Journal: Oncogene

Article Title: AP2α controls the dynamic balance between miR-126&126* and miR-221&222 during melanoma progression

doi: 10.1038/onc.2015.357

Figure Lengend Snippet: AP2α targeting by miR-221&222. ( a ) WB analysis of AP2α with relative densitometric evaluation and miR-221 and -222 levels in normal melanocytes (NHEM) and melanoma cell lines. ( b ) Nucleotide pairing between AP2α 3′UTR and miR-221 and -222 is shown by bars. The seed sequence is indicated in bold, while lower case letters with asterisks represent the mutated nucleotides. ( c ) Luciferase reporter assay performed in 293FT (left) and Me1007 (right) cell lines by cotransfecting miR-221 or -222 in the presence of the AP2α 3′UTR. As controls, mutated 3′UTR sequences and a non-targeting oligomer were also included. ( d ) qRT–PCR (left) and representative WB analysis (right) of AP2α in Me1007 (top) and Me1402/R (bottom) control Tween vector vs miR-221- or 222-transduced cells. ( e ) Representative WB analysis of AP2α in anti-miR-221&222-transfected Me1402/R and A375M melanoma cells. GAPDH and actin are the internal controls. Non-targeting oligomers were also included as negative controls. Columns, mean±s.d. of at least three independent experiments. * P <0.05, ** P <0.01. LUC, luciferase; Mut, mutated; non targ, non-targeting; WB, western blot.

Article Snippet: The polyclonal rabbit anti -AP2α (Santa Cruz) was used as primary antibody at a dilution of 1:200 and the goat anti-rabbit IgG-HRP (656120; Life Technologies) at 1:3000 dilution as secondary one.

Techniques: Sequencing, Luciferase, Reporter Assay, Quantitative RT-PCR, Control, Plasmid Preparation, Transfection, Western Blot

Journal: Oncogene

Article Title: AP2α controls the dynamic balance between miR-126&126* and miR-221&222 during melanoma progression

doi: 10.1038/onc.2015.357

Figure Lengend Snippet: EGFL7/miR-126&126* promoter regulation. ( a ) Schematic illustration of the genomic region representing the promoter 1 of EGFL7. BS1-2, BS3, BS4 and BS5 indicate the AP2α BSs. ( b ) Promoter luciferase assays performed in the 293FT and Me1402/R cells transfected with AP2α. As control, the empty vector Tween was included. ( c ) Promoter luciferase assays performed in the 293FT and Me1402/R cells transfected with either Dsi-AP2α or Dsi-scr negative control. ( d ) As control of the APα BSs specificity, point mutations were inserted in the core-binding sequences and promoter luciferase assay performed. ( e ) Chromatin immunoprecipitation assay performed in Me1402/R cells with anti-AP2α antibodies and analyzed by semiquantitative PCR. Columns, mean±s.d. of at least three independent experiments. * P <0.05, ** P <0.01, *** P <0.001. Mut, mutated.

Article Snippet: The polyclonal rabbit anti -AP2α (Santa Cruz) was used as primary antibody at a dilution of 1:200 and the goat anti-rabbit IgG-HRP (656120; Life Technologies) at 1:3000 dilution as secondary one.

Techniques: Luciferase, Transfection, Control, Plasmid Preparation, Negative Control, Binding Assay, Chromatin Immunoprecipitation

Journal: Oncogene

Article Title: AP2α controls the dynamic balance between miR-126&126* and miR-221&222 during melanoma progression

doi: 10.1038/onc.2015.357

Figure Lengend Snippet: AP2α-dependent regulation of miR-126&126* and miR-221&222. qRT–PCR evaluations of ( a ) miR-126&126* (left), and miR-221&-222 (middle) in AP2α-overexpressing Me1402/R cell line and miR-126&126* after AP2α silencing (right). ( b ) miR-126&126* as a consequence of AP2α enforced expression or silencing in Me1007. ( c ) miR-126&126* in AP2α-transduced A375M cells. Samples were normalized on RNU6B levels. Columns, mean±s.d. of at least three independent experiments. ** P <0.01, *** P <0.001. ( d ) In situ hybridization of miR-221, miR-222 and miR-126 and immunohistochemistry of AP2α. Bar, 50 μm. Representative sections from one primary and one metastatic melanoma specimen are shown. Scrambled and RNU6B correspond to negative and positive controls, respectively. * P <0.05, ** P <0.01.

Article Snippet: The polyclonal rabbit anti -AP2α (Santa Cruz) was used as primary antibody at a dilution of 1:200 and the goat anti-rabbit IgG-HRP (656120; Life Technologies) at 1:3000 dilution as secondary one.

Techniques: Quantitative RT-PCR, Expressing, In Situ Hybridization, Immunohistochemistry

Journal: Oncogene

Article Title: AP2α controls the dynamic balance between miR-126&126* and miR-221&222 during melanoma progression

doi: 10.1038/onc.2015.357

Figure Lengend Snippet: AP2α expression is directly related with EGFL7 and miR-126&126* and inversely with miR-221&222. ( a ) Evaluation of EGFL7 in AP2α-transduced Me1402/R and A375M cells at mRNA (left) and protein (right) levels (** P <0.01). ( b ) Schematic depiction of miR-126&126* and miR-221&222 coregulatory pathways. The AP2α dependent activation of miR-126&126* and the consequential ADAM9 and MMP7-targeted downregulation prevent the pro-HB-EGF shedding in normal melanocytes (left). In melanoma the low levels of miR-126&126* unblock ADAM9 and MMP7 resulting in pro-HB-EGF shedding. This proteolytic cleavage originates the intracellular HB-EGF-C fragment that, entering the nucleus, binds and delocalizes the PLZF transcription factor, thus preventing its repressive function on miR-221&222 transcription. High amounts of miR-221&222 downregulate AP2α and consequently miR-126&126*, thereby contributing to close the circuitry maintaining advanced melanoma traits (right).

Article Snippet: The polyclonal rabbit anti -AP2α (Santa Cruz) was used as primary antibody at a dilution of 1:200 and the goat anti-rabbit IgG-HRP (656120; Life Technologies) at 1:3000 dilution as secondary one.

Techniques: Expressing, Activation Assay

Journal: Breast Cancer Research : BCR

Article Title: The combined immunodetection of AP-2α and YY1 transcription factors is associated with ERBB2 gene overexpression in primary breast tumors

doi: 10.1186/bcr1851

Figure Lengend Snippet: Sequences of small interfering RNAs and primers for reverse transcription-polymerase chain reaction

Article Snippet: Sections of breast biopsy specimens underwent immunoperoxidase staining using antibodies directed against AP-2α (1:100) (#39001; Active Motif, Carlsbad, CA, USA), against YY1 (1:50) (H-10; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), or against ERBB2 (1:250) (A0485; Dako A/S, Glostrup, Denmark).

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: The combined immunodetection of AP-2α and YY1 transcription factors is associated with ERBB2 gene overexpression in primary breast tumors

doi: 10.1186/bcr1851

Figure Lengend Snippet: Detection of activator protein 2 alpha (AP-2α), Yin Yang 1 (YY1), and ERBB2 by immunohistochemistry in breast tumors. (a) Case with low immunoreactivity for AP-2α. (b) Tumor sample expressing high level of AP-2α protein in more than 80% of the nuclei. (c) Tumor with low immunoreactivity for YY1. (d) Case expressing high level of YY1 protein in more than 80% of the nuclei. (e) Case with no ERBB2 membrane staining, scored as IHC 0. (f) Tumor with partial weak membrane staining, scored as IHC 1+. (g) Case with ERBB2 score of 2+. (h) Tumor with thick circumferential ERBB2 membrane staining, scored as IHC 3+.

Article Snippet: Sections of breast biopsy specimens underwent immunoperoxidase staining using antibodies directed against AP-2α (1:100) (#39001; Active Motif, Carlsbad, CA, USA), against YY1 (1:50) (H-10; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), or against ERBB2 (1:250) (A0485; Dako A/S, Glostrup, Denmark).

Techniques: Immunohistochemistry, Expressing, Membrane, Staining

Journal: Breast Cancer Research : BCR

Article Title: The combined immunodetection of AP-2α and YY1 transcription factors is associated with ERBB2 gene overexpression in primary breast tumors

doi: 10.1186/bcr1851

Figure Lengend Snippet: Associations between ERBB2 and AP-2α and/or YY1 levels determined by immunohistochemistry

Article Snippet: Sections of breast biopsy specimens underwent immunoperoxidase staining using antibodies directed against AP-2α (1:100) (#39001; Active Motif, Carlsbad, CA, USA), against YY1 (1:50) (H-10; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), or against ERBB2 (1:250) (A0485; Dako A/S, Glostrup, Denmark).

Techniques: Expressing

Journal: Breast Cancer Research : BCR

Article Title: The combined immunodetection of AP-2α and YY1 transcription factors is associated with ERBB2 gene overexpression in primary breast tumors

doi: 10.1186/bcr1851

Figure Lengend Snippet: Inverse associations between FISH results and AP-2α and/or YY1 levels in ERBB2 expressing group (1+, 2+, 3+)

Article Snippet: Sections of breast biopsy specimens underwent immunoperoxidase staining using antibodies directed against AP-2α (1:100) (#39001; Active Motif, Carlsbad, CA, USA), against YY1 (1:50) (H-10; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), or against ERBB2 (1:250) (A0485; Dako A/S, Glostrup, Denmark).

Techniques: Expressing

Journal: Breast Cancer Research : BCR

Article Title: The combined immunodetection of AP-2α and YY1 transcription factors is associated with ERBB2 gene overexpression in primary breast tumors

doi: 10.1186/bcr1851

Figure Lengend Snippet: Associations between ERBB2 and AP-2α and/or YY1 levels (immunohistochemistry) in the FISH-negative group

Article Snippet: Sections of breast biopsy specimens underwent immunoperoxidase staining using antibodies directed against AP-2α (1:100) (#39001; Active Motif, Carlsbad, CA, USA), against YY1 (1:50) (H-10; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), or against ERBB2 (1:250) (A0485; Dako A/S, Glostrup, Denmark).

Techniques: Immunohistochemistry, Expressing

Journal: Breast Cancer Research : BCR

Article Title: The combined immunodetection of AP-2α and YY1 transcription factors is associated with ERBB2 gene overexpression in primary breast tumors

doi: 10.1186/bcr1851

Figure Lengend Snippet: Suppression of AP-2α, AP-2γ, and YY1 expression downregulates ERBB2 transcript levels in BT-474 cells. (a) Cells were transfected on days 0 and 2 by small interfering RNAs (siRNAs) directed against AP-2α ( siAP-2α ) and/or AP-2γ ( siAP-2γ ) or against luciferase mRNA as control. Graphic shows real-time reverse transcription-polymerase chain reaction (RT-PCR) for AP-2α transcripts on total RNA extracted after 2, 3, or 4 days of treatment with indicated siRNAs. Results are presented as percentages of mRNA level as compared with control cells transfected with luciferase siRNA. Data are means ± standard deviation of three experiments. (b) RT-PCR for AP-2γ transcripts on total RNA. Cells were transfected like in (a). (c) Detection by Western blotting of AP-2α and AP-2γ levels at day 3. Ku70 protein served as control. Cells were transfected like in (a). (d) RT-PCR for ERBB2 transcripts on total RNA. Cells were transfected like in (a). (e) Cells were transfected on day 0 with 30 nM siRNA directed against YY1 ( siYY1 ) or 100 nM combined siRNAs against AP-2α and AP-2γ transcripts (siAP-2α+γ) or both as indicated. Control cells were transfected with a commercially available negative control siRNA ( control ). Proteins extracted after 24 hours of treatment were detected by Western blotting. Ku70 protein served as control for the protein amount charged on the gel. The percentage of ERBB2 protein level compared with transfection of control siRNA is shown in brackets. AP-2, activator protein 2; YY1, Yin Yang 1.

Article Snippet: Sections of breast biopsy specimens underwent immunoperoxidase staining using antibodies directed against AP-2α (1:100) (#39001; Active Motif, Carlsbad, CA, USA), against YY1 (1:50) (H-10; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), or against ERBB2 (1:250) (A0485; Dako A/S, Glostrup, Denmark).

Techniques: Expressing, Transfection, Luciferase, Control, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Western Blot, Negative Control

Journal: Breast Cancer Research : BCR

Article Title: The combined immunodetection of AP-2α and YY1 transcription factors is associated with ERBB2 gene overexpression in primary breast tumors

doi: 10.1186/bcr1851

Figure Lengend Snippet: Association between AP-2α and YY1 levels determined by immunohistochemistry on breast tumor specimens

Article Snippet: Sections of breast biopsy specimens underwent immunoperoxidase staining using antibodies directed against AP-2α (1:100) (#39001; Active Motif, Carlsbad, CA, USA), against YY1 (1:50) (H-10; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), or against ERBB2 (1:250) (A0485; Dako A/S, Glostrup, Denmark).

Techniques: Immunohistochemistry, Expressing