Journal: Journal of Cellular and Molecular Medicine

Article Title: MATN1‐AS1 Promotes Tumour Metastasis and Sunitinib Resistance via E2F2 in Clear Cell Renal Cell Carcinoma

doi: 10.1111/jcmm.70428

Figure Lengend Snippet: MATN1‐AS1 regulates E2F2 expression in ccRCC. (A) Heatmap showing DEGs expression profiles of 786‐O cells transfected with control (Control 1–3) or MATN1‐AS1 targeted sequence (sh 1–3). Data were normalised with FPKM. Results were filtered with p < 0.05, |log 2 FC| > 1. (B, C) GSEA of Control and MATN1‐AS1 knocked down groups. (D) Heatmap showing the expression level changes of the E2F family after knocking down MATN1‐AS1. Data were normalised with FPKM. (E) Co‐expression analysis of E2Fs with MATN1‐AS1 in TCGA‐KIRC dataset (Pearson correlation test). (F) Expression correlation between E2F2 and MATN1‐AS1 in TCGA‐KIRC dataset (Pearson correlation test). (G) E2F2 expression levels in ccRCC and normal tissues in the TCGA‐KIRC dataset (Data were normalised with log 2 (TPM + 1), Mean ± SEM, Wilcoxon test, *** p < 0.001). (H) E2F2 expression levels in ccRCC and normal tissues in the ICGC‐RECA dataset (Data were normalised with log 2 (TPM + 1), Mean ± SEM, Wilcoxon test, *** p < 0.001). (I) Overall survival (OS) curves of E2F2 in ccRCC (Log‐rank test, * p < 0.05). (J, K) GSEA between High‐ and Low‐E2F2 expression individuals from TCGA‐KIRC dataset. (L) E2F2 expression level changes after knocking down MATN1‐AS1. (M) Potential micro‐RNA links between MATN1‐AS1 and E2F2. (N) miR‐214‐5p expression levels in ccRCC and renal tissue from TCGA‐KIRC dataset ( p = 2.2 × 10 −13 ). (O, P) Binding sites schematic diagrams of miR‐214‐5p with E2F2 and MATN1‐AS1. (Q) Dual‐luciferase reporter assay results of MATN1‐AS1 and E2F2 (Mean ± SEM, Student's t ‐test, ns, no significance, ** p < 0.01, *** p < 0.001). (R) E2F2 expression level changes after being treated with miR‐214‐5p mimics.

Article Snippet: Antibody reagents were anti‐E2F2 (1:1000; bsm‐52641R; Bioss), anti‐SNAI1 (1:1000; 13099‐1‐AP; Proteintech), anti‐SNAI2 (1:1000; 12129‐1‐AP; Proteintech), anti‐Vimentin (1:1000; 10366‐1‐AP; Proteintech), anti‐N‐cadherin (1:1000; 22018‐1‐AP; Proteintech), anti‐E‐cadherin (1:1000; no. 3195; CST), and anti‐GAPDH (1:2000; no. 2118; CST).

Techniques: Expressing, Transfection, Control, Sequencing, Binding Assay, Luciferase, Reporter Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: MATN1‐AS1 Promotes Tumour Metastasis and Sunitinib Resistance via E2F2 in Clear Cell Renal Cell Carcinoma

doi: 10.1111/jcmm.70428

Figure Lengend Snippet: MATN1‐AS1/miR‐214‐5p/E2F2 axis regulated EMT in ccRCC. (A, B) Cell viability of indicated groups measured with CCK8 assay (Mean ± SEM, Two‐way repeated‐measures analysis of ANOVA with Geisser–Greenhouse correction, ns, no significance, ** p < 0.01, *** p < 0.001). (C) Cell migration and invasion abilities of corresponding groups as measured with the Transwell assay. (D, E) RNA expression level changes of E2F2 after overexpression miR‐214‐5p in 786‐O and A‐498 cell lines. (F, G) RNA expression levels of E2F2 and MATN1‐AS1 in indicated cells. (H, I) E2F2 and EMT biomarkers expression changes across four groups.

Article Snippet: Antibody reagents were anti‐E2F2 (1:1000; bsm‐52641R; Bioss), anti‐SNAI1 (1:1000; 13099‐1‐AP; Proteintech), anti‐SNAI2 (1:1000; 12129‐1‐AP; Proteintech), anti‐Vimentin (1:1000; 10366‐1‐AP; Proteintech), anti‐N‐cadherin (1:1000; 22018‐1‐AP; Proteintech), anti‐E‐cadherin (1:1000; no. 3195; CST), and anti‐GAPDH (1:2000; no. 2118; CST).

Techniques: CCK-8 Assay, Migration, Transwell Assay, RNA Expression, Over Expression, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: MATN1‐AS1 Promotes Tumour Metastasis and Sunitinib Resistance via E2F2 in Clear Cell Renal Cell Carcinoma

doi: 10.1111/jcmm.70428

Figure Lengend Snippet: MATN1‐AS1/miR‐214‐5p/E2F2 axis affects tumour metastasis in ccRCC. (A) Four groups of nude mice were subcutaneously injected with response cells, and tumours were removed and imaged. (B) Tumour volumes were measured every 3 days and drawn as a curve plot (Mean ± SEM, Two‐way repeated‐measures ANOVA with Dunnett multiple comparisons test correction, ns, no significance, *** p < 0.001). (C, D) Four groups of nude mice were tail vein‐injected corresponsive cells and imaged after 6 months (Signals were normalised as p/s/cm2/sr, Mean ± SEM, Student's t ‐test, ns, no significance, * p < 0.05). (E) E2F2, N‐cadherin, and E‐cadherin expression levels in four groups of subcutaneously generated tumours as detected by IHC using serial slices.

Article Snippet: Antibody reagents were anti‐E2F2 (1:1000; bsm‐52641R; Bioss), anti‐SNAI1 (1:1000; 13099‐1‐AP; Proteintech), anti‐SNAI2 (1:1000; 12129‐1‐AP; Proteintech), anti‐Vimentin (1:1000; 10366‐1‐AP; Proteintech), anti‐N‐cadherin (1:1000; 22018‐1‐AP; Proteintech), anti‐E‐cadherin (1:1000; no. 3195; CST), and anti‐GAPDH (1:2000; no. 2118; CST).

Techniques: Injection, Expressing, Generated

Journal: Journal of Cellular and Molecular Medicine

Article Title: MATN1‐AS1 Promotes Tumour Metastasis and Sunitinib Resistance via E2F2 in Clear Cell Renal Cell Carcinoma

doi: 10.1111/jcmm.70428

Figure Lengend Snippet: Knocking down MATN1‐AS1 reversed drug resistance in sunitinib resistance ccRCC. (A, B) GSEA of drug resistance signalling pathway. (C) DEGs between sunitinib resistance ccRCC cells and non‐treated cells of the GSE216494 dataset (|FDR| < 1, p < 0.05 was defined as non‐significance expressed genes filter criteria). (D) MATN1‐AS1 expression levels between sunitinib resistance ccRCC cells and non‐treated cells. (E) Gene ontology analysis of DEGs between sunitinib resistance ccRCC cells and non‐treated cells. (F) Graph diagram of sunitinib resistance A‐498 cell line (A‐498‐R) establishing process. (G) IC50 curves of sunitinib in A‐498 and A‐498‐R cell lines. (Data were presented as mean ± SEM). (H, I) MATN1‐AS1 and E2F2 RNA expression values between A‐498 and A‐498‐R cell lines (Mean ± SEM, Student's t ‐test, ** p < 0.01, *** p < 0.001). (J) E2F2 expression levels of A‐498 and A‐498‐R cells. (K) Knocking down MATN1‐AS1 inhibited cell viability of the A‐498‐R cell line cultured in 10 μM sunitinib (Mean ± SEM, Two‐way ANOVA, *** p < 0.001). (L) Drug‐response curves of sunitinib in MATN1‐AS1 knocked down A‐498‐R cells and non‐treated A‐498‐R cells (Mean ± SEM, Two‐way ANOVA test, *** p < 0.001). (M) Overexpression E2F2 reversed cell viability of the A‐498‐R cell line treated with 10 μM sunitinib (Mean ± SEM, Two‐way ANOVA, ** p < 0.01). (N) Drug‐response curves of sunitinib in E2F2 overexpressed A‐498‐R cells and non‐treated A‐498‐R cells (Mean ± SEM, Two‐way ANOVA test, *** p < 0.001). (O) Drug‐response curves of sunitinib in indicated groups (Mean ± SEM, Two‐way ANOVA test, ns, no significance, *** p < 0.001).

Article Snippet: Antibody reagents were anti‐E2F2 (1:1000; bsm‐52641R; Bioss), anti‐SNAI1 (1:1000; 13099‐1‐AP; Proteintech), anti‐SNAI2 (1:1000; 12129‐1‐AP; Proteintech), anti‐Vimentin (1:1000; 10366‐1‐AP; Proteintech), anti‐N‐cadherin (1:1000; 22018‐1‐AP; Proteintech), anti‐E‐cadherin (1:1000; no. 3195; CST), and anti‐GAPDH (1:2000; no. 2118; CST).

Techniques: Expressing, RNA Expression, Cell Culture, Over Expression

Journal: Cold Spring Harbor symposia on quantitative biology

Article Title: Regulation of the Arf/p53 tumor surveillance network by E2F.

doi: 10.1101/sqb.2005.70.050

Figure Lengend Snippet: Figure 5. The activating E2Fs bind to the Arf promoter in re- sponse to oncogenic stress. Wild-type MEFs were infected with retrovirus overexpressing E1A, or an empty virus (vector) and subjected to western (A) or ChIP (B) analyses with the indicated antisera. MEFs expressing E1A exhibit dramatically increased levels of p19Arf, coincident with recruitment of activating E2Fs (E2F1, E2F2, and E2F3A) to the Arf promoter.

Article Snippet: Sonicated, cross-linked chromatin corresponding to approximately 3 x 106 cells was immunoprecipitated with the following antibodies: normal rabbit IgG (control), sc-2027; E2F1, sc-193; E2F2, sc-633x; E2F3A, sc-879x; E2F3A+B, sc-878x; E2F4, sc-1082x; p130, sc-317x (all from Santa Cruz Biotechnology).

Techniques: Infection, Virus, Plasmid Preparation, Western Blot, Expressing

Journal: PPAR Research

Article Title: PPAR- γ Ligand Inhibits Nasopharyngeal Carcinoma Cell Proliferation and Metastasis by Regulating E2F2

doi: 10.1155/2019/8679271

Figure Lengend Snippet: Expressions of E2F2 and PPAR- γ in clinical samples of undifferentiated NPC and NPG and associations with clinicopathological characteristics of undifferentiated NPC samples.

Article Snippet: After three washes with phosphate-buffered saline (PBS), the sections were incubated in a blocking solution containing bovine serum albumin (BSA) for 20 min, followed by exposure to primary antibodies against E2F2 (YT1443, Immunoway, rabbit polyclonal, 1:200 dilution) and PPAR- γ (Novusbio, rabbit polyclonal, 1:100 dilution) at 4°C overnight.

Techniques: Expressing

Journal: PPAR Research

Article Title: PPAR- γ Ligand Inhibits Nasopharyngeal Carcinoma Cell Proliferation and Metastasis by Regulating E2F2

doi: 10.1155/2019/8679271

Figure Lengend Snippet: E2F2 and PPAR-γ protein expression in nonkeratinizing nasopharyngeal carcinoma (NPC) and nasopharyngitis (NPG) tissues . Immunohistochemistry was performed to detect E2F2 and PPAR- γ protein expression in nonkeratinizing NPC tissues (a, b) and NPG tissues (c, d). Scale bar, 50 μ m (e, f). The immunohistochemistry data were analyzed semiquantitatively to determine the E2F2 and PPAR- γ expression levels in nonkeratinizing NPC and NPG tissues. Data are shown as means ± standard deviations. ∗∗ P <0.01.

Article Snippet: After three washes with phosphate-buffered saline (PBS), the sections were incubated in a blocking solution containing bovine serum albumin (BSA) for 20 min, followed by exposure to primary antibodies against E2F2 (YT1443, Immunoway, rabbit polyclonal, 1:200 dilution) and PPAR- γ (Novusbio, rabbit polyclonal, 1:100 dilution) at 4°C overnight.

Techniques: Expressing, Immunohistochemistry

Journal: PPAR Research

Article Title: PPAR- γ Ligand Inhibits Nasopharyngeal Carcinoma Cell Proliferation and Metastasis by Regulating E2F2

doi: 10.1155/2019/8679271

Figure Lengend Snippet: E2F2 and PPAR-γ protein expression in nonkeratinizing nasopharyngeal carcinoma (NPC) and nasopharyngitis (NPG) tissue lysates . (a, b) RT-PCR and Western blotting were performed to detect the expression of E2F2, PPAR- γ , and GAPDH in lysates of nonkeratinizing NPC and NPG tissues. (c, d) Quantitative analyses of the E2F2 and PPAR- γ expression levels in nonkeratinizing NPC and NPG tissues. Data are expressed as means ± standard deviations. ∗ P <0.05; ∗∗ P <0.01.

Article Snippet: After three washes with phosphate-buffered saline (PBS), the sections were incubated in a blocking solution containing bovine serum albumin (BSA) for 20 min, followed by exposure to primary antibodies against E2F2 (YT1443, Immunoway, rabbit polyclonal, 1:200 dilution) and PPAR- γ (Novusbio, rabbit polyclonal, 1:100 dilution) at 4°C overnight.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: PPAR Research

Article Title: PPAR- γ Ligand Inhibits Nasopharyngeal Carcinoma Cell Proliferation and Metastasis by Regulating E2F2

doi: 10.1155/2019/8679271

Figure Lengend Snippet: E2F2 and PPAR-γ expression in CNE1 and CNE2 nasopharyngeal carcinoma cells after treatment with PPAR-γ ligand . (a, b) Western blotting was performed to detect the expression of E2F2, PPAR- γ , and GAPDH in CNE1 and CNE2 cells after treatment with dose-dependent PPAR- γ agonist Rog. (c, d) Western blotting was performed to detect the expression of E2F2, PPAR- γ , and GAPDH in CNE1 and CNE2 cells after treatment with PPAR- γ agonist (rosiglitazone, Rog) and PPAR- γ antagonist (GW9662).

Article Snippet: After three washes with phosphate-buffered saline (PBS), the sections were incubated in a blocking solution containing bovine serum albumin (BSA) for 20 min, followed by exposure to primary antibodies against E2F2 (YT1443, Immunoway, rabbit polyclonal, 1:200 dilution) and PPAR- γ (Novusbio, rabbit polyclonal, 1:100 dilution) at 4°C overnight.

Techniques: Expressing, Western Blot

Journal:

Article Title: CDC25A Phosphatase Is a Target of E2F and Is Required for Efficient E2F-Induced S Phase

doi:

Figure Lengend Snippet: Subcellular localization of ERE2F-1. (A and B) Immunofluorescence of U2OS cells transiently transfected with pCMVHAERE2F-1, detected by using an anti-E2F-1 monoclonal antibody (KH95), in the absence (A) or in the presence (B) of OHT. (C to F) A selected pool of Rat1 clones expressing ERE2F-1, stained with an anti-E2F-1 monoclonal antibody, in the absence (C) or in the presence (D) of OHT and the corresponding DAPI staining (E and F).

Article Snippet: Stable transfected Rat1 cells expressing a low level of protein were stained with the E2F-1-, E2F-2-, or E2F-3-specific antibodies as described above and developed by using a TSA-Direct kit (NEN Life Science Products) according to the manufacturer’s suggestions.

Techniques: Immunofluorescence, Transfection, Clone Assay, Expressing, Staining

Journal:

Article Title: CDC25A Phosphatase Is a Target of E2F and Is Required for Efficient E2F-Induced S Phase

doi:

Figure Lengend Snippet: E2F transactivation of the CDC25A promoter. (A and B) Activation of the CDC25A promoter by transient transfection of pCMVE2F-1, pCMVE2F-2, and pCMVE2F-3. U2OS cells were transiently transfected with pCMVE2F-1, pCMVE2F-2, or pCMVE2F-3 together with CDC25A(−755/+423) luc (A) or with a synthetic E2F-responsive promoter, 6× E2Fluc (B). −, transfected with empty pcnv expression vector. (C and D) Activation of the CDC25A promoter in the ERE2F-expressing cell lines. Rat1 cells expressing ERE2F-1, ERE2F-2, or ERE2F-3 were transfected with CDC25A(−755/+423) luc (C) or with 6× E2Fluc (D). +, addition of OHT; −, without OHT. pCMVβ-gal was cotransfected in all experiments, and β-galactosidase activity served as a control for transfection efficiency. Numbers indicate fold induction relative to the sample transfected with empty pCMV (A and B) or fold induction after addition of OHT (C and D). The luciferase counts are normalized for β-galactosidase activity. The presented data are representative of at least three different experiments.

Article Snippet: Stable transfected Rat1 cells expressing a low level of protein were stained with the E2F-1-, E2F-2-, or E2F-3-specific antibodies as described above and developed by using a TSA-Direct kit (NEN Life Science Products) according to the manufacturer’s suggestions.

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Activity Assay, Luciferase

Journal:

Article Title: CDC25A Phosphatase Is a Target of E2F and Is Required for Efficient E2F-Induced S Phase

doi:

Figure Lengend Snippet: Activation of E2F-1 by OHT is sufficient for S-phase induction a

Article Snippet: Stable transfected Rat1 cells expressing a low level of protein were stained with the E2F-1-, E2F-2-, or E2F-3-specific antibodies as described above and developed by using a TSA-Direct kit (NEN Life Science Products) according to the manufacturer’s suggestions.

Techniques: Activation Assay

Journal:

Article Title: CDC25A Phosphatase Is a Target of E2F and Is Required for Efficient E2F-Induced S Phase

doi:

Figure Lengend Snippet: Activation of E2F-1, E2F-2, or E2F-3 results in S-phase entry and apoptosis. (A) E2F-1, E2F-2, or E2F-3 activation is sufficient for S-phase induction. Rat1 cells expressing ERE2F-1, ERE2F-2, or ERE2F-3 were grown in medium containing 0.1% serum for 48 h and subsequently induced with OHT. Cells were harvested for FACS analysis at the indicated times after addition of OHT, and the cell cycle profile was determined. (B) E2F-1, E2F-2, and E2F-3 induce cell death. Cells were serum starved for 48 h in medium with 0.1% serum, and OHT was subsequently added to the starvation medium. At the indicated times, cells were harvested and living cells were counted after being stained with Trypan blue. The percentage of surviving cells was calculated by using the number serum-starved cells in the absence of OHT as the reference point. (C) Activation of E2F-1, E2F-2, or E2F-3 induces apoptosis. FACS analysis of Rat1 cells expressing ERE2F-1, ERE2F-2, or ERE2F-3, showing the percentage of apoptotic cells (sub-G1 fraction) at 0 and 24 h after OHT addition. The cells were grown in medium with 0.1% serum for 48 h prior to addition of OHT.

Article Snippet: Stable transfected Rat1 cells expressing a low level of protein were stained with the E2F-1-, E2F-2-, or E2F-3-specific antibodies as described above and developed by using a TSA-Direct kit (NEN Life Science Products) according to the manufacturer’s suggestions.

Techniques: Activation Assay, Expressing, Staining

Journal:

Article Title: CDC25A Phosphatase Is a Target of E2F and Is Required for Efficient E2F-Induced S Phase

doi:

Figure Lengend Snippet: Cyclin E is a direct target of E2F-1. ERE2F-1 clone D cells were made quiescent by growing the cells in medium with 0.1% serum for 48 h. RT-PCR was performed on samples of total RNA from ERE2F-1 clone D that had been kept for 48 h in medium containing 0.1% serum and induced for different lengths of time by addition of OHT, serum, or OHT plus CHX. Primers specific for the amplification of cyclin E mRNA were used as described in Materials and Methods.

Article Snippet: Stable transfected Rat1 cells expressing a low level of protein were stained with the E2F-1-, E2F-2-, or E2F-3-specific antibodies as described above and developed by using a TSA-Direct kit (NEN Life Science Products) according to the manufacturer’s suggestions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification

Journal:

Article Title: CDC25A Phosphatase Is a Target of E2F and Is Required for Efficient E2F-Induced S Phase

doi:

Figure Lengend Snippet: CDC25A is a direct target of E2F-1. (A and B) RT-PCR performed on RNA samples from ERE2F-1 clone D cells that were starved and induced with OHT or serum (A) or with OHT alone, CHX plus OHT, CHX alone, or serum alone (B). Primers for specific amplification of cdc25A, the cyclin E gene, and GAPDH were used as indicated and as described in Materials and Methods. (C) Activation of E2F-1 leads to accumulation of the cdc25A protein. Immunoprecipitation followed by Western blotting with cdc25A-specific antibodies was performed with 200-μg quantities of cell lysate at the indicated time points after addition of OHT or serum. The masses of molecular size markers are indicated to the left in kilodaltons. (D) Activation of E2F-2 and E2F-3 is not sufficient to induce transcription of CDC25A. RT-PCR was performed on RNA prepared from ERE2F-1, ERE2F-2, and ERE2F-3 pools as described above. Cyclin E served as an internal positive control. The cells were starved and induced for 4 h in the presence of CHX, CHX plus OHT, or OHT alone.

Article Snippet: Stable transfected Rat1 cells expressing a low level of protein were stained with the E2F-1-, E2F-2-, or E2F-3-specific antibodies as described above and developed by using a TSA-Direct kit (NEN Life Science Products) according to the manufacturer’s suggestions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Activation Assay, Immunoprecipitation, Western Blot, Positive Control

Journal:

Article Title: CDC25A Phosphatase Is a Target of E2F and Is Required for Efficient E2F-Induced S Phase

doi:

Figure Lengend Snippet: Genes activated by E2F-1. (A and B) RT-PCR was performed on RNA samples prepared from ERE2F-1 clone D (A) and from an ERE2F-1 pool (B). Cells were kept for 48 h in medium containing 0.1% serum and induced for different lengths of time with OHT or serum (A) or for 4 h in the presence of CHX, CHX plus OHT, or OHT alone (B). Primers specific for the cDNA amplification of the indicated genes were used as described in Materials and Methods. (C) Genes activated directly by E2F-1 expression. ++, upregulation over 10-fold; +, upregulation between 5- and 10-fold; +/−, upregulation between 1- and 5-fold; −, no upregulation observed; ∗, data not shown.

Article Snippet: Stable transfected Rat1 cells expressing a low level of protein were stained with the E2F-1-, E2F-2-, or E2F-3-specific antibodies as described above and developed by using a TSA-Direct kit (NEN Life Science Products) according to the manufacturer’s suggestions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Expressing

Journal:

Article Title: CDC25A Phosphatase Is a Target of E2F and Is Required for Efficient E2F-Induced S Phase

doi:

Figure Lengend Snippet: Subcellular localization of ERE2F-2 and ERE2F-3 in the presence or absence of OHT. (A and B) A pool of puromycin-resistant Rat1 cells infected with retroviruses expressing ERE2F-2, stained with an anti-E2F-2 monoclonal antibody (TFE22), in the absence (A) or in the presence (B) of OHT. (C and D) A pool of puromycin-resistant Rat1 cells infected with retroviruses expressing ERE2F-3, stained with an anti-E2F-3 monoclonal antibody (TFE31), in the absence (C) or in the presence (D) of OHT.

Article Snippet: Stable transfected Rat1 cells expressing a low level of protein were stained with the E2F-1-, E2F-2-, or E2F-3-specific antibodies as described above and developed by using a TSA-Direct kit (NEN Life Science Products) according to the manufacturer’s suggestions.

Techniques: Infection, Expressing, Staining

Journal:

Article Title: CDC25A Phosphatase Is a Target of E2F and Is Required for Efficient E2F-Induced S Phase

doi:

Figure Lengend Snippet: Genes activated by E2F-2 and E2F-3. (A) RT-PCR was performed on RNA samples prepared from ERE2F-2 and ERE2F-3. The cells were grown in medium containing 0.1% serum for 48 h, and OHT was subsequently added for 4 h in the presence of CHX, CHX plus OHT, or OHT alone. (B) Summary of genes activated directly by E2F-2 and E2F-3 expression. ++, upregulation over 10-fold; +, upregulation between 5- and 10-fold; +/−, upregulation between 1- and 5-fold; −, no upregulation observed; ?, increased levels in serum-starved cells.

Article Snippet: Stable transfected Rat1 cells expressing a low level of protein were stained with the E2F-1-, E2F-2-, or E2F-3-specific antibodies as described above and developed by using a TSA-Direct kit (NEN Life Science Products) according to the manufacturer’s suggestions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal:

Article Title: CDC25A Phosphatase Is a Target of E2F and Is Required for Efficient E2F-Induced S Phase

doi:

Figure Lengend Snippet: Sequence and schematic representation of the human CDC25A promoter. (A) Nucleotide sequence of the 1,178-bp CDC25A promoter SacI fragment containing 755 nucleotides upstream of the transcription initiation site and 423 nucleotides of the 5′ untranslated region of the human CDC25A cDNA. Two E2F DNA binding sites are underlined. The transcription initiation site, which coincides with the longest cDNA clone isolated, is indicated with an arrow. (B) RNase protection assay. RNA was prepared from HeLa and U2OS cells and processed for RNase protection, using a 258-nucleotide probe containing 237 nucleotides of the CDC25A gene surrounding the putative transcription initiation site. The longest protected fragment, corresponding to approximately 120 nucleotides, is indicated with an arrow. The length of the probe is indicated to the right, and the number of nucleotides in a double-stranded DNA molecular size marker is indicated to the left. RNA has a slower mobility than DNA, and it is estimated to be 5 to 10% different from DNA. (C) Schematic representation of transcription factor binding sites in the 1,178-bp human CDC25A promoter. The transcription start site is depicted with an arrow. The two E2F sites are indicated, as are DNA consensus binding sites for AP-2, RBPJ-κ, bovine papillomavirus E2 (BPVE2), CCAAT-box binding proteins, Sp-1, and bHLH (E-box).

Article Snippet: Stable transfected Rat1 cells expressing a low level of protein were stained with the E2F-1-, E2F-2-, or E2F-3-specific antibodies as described above and developed by using a TSA-Direct kit (NEN Life Science Products) according to the manufacturer’s suggestions.

Techniques: Sequencing, Binding Assay, Isolation, Rnase Protection Assay, Marker

Journal:

Article Title: CDC25A Phosphatase Is a Target of E2F and Is Required for Efficient E2F-Induced S Phase

doi:

Figure Lengend Snippet: E2F-dependent cell cycle regulation of the CDC25A promoter. (A) Sequences of the putative E2F DNA binding sites in the CDC25A promoter. These sites were mutated to the indicated sequences by PCR–site-directed mutagenesis as described in Materials and Methods. (B) Both E2F DNA binding sites respond to E2F transactivation. Transfection of U2OS cells with CDC25A(−755/+423) luc (wild type) and mutants thereof, with (+) or without (−) pCMVE2F-1. The adjusted luciferase activities are the relative luciferase activities after normalization for the activity of cotransfected pCMVβ-gal. (C and D) The activity of the CDC25A promoter during the cell cycle. Rat1 cells transfected with CDC25A(−755/+422) luc wild type (wt), m1, or m2 were starved for 48 h and subsequently induced with serum. Samples were collected at different time points to evaluate the luciferase activity and the cell cycle profile as determined by FACS analysis. The adjusted luciferase activity in panel C indicates fold induction relative to the activity of the wild-type promoter at time 0 h, obtained by first normalizing the luciferase counts for the β-galactosidase activity of cotransfected pCMVβ-gal. In panel D are shown the percentages of S-phase cells for each time point. The data presented are representative of at least three different experiments.

Article Snippet: Stable transfected Rat1 cells expressing a low level of protein were stained with the E2F-1-, E2F-2-, or E2F-3-specific antibodies as described above and developed by using a TSA-Direct kit (NEN Life Science Products) according to the manufacturer’s suggestions.

Techniques: Binding Assay, Mutagenesis, Transfection, Luciferase, Activity Assay

Journal:

Article Title: CDC25A Phosphatase Is a Target of E2F and Is Required for Efficient E2F-Induced S Phase

doi:

Figure Lengend Snippet: CDC25A cooperates with cyclin E in inducing S phase and is required for efficient E2F-1-induced S-phase entry. (A) Cyclin E and CDC25A cooperate to induce S phase in serum-starved Rat1 fibroblasts. E2F-1, cyclin E, CDC25A, or cyclin E plus CDC25A expression vectors (20 ng/μl each) were injected into serum-starved Rat1 fibroblasts. pCMVEGFP (100 ng/μl) was coinjected as an injection marker. At 16 h after microinjection, the percentage of BrdU-positive microinjected cells was determined. GFP, green fluorescent protein. (B) CDC25A is necessary for efficient E2F-1-induced S-phase entry. Rat1 ERE2F-1 cells were incubated in DMEM containing 0.1% serum for 48 h. The indicated antibodies were then microinjected (300 ng/ml) along with rabbit IgG as a microinjection marker (2 μg/μl). At 3 h after microinjection, OHT was added (300 nM) to induce E2F-1 activity. At 12 h after the induction of E2F-1, cells were harvested and processed for immunofluorescence analysis, and the percentage of BrdU in microinjected cells was determined. rIgG, rabbit IgG; pept., peptide. (C) An example of an antibody microinjection experiment. The percentage of BrdU in injected cells is lower in samples injected with antibodies against CDC25A.

Article Snippet: Stable transfected Rat1 cells expressing a low level of protein were stained with the E2F-1-, E2F-2-, or E2F-3-specific antibodies as described above and developed by using a TSA-Direct kit (NEN Life Science Products) according to the manufacturer’s suggestions.

Techniques: Expressing, Injection, Marker, Incubation, Activity Assay, Immunofluorescence

Journal:

Article Title: CDC25A Phosphatase Is a Target of E2F and Is Required for Efficient E2F-Induced S Phase

doi:

Figure Lengend Snippet: Model for the central role of the E2F transcription factors in cell proliferation and apoptosis. The stimulation of resting cells with growth factors results in the induction of a signal cascade leading to the phosphorylation of the retinoblastoma protein (pRB) by D-type cyclins in association with CDK4 or CDK6, as well as the activation of the E2F transcription factors. The E2F transcription factors are essential for controlling several genes whose products are required for DNA replication, cell cycle progression, and/or growth. Deregulation of E2F activity as a result of upstream mutations in the pathway leads to premature entry into S phase of the cell cycle and—depending on the genetic status of the cell—to hyperproliferation or apoptosis. E2F-induced apoptosis occurs via p53-dependent and -independent pathways. One of the connections between the pRB and p53 pathways is provided by ARF, and the transactivation of ARF by the E2Fs may be involved in p53-dependent apoptosis. Presently we do not know which gene products are implicated in p53-independent E2F-induced apoptosis. PIGs, p53-induced genes; IGF-BP3, insulin growth factor-binding protein 3.

Article Snippet: Stable transfected Rat1 cells expressing a low level of protein were stained with the E2F-1-, E2F-2-, or E2F-3-specific antibodies as described above and developed by using a TSA-Direct kit (NEN Life Science Products) according to the manufacturer’s suggestions.

Techniques: Activation Assay, Activity Assay, Binding Assay