Journal: Journal of Translational Medicine

Article Title: Exosomes loaded with the anti-cancer molecule mir-1-3p inhibit intrapulmonary colonization and growth of human esophageal squamous carcinoma cells

doi: 10.1186/s12967-024-05997-9

Figure Lengend Snippet: miR-1-3p directly targets and down-regulates E2F5 expression. The differentially expressed genes in the Exo-miR-1-3p group compared with the Exo-NC group, shown in the volcano plot ( A ), and the KEGG pathway analysis of differentially expressed genes ( B ). ( C ) Predicted target genes of miR-1-3p with TargetScan and miRWalk were cross-analyzed with significantly expression down-regulated genes. ( D ) E2F5 mRNA level after Exo-miR-1-3p incubation. ( E ) Putative binding sequence of miR-1-3p to the E2F5 mRNA 3’UTR and results of dual luciferase reporter assays. E2F5 protein expression of KYSE150 and Eca109 cells transfected with miR-1-3p mimics ( F ) or miR-1-3p inhibitor ( G ). Immunohistochemical staining ( H ) and H-scores ( I ) of E2F5 in mouse lung tissues. * P < 0.05, *** P < 0.001

Article Snippet: Antibodies information used in this study is provided below: anti-TSG101 (1:2000, 14497-1-AP, Proteintech), anti-CD81 (66866-1-Ig), anti-CD9 (60232-1-Ig), anti-E2F5 (1:2000, bs-1734R, Bioss), anti-GAPDH (1:5000, 60004-1-Ig, Proteintech), anti-p-p38 (1:1000, 4511, CST), anti-p38 (1:5000, 66234-1-lg, Proteintech), anti-p-ERK (1:1000, AP0974, ABclonal), anti-ERK (1:700, A4782, ABclonal), anti-p-Akt (1:2000, 28731-1-AP, Proteintech), anti-p-mTOR (1:500, AF3308, Affinity), HRP Goat anti-Rabbit IgG (1: 5000, AS014, ABclonal), HRP Goat anti-Mouse IgG (1: 5000, AS003, ABclonal).

Techniques: Expressing, Incubation, Binding Assay, Sequencing, Luciferase, Transfection, Immunohistochemical staining, Staining

Journal: Journal of Translational Medicine

Article Title: Exosomes loaded with the anti-cancer molecule mir-1-3p inhibit intrapulmonary colonization and growth of human esophageal squamous carcinoma cells

doi: 10.1186/s12967-024-05997-9

Figure Lengend Snippet: miR-1-3p inhibits proliferation and migration of ESCC cells in vitro by down-regulating E2F5. The miR-1-3p levels ( A ), E2F5 mRNA levels ( B ), and E2F5 protein levels in KYSE150 and Eca109 cells in vitro co-transfected with miR-1-3p mimics and E2F5 expression plasmids. The proliferation ( D ) and migration ( E and F ) of transfected cells. ns, no significance; * P < 0.05, *** P < 0.001

Article Snippet: Antibodies information used in this study is provided below: anti-TSG101 (1:2000, 14497-1-AP, Proteintech), anti-CD81 (66866-1-Ig), anti-CD9 (60232-1-Ig), anti-E2F5 (1:2000, bs-1734R, Bioss), anti-GAPDH (1:5000, 60004-1-Ig, Proteintech), anti-p-p38 (1:1000, 4511, CST), anti-p38 (1:5000, 66234-1-lg, Proteintech), anti-p-ERK (1:1000, AP0974, ABclonal), anti-ERK (1:700, A4782, ABclonal), anti-p-Akt (1:2000, 28731-1-AP, Proteintech), anti-p-mTOR (1:500, AF3308, Affinity), HRP Goat anti-Rabbit IgG (1: 5000, AS014, ABclonal), HRP Goat anti-Mouse IgG (1: 5000, AS003, ABclonal).

Techniques: Migration, In Vitro, Transfection, Expressing

Journal: Journal of Translational Medicine

Article Title: Exosomes loaded with the anti-cancer molecule mir-1-3p inhibit intrapulmonary colonization and growth of human esophageal squamous carcinoma cells

doi: 10.1186/s12967-024-05997-9

Figure Lengend Snippet: miR-1-3p inhibits invasion of ESCC cells in vitro by down-regulating E2F5, which may involve the MAPK/ERK signaling pathway. ( A and B ) The invasion of KYSE150 and Eca109 cells in vitro co-transfected with miR-1-3p mimics and E2F5 expression plasmids. ( C ) The protein levels of p-p38, p38, p-ERK, ERK in transfected cells, detected with western blot. ns, no significance; *** P < 0.001

Article Snippet: Antibodies information used in this study is provided below: anti-TSG101 (1:2000, 14497-1-AP, Proteintech), anti-CD81 (66866-1-Ig), anti-CD9 (60232-1-Ig), anti-E2F5 (1:2000, bs-1734R, Bioss), anti-GAPDH (1:5000, 60004-1-Ig, Proteintech), anti-p-p38 (1:1000, 4511, CST), anti-p38 (1:5000, 66234-1-lg, Proteintech), anti-p-ERK (1:1000, AP0974, ABclonal), anti-ERK (1:700, A4782, ABclonal), anti-p-Akt (1:2000, 28731-1-AP, Proteintech), anti-p-mTOR (1:500, AF3308, Affinity), HRP Goat anti-Rabbit IgG (1: 5000, AS014, ABclonal), HRP Goat anti-Mouse IgG (1: 5000, AS003, ABclonal).

Techniques: In Vitro, Transfection, Expressing, Western Blot

Journal:

Article Title: Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression

doi:

Figure Lengend Snippet: In vivo detection of promoter occupancy by the E2F and pRB family using chromatin immunoprecipitations. (A) Outline of the in vivo cross-linking and chromatin immunoprecipitation (IP) protocol. (B) Promoters examined in this study. Solid ovals represent E2F-binding sites on the basis of previous studies; small arrows indicate primers used in PCR amplification reactions. Large arrows represent published major transcription start site. (C) Chromatin immunoprecipitations from asynchronously growing T98G cells. Input corresponds to PCR reactions containing 0.5% of total amount of chromatin used in immunoprecipitation reactions. Mock immunoprecipitations performed with irrelevant, control antibodies (anti-T Antigen pAb101) and control reactions lacking antibodies (No Ab) are shown. Amplified products were detected by autoradiography.

Article Snippet: Antibodies that recognize E2F-1 (sc-193), E2F-2 (sc-633x), E2F-3 (sc-878x), E2F-4 (sc-1082x), E2F-5 (sc-999, sc-968), p107 (sc-318), pRB (sc-50, G99-549, G3-245), p130 (sc-317, Rb2), cdk2 (sc-163), and 12CA5 anti-flu hemagglutinin were obtained from Santa Cruz Biotechnology, Neomarkers, Pharmingen, Transduction Labs, and BabCo.

Techniques: In Vivo, Chromatin Immunoprecipitation, Binding Assay, Amplification, Immunoprecipitation, Autoradiography

Journal:

Article Title: Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression

doi:

Figure Lengend Snippet: Cell cycle synchronization and gene expression analysis. (A) FACS analysis of T98G cells that were rendered quiescent by serum starvation (0 hr) and were subsequently restimulated with serum for the indicated lengths of time and allowed to enter the cell cycle. DNA content (propidium iodide intensity) is plotted vs. cell number. (B) Northern blot analysis of expression of the indicated genes during the synchronization experiment in A. To ensure equal loading of RNA, each blot was subsequently stripped and rehybridized with an ARPP P0 gene fragment, and a representative filter is shown. (C) Gel mobility shift analysis of E2F and pRB complexes. Whole cell extracts at each stage of the cell cycle (indicated at top) were incubated with a labeled probe containing an E2F site and each of the designated antibodies. The positions of E2F-pRB/p107/p130 complexes and free E2F (E2F) are shown at right. Each of these complexes was abrogated by a specific competitor oligonucleotide (data not shown). Asynchronous (AS) samples were prepared from cells entering a second cell cycle (with roughly equal percentages of G1, S, and G2 cells). Lanes marked − and control contained no antibody and 12CA5 (anti-flu hemagglutinin antibody), respectively. α-E2F-4 lanes contain two different monoclonal antibodies specific for E2F-4 (WUF3 and LLF4, respectively).

Article Snippet: Antibodies that recognize E2F-1 (sc-193), E2F-2 (sc-633x), E2F-3 (sc-878x), E2F-4 (sc-1082x), E2F-5 (sc-999, sc-968), p107 (sc-318), pRB (sc-50, G99-549, G3-245), p130 (sc-317, Rb2), cdk2 (sc-163), and 12CA5 anti-flu hemagglutinin were obtained from Santa Cruz Biotechnology, Neomarkers, Pharmingen, Transduction Labs, and BabCo.

Techniques: Expressing, Northern Blot, Mobility Shift, Incubation, Labeling

Journal:

Article Title: Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression

doi:

Figure Lengend Snippet: The binding of E2F family of proteins changes during the cell cycle. Chromatin immunoprecipitations were performed with antibodies specific for individual E2F family members as indicated, and the resulting immunoprecipitates were amplified with primer pairs corresponding to the genes shown. G0, serum-starved cells; early G1, late G1, G1/S, and S-phase cells were stimulated with serum 4–8, 16, 20, and 24 hr, respectively. Antibodies tested in each case are listed in Materials and Methods.

Article Snippet: Antibodies that recognize E2F-1 (sc-193), E2F-2 (sc-633x), E2F-3 (sc-878x), E2F-4 (sc-1082x), E2F-5 (sc-999, sc-968), p107 (sc-318), pRB (sc-50, G99-549, G3-245), p130 (sc-317, Rb2), cdk2 (sc-163), and 12CA5 anti-flu hemagglutinin were obtained from Santa Cruz Biotechnology, Neomarkers, Pharmingen, Transduction Labs, and BabCo.

Techniques: Binding Assay, Amplification

Journal:

Article Title: Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression

doi:

Figure Lengend Snippet: Antibodies against each pRB family member immunoprecipitate their chromatin-bound targets. (A) Schematic representing the position of chromatin in gradients relative to free DNA and protein under conditions in which cells were either fixed with formaldehyde (+ cross-linker) or left untreated (− cross-linker). Brackets indicate chromatin fractions selected for further analysis in B. (B) Fractions in A were immunoprecipitated with anti-pRB, anti-p107, and p130 antibodies as indicated and the resulting precipitates were probed with the same antibodies as indicated. (C) p107 and p130 are detected at similar levels in T98G cells entering a second cell cycle (32-hr time point in Fig. ​Fig.2).2). Chromatin immunoprecipitations were performed with the indicated antibodies, and E2F-1, cyclin A, and actin promoter fragments were amplified as described in the legend to Fig. ​Fig.1.1. Distinct monoclonal and polyclonal anti-pRB antibodies were tested as shown.

Article Snippet: Antibodies that recognize E2F-1 (sc-193), E2F-2 (sc-633x), E2F-3 (sc-878x), E2F-4 (sc-1082x), E2F-5 (sc-999, sc-968), p107 (sc-318), pRB (sc-50, G99-549, G3-245), p130 (sc-317, Rb2), cdk2 (sc-163), and 12CA5 anti-flu hemagglutinin were obtained from Santa Cruz Biotechnology, Neomarkers, Pharmingen, Transduction Labs, and BabCo.

Techniques: Immunoprecipitation, Amplification

Journal:

Article Title: Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression

doi:

Figure Lengend Snippet: Histone acetylation levels of E2F-responsive genes change during the cell cycle. (A) Chromatin was prepared from synchronized T98G cells (as described in Fig. ​Fig.2A)2A) and immunoprecipitated with antibodies specific for acetylated histone H3 and acetylated H4 as described in the legend to Fig. ​Fig.1.1. Cell cycle stages were identical to those described in Fig. ​Fig.3.3. Parallel immunoprecipitations without antibody or with an irrelevant antibody control failed to yield detectable signals after an equivalent autoradiographic exposure (data not shown). (B) Data in A were quantitated by PhosphorImager analysis and normalized vs. input levels. The y-axis indicates fold acetylation (in arbitrary units). (Light-colored bars) acetylated H3; (dark bars) acetylated H4.

Article Snippet: Antibodies that recognize E2F-1 (sc-193), E2F-2 (sc-633x), E2F-3 (sc-878x), E2F-4 (sc-1082x), E2F-5 (sc-999, sc-968), p107 (sc-318), pRB (sc-50, G99-549, G3-245), p130 (sc-317, Rb2), cdk2 (sc-163), and 12CA5 anti-flu hemagglutinin were obtained from Santa Cruz Biotechnology, Neomarkers, Pharmingen, Transduction Labs, and BabCo.

Techniques: Immunoprecipitation

Journal:

Article Title: Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression

doi:

Figure Lengend Snippet: Model for in vivo occupancy by E2F and pRB family members during cell cycle progression. (A) In vivo occupation of E2F-1, Cdc25A, cyclin A, Cdc6, and p107 promoters by the E2F and pRB family during cell cycle progression and transcriptional consequences. (B) In vivo binding of B-myb promoter. In A and B, promoter binding by E2F-4 correlates with transcriptional repression, whereas binding by E2F-1 and E2F-3 occurs at a time when each promoter is activated. However, the B-myb promoter is transcriptionally active after E2F is no longer bound, consistent with Zwicker et al. (1996). (D) HDAC activity; (H) nucleosomes; (Ac) acetylated histones; (HAT) histone acetyltransferase. These promoters are likely to be regulated by additional trans-activator proteins proximal to E2F, including Sp1. Recruitment of deacetylase and HAT activities could occur by direct interactions with E2F complexes or could require additional factors (X) yet to be defined.

Article Snippet: Antibodies that recognize E2F-1 (sc-193), E2F-2 (sc-633x), E2F-3 (sc-878x), E2F-4 (sc-1082x), E2F-5 (sc-999, sc-968), p107 (sc-318), pRB (sc-50, G99-549, G3-245), p130 (sc-317, Rb2), cdk2 (sc-163), and 12CA5 anti-flu hemagglutinin were obtained from Santa Cruz Biotechnology, Neomarkers, Pharmingen, Transduction Labs, and BabCo.

Techniques: In Vivo, Binding Assay, Activity Assay, Histone Deacetylase Assay

Journal: BMC Cancer

Article Title: E2F5 status significantly improves malignancy diagnosis of epithelial ovarian cancer

doi: 10.1186/1471-2407-10-64

Figure Lengend Snippet: Clinical characteristics and age distribution of study samples (serum samples) used for E2F5 and CA125 expression studies.

Article Snippet: Primary antibodies directed against the AREB6 (AVIVA Systems Biology, San Diego, California, USA), E2F5 (Acris GmbH, Hiddenhausen, Germany), PAX8 (Abcam Limited, Cambridge, UK), and ELF3 (Orbigen Inc, San Diego, CA) were chosen as the four TFs suggested by the bioinformatics analysis.

Techniques: Expressing

Journal: BMC Cancer

Article Title: E2F5 status significantly improves malignancy diagnosis of epithelial ovarian cancer

doi: 10.1186/1471-2407-10-64

Figure Lengend Snippet: Summary of Immunohistochemistry results for E2F5 antibody tested on OEC

Article Snippet: Primary antibodies directed against the AREB6 (AVIVA Systems Biology, San Diego, California, USA), E2F5 (Acris GmbH, Hiddenhausen, Germany), PAX8 (Abcam Limited, Cambridge, UK), and ELF3 (Orbigen Inc, San Diego, CA) were chosen as the four TFs suggested by the bioinformatics analysis.

Techniques: Immunohistochemistry

Journal: BMC Cancer

Article Title: E2F5 status significantly improves malignancy diagnosis of epithelial ovarian cancer

doi: 10.1186/1471-2407-10-64

Figure Lengend Snippet: Validation of potential target (AREB6, PAX8, ELF3) using western blots (A) and expression of E2F5 in serum from normal, benign and, malignant patient samples (B) . (A) Target validation using western blots of AREB6, PAX8, ELF3, in serum of healthy volunteers (N) (n = 2), patients with benign ovarian cysts (B1, B2) (n = 2), and patients with late- (L1, L2) (n = 2) and early-stage (E1-E3) (n = 3) serous adenocarcinoma. AREB6 was overexpressed in late stage disease, but was not discriminatory for early cancer. PAX8 and ELF3 were equally expressed in all serum samples. (B) Expression of E2F5 in serum from normal (n = 56), benign (n = 40) and, malignant (n = 48) serum obtained from patients with cancer of the ovary.

Article Snippet: Primary antibodies directed against the AREB6 (AVIVA Systems Biology, San Diego, California, USA), E2F5 (Acris GmbH, Hiddenhausen, Germany), PAX8 (Abcam Limited, Cambridge, UK), and ELF3 (Orbigen Inc, San Diego, CA) were chosen as the four TFs suggested by the bioinformatics analysis.

Techniques: Biomarker Discovery, Western Blot, Expressing

Journal: BMC Cancer

Article Title: E2F5 status significantly improves malignancy diagnosis of epithelial ovarian cancer

doi: 10.1186/1471-2407-10-64

Figure Lengend Snippet: Details of E2F5/CA125 expression pattern on different subtypes of OEC performed using western blotting technique.

Article Snippet: Primary antibodies directed against the AREB6 (AVIVA Systems Biology, San Diego, California, USA), E2F5 (Acris GmbH, Hiddenhausen, Germany), PAX8 (Abcam Limited, Cambridge, UK), and ELF3 (Orbigen Inc, San Diego, CA) were chosen as the four TFs suggested by the bioinformatics analysis.

Techniques: Expressing, Western Blot

Journal: BMC Cancer

Article Title: E2F5 status significantly improves malignancy diagnosis of epithelial ovarian cancer

doi: 10.1186/1471-2407-10-64

Figure Lengend Snippet: Diagnostic accuracy developed for ovarian cancer detection with 13 features using Kstar classification based on SMOTE algorithms.

Article Snippet: Primary antibodies directed against the AREB6 (AVIVA Systems Biology, San Diego, California, USA), E2F5 (Acris GmbH, Hiddenhausen, Germany), PAX8 (Abcam Limited, Cambridge, UK), and ELF3 (Orbigen Inc, San Diego, CA) were chosen as the four TFs suggested by the bioinformatics analysis.

Techniques: Diagnostic 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