (1:1000, CST, #9101), rabbit anti-ERK1/2 antibody (1:1000, Proteintech, #11257-1-AP).

Techniques: Pull Down Assay, Activity Assay, Over Expression, Mutagenesis, Control, Western Blot, Knockdown, Ubiquitin Proteomics, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Arthrocolin B Impairs Adipogenesis via Delaying Cell Cycle Progression During the Mitotic Clonal Expansion Period

doi: 10.3390/ijms26041474

Figure Lengend Snippet: Arthrocolin B reduces the expression of key regulators associated with the G0/G1 phase. ( A ) 3T3-L1 preadipocytes were differentiated with MDI for 24 h or 48 h, and concurrently treated with 10 μM or 20 μM arthrocolin B. After that, cell lysates were collected, and the protein levels of CDK4, Cyclin D1, CDK2, Cyclin E1, p-Rb, and E2F1 were determined by Western blotting. ( B – G ) The intensity of the bands in panel A was quantified to determine the relative protein expression levels, with β-tubulin as an internal control. Data are shown as the mean ± SEM from three independent experiments. Significance is presented as * p < 0.05, ** p < 0.01 between the indicated groups. ns: no significance. Und, undifferentiated preadipocytes; Ctrl, normally differentiated preadipocytes; Veh, normally differentiated preadipocytes treated with dimethyl sulfoxide (DMSO); Acn B, arthrocolin B.

Article Snippet: Rabbit monoclonal antibody against E2F1 was purchased from Boster Biological Technology (Wuhan, China).

Techniques: Expressing, Western Blot, Control

Journal: bioRxiv

Article Title: Impaired Myelination in Multiple Sclerosis Organoids: p21 Links Oligodendrocyte Dysfunction to Disease Subtype

doi: 10.1101/2025.01.08.631924

Figure Lengend Snippet: A) Schematic representation of the involvement of PAK1 and E2F1 in p21 regulation, but also in oligodendrocyte differentiation, maturation, and myelination capacity. B) Immunofluorescence for PAK1 co-stained with neuroblast marker DCX (Top) and E2F1 co-stained with stem cell marker SOX2. C) Representative images of an immunofluorescence for PAK1 and E2F1 in organoids at D42 in control, PPMS, RRMS and SPMS samples. D) Quantifications for E2F1 in c-organoids at D42 (Kruskal-Wallis, p = 0.0898), for E2F1 in c-organoids at D120 (Kruskal-Wallis, p < 0.0001) and for PAK1 at D42 (Kruskal-Wallis, p < 0.0001). Each dot represents an organoid generated from different batches and patient cell lines. 4-6 images were taken per organoid.

Article Snippet: Slides were then incubated overnight at 4°C with the following primary antibodies diluted in blocking solution: mouse anti-APC (1:100, Calbiochem), mouse anti-ChAT (1:200, ThermoFisher), rat anti-CTIP2 (1:500, Abcam), guinea pig anti-DCX (1:500, Millipore), Rabbit anti-E2F1 (1:400, ThermoFisher), mouse anti-EOMES (1:100, ThermoFisher), mouse anti-GAD67 (1:200, Abcam), mouse anti-GFAP (1:500, Novus Biologicals), mouse anti-Ki67 (1:400, Millipore), chicken anti-MBP (1:500, ThermoFisher), mouse anti-Nanog (1:400, Abcam), mouse anti-O4 (1:200, R&D systems), rabbit anti-Oct4 (1:400, Abcam), rabbit anti-Olig2 (1:200, Abcam), rabbit anti-p16 (1:200, ThermoFisher), rabbit anti-p21 (1:200, ThermoFisher), rabbit anti-p27 (1:400, ThermoFisher), rabbit anti-p57 (1:400, ThermoFisher), mouse anti-Pax6 (1:100, Abcam), rabbit anti-PAK1 (1:100, ThermoFisher), mouse anti-SOX2 (1:400, Abcam), rabbit anti-TBR1 (1:400, Abcam), rabbit anti-vGluT1 (1:200, Abcam).

Techniques: Immunofluorescence, Staining, Marker, Control, Generated

Journal: The FASEB Journal

Article Title: The in vivo effects of knockdown of long non‐coding RNA XIST on fibroid growth and gene expression

doi: 10.1096/fj.202401982r

Figure Lengend Snippet: FIGURE 2 Relative mRNA levels of CDK2, SPARC, COL3A1, FN1, TGF-β3, TDO2, CYP1B1, E2F1, PRL, and EZH2 in xenografts following 4 weeks of implantation (n = 8). The results are presented as mean ± SEM with P values indicated at the corresponding line. *p < .05; **p < .01.

Article Snippet: Masson's trichrome staining (HT15- 1KT, SigmaAldrich) and immunohistochemical analysis were carried out as detailed in prior protocols.10,15 The primary antibodies used in this study were rabbit anti- Ki67 (dilution 1:250, 27 309- 1- AP, RRID:AB_2756525, Proteintech Group), rabbit anti- cleaved caspase- 3 (dilution 1:50, #9664, RRID:AB_2070042, Cell Signaling Technology, Danvers, MA, USA), rabbit anti- COL3A1 (dilution 1:1000, 22 734- 1- AP, RRID:AB_2879158, Proteintech Group), and mouse anti- E2F1 (dilution 1:50, sc- 251, RRID:AB_627476, Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques:

Journal: The FASEB Journal

Article Title: The in vivo effects of knockdown of long non‐coding RNA XIST on fibroid growth and gene expression

doi: 10.1096/fj.202401982r

Figure Lengend Snippet: FIGURE 4 Representative IHC staining images of fibroid xenografts from the control (siNC) and XIST knockdown treatment groups (n = 5) are shown, along with the quantification of staining intensity using Halo software for Ki67 (A and B), E2F1 (C and D), COL3A1 (E and F), and cleaved caspase 3 (G and H). (I) Representative histopathological images of fibroid xenografts from the control (siNC) and XIST knockdown treatment groups, determined by Masson's trichrome staining, where blue indicates collagen fibers and red denotes smooth muscle cells. (J) Quantification of staining intensity by Halo software. Data are presented as mean ± SEM, with p-values indicated at the corresponding lines. *p < .05.

Article Snippet: Masson's trichrome staining (HT15- 1KT, SigmaAldrich) and immunohistochemical analysis were carried out as detailed in prior protocols.10,15 The primary antibodies used in this study were rabbit anti- Ki67 (dilution 1:250, 27 309- 1- AP, RRID:AB_2756525, Proteintech Group), rabbit anti- cleaved caspase- 3 (dilution 1:50, #9664, RRID:AB_2070042, Cell Signaling Technology, Danvers, MA, USA), rabbit anti- COL3A1 (dilution 1:1000, 22 734- 1- AP, RRID:AB_2879158, Proteintech Group), and mouse anti- E2F1 (dilution 1:50, sc- 251, RRID:AB_627476, Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques: Immunohistochemistry, Control, Knockdown, Staining, Software

Journal: Pharmacological research

Article Title: BET degrader exhibits lower antiproliferative activity than its inhibitor via EGR1 recruiting septins to promote E2F1-3 transcription in triple-negative breast cancer.

doi: 10.1016/j.phrs.2024.107377

Figure Lengend Snippet: Fig. 3. The dissociation of BETs from E2F1–3 promoters upon MS645 and ARV771 treatments. a. Heat map of the normalized density of BRD4 in HCC1806 cells treated with MS645 or ARV771, centered at the TSS in a ±3 kb window. b. Genome browser views of the BRD4 peaks at E2F1–3 gene loci in HCC1806 cells treated with MS645 or ARV771. c. ChIP-qPCR analysis of BRD2, BRD3 and BRD4 occupancy at E2F1–3 gene loci in HCC1806 cells. d. ChIP-qPCR analysis of BRD2 and BRD4 occupancy at E2F1–3 gene loci in HCC1806 cells treated with MS645 or ARV771. e. Genome browser views of the H3K27ac peaks at E2F1–3 gene loci in HCC1806 cells treated with MS645 or ARV771. f and g. ChIP-qPCR analysis of H3K27ac (f), RNA polymerase II and MED1 (g) occupancy at E2F1–3 gene loci in HCC1806 cells treated with MS645 or ARV771. Statistical analysis was calculated by comparing DMSO to MS645 and ARV771 respectively. The data are shown as mean ± SEM from three biological replicates. Statistical analysis was performed using a two-tailed paired Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.

Article Snippet: Antibodies against EGR1 (4154), E2F1 (3742), CDK4 (12790 T) and normal rabbit IgG (2729) were purchased from Cell Signaling Technology.

Techniques: ChIP-qPCR, Two Tailed Test