Journal: Cancer Research

Article Title: The Myc–miR-17∼92 Axis Blunts TGFβ Signaling and Production of Multiple TGFβ-Dependent Antiangiogenic Factors

doi: 10.1158/0008-5472.can-10-2412

Figure Lengend Snippet: Figure 1. Clusterin inhibits tumor growth and angiogenesis. A, growth of subcutaneous tumors derived from RasMyc cells transduced with a retrovirus expressing murine clusterin and/or the puromycin resistance gene. Inset, immunoblot of clusterin levels in Ras and RasMyc colonocytes. B, growth of subcutaneous tumors derived from HCT116 cells transduced with an empty vector or the retrovirus expressing human clusterin. Regression lines represent average rates of growth. The P value refers to the difference in regression coefficients. Inset, immunoblot of clusterin levels in transduced cells. C, in vivo bioluminescent imaging of representative animals from the experiment in B. Mice were photographed at days 3 and 21 after injection. No less than five animals per group were used in all these experiments. D, immunohistochemical staining of Ki-Ras and Ki-Ras/Myc tumors with an anti-clusterin antibody. Cytoplasmic staining for clusterin is depicted in brown whereas nuclei are counterstained in blue. E, alignment of the second TSR of murine thrombospondin-1 with the C9 protein and clusterin. F, hemoglobin content of Matrigel pellets 7 d after injection. Matrigels were admixed with p53-null mouse colonocytes transduced with either empty vector (blue bar) or clusterin retrovirus (red bar) and injected s.c. into syngeneic host animals. G, H&E staining of RasMyc/vector (top) and RasMyc/clusterin (bottom) tumor sections. Perfused blood vessels contain numerous RBC, which could be clearly seen under higher magnification (inset). The scatter plot represents densities of perfused blood vessels in four individual tumors. H, immunohistochemical staining of control (top) and clusterin-overexpressing (bottom) HCT116 tumor sections with antibody recognizing the endothelial cell surface antigen CD31. The scatter plot represents the densities of CD31-positive blood vessels in four individual tumors.

Article Snippet: For thrombospondin-1 expression is, either cell lysates or conditioned media were used. ranes were probed with antibodies to clusterin, , TGFBR2 (Santa Cruz Biotechnology and Abcam), 4 (Santa Cruz Biotechnology), Smad2 and Smad3 ogen), phosphorylated Smad3 (Cell Signaling), and (Ab-11, Lab Vision) according to the recommendaof the manufacturer.

Techniques: Derivative Assay, Transduction, Expressing, Western Blot, Plasmid Preparation, In Vivo, Imaging, Injection, Immunohistochemical staining, Staining, Control

Journal: Cancer Research

Article Title: The Myc–miR-17∼92 Axis Blunts TGFβ Signaling and Production of Multiple TGFβ-Dependent Antiangiogenic Factors

doi: 10.1158/0008-5472.can-10-2412

Figure Lengend Snippet: Figure 2. Clusterin is regulated by miR-17~92 via the TGFβ pathway. A, immunoblotting analysis of clusterin expression levels in the following cell lines. Left, Ras-only mouse colonocytes transduced with either empty vector (Ras/Puro) or the miR-17~92–encoding retrovirus (Ras/miR-17~92). Right, RasMyc cells transfected with scrambled or anti–miR-17~92 2′-O-methyl oligoribonucleotides. B, changes in expression levels of thrombospondin-1 (THBS1) and clusterin mRNAs in HCT116 Dicerhypo (left) and A172 (right) cells after transfection with the indicated microRNA mimics. mRNA levels in HCT116 and A172 cells were profiled using Affymetrix microarrays and qPCR as described in Materials and Methods. C, activation of TGFβ signaling in Ras colonocytes. Ras cells treated with vehicle or 10 ng/mL of TGFβ1 for 30 min were analyzed by immunoblotting for phosphorylated Smad3 (pSmad3) and total Smad3. D, measurement of TGFβ effects on TSR proteins. Ras cells were treated with increasing doses of TGFβ1 for 48 h and lysates were immunoblotted for clusterin and CTGF proteins. Tsp-1 was detected in conditioned medium. E, immunoblotting analysis of TSR proteins in Ras/vector, Ras/miR-17~92 or c-Myc cells cultured in the absence or presence of TGFβ1 (5 ng/mL) for 48 h. F, CLU mRNA levels in Ras/vector and Ras/17~92 cells before and after (24 h) stimulation with TGFβ, as measured by qPCR. Expression levels of CLU are adjusted to those of glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: For thrombospondin-1 expression is, either cell lysates or conditioned media were used. ranes were probed with antibodies to clusterin, , TGFBR2 (Santa Cruz Biotechnology and Abcam), 4 (Santa Cruz Biotechnology), Smad2 and Smad3 ogen), phosphorylated Smad3 (Cell Signaling), and (Ab-11, Lab Vision) according to the recommendaof the manufacturer.

Techniques: Western Blot, Expressing, Transduction, Plasmid Preparation, Transfection, Activation Assay, Cell Culture

Journal: Cancer Research

Article Title: The Myc–miR-17∼92 Axis Blunts TGFβ Signaling and Production of Multiple TGFβ-Dependent Antiangiogenic Factors

doi: 10.1158/0008-5472.can-10-2412

Figure Lengend Snippet: Figure 3. miR-17~92 targets endogenous TGFβ receptor II. A, luciferase sensor assay. Constructs tested were psiCHECK-2 derivatives containing a single miR-17/20a binding site from TGFBR2 3′-UTR in either wild-type (wt) or seed-mutated (mut) conformation. Cells were additionally cotransfected with miR-17 or control mimic. Results are expressed as ratios of renilla to firefly luciferase, the latter being constitutively expressed from the same vector and serving as a control for transfection efficiency. Sequence alignment corresponds to positions 268 to 274 of TGFBR2 3′-UTR and mature hsa-miR-17. Arrows indicate mutated nucleotides. B, expression levels of TGFBR2 mRNA in DLD1 Dicerhypo cells 10 h after transfection with microRNA mimics (25 nmol/L). mRNA levels were quantified by microarray. C, immunoblotting analysis of TGFBR2 and clusterin expression levels in Ras cells transfected with 10 nmol/L of nontargeting siRNA pool or siRNA pool targeting mouse TGFBR2. D and E, immunoblotting analysis of TGFBR2 and Smad3 in Ras cells 20 h after transfection with microRNA mimics (25 nmol/L). Bottom, quantitations of Western blots above.

Article Snippet: For thrombospondin-1 expression is, either cell lysates or conditioned media were used. ranes were probed with antibodies to clusterin, , TGFBR2 (Santa Cruz Biotechnology and Abcam), 4 (Santa Cruz Biotechnology), Smad2 and Smad3 ogen), phosphorylated Smad3 (Cell Signaling), and (Ab-11, Lab Vision) according to the recommendaof the manufacturer.

Techniques: Luciferase, Construct, Binding Assay, Control, Plasmid Preparation, Transfection, Sequencing, Expressing, Microarray, Western Blot

Journal: Leukemia

Article Title: Hypoxia-microRNA-16 downregulation induces VEGF expression in anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphomas.

doi: 10.1038/leu.2011.168

Figure Lengend Snippet: Figure 4 In vivo repression of tumor growth and angiogenesis by miR-16 gain of function. TPM3-ALK MEF cells, transfected with pre-miR-16 (miR-16) or scrambled pre-miRNA control (Sc), were injected subcutaneously into five female Balb/c nude mice for each tested condition. (a) Tumor volume was measured every 2 days using callipers. (b) Macroscopic observation of tumor-bearing mice 27 days post subcutaneous injection. (c) Tumor weight at necropsy. (d) ELISA assays were performed on mice sera to measure VEGF levels. (c, d) Bars represent SD and asterisks depict statistically significant differences compared with scrambled control (*Po0.05, **Po0.01 and ***Po0.001; Student’s t-test). (e) Microvessel density was evaluated by CD34 immunoperoxidase staining 27 days post subcutaneous injection. (-) collapsed vessel, (*) open lumen vessel. Original magnification 400 0.85.

Article Snippet: VEGF and HIF1a immunohistochemistry Sections (5mM) were cut from each tissue microarray, deparaffinized, subjected to heat antigen retrieval and stained with rabbit polyclonal anti-VEGF antibody (A20, 1/40 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-HIF-1a antibody (clone H1a67, 1/100, Novus Biologicals, Cambridge, UK).

Techniques: In Vivo, Transfection, Control, Injection, Enzyme-linked Immunosorbent Assay, Immunoperoxidase Staining

Journal: Leukemia

Article Title: Hypoxia-microRNA-16 downregulation induces VEGF expression in anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphomas.

doi: 10.1038/leu.2011.168

Figure Lengend Snippet: Figure 6 Inverse correlation between VEGF expression and relative amount of endogenous miR-16 in ALK-positive ALCL. (a) Immuno- histochemistry (immunoperoxidase staining, original magnification, 400 0.85) and RT-qPCR were performed to determine VEGF and miR-16 expression in 8 human ALK þ lymphomas (ALCL-10, -21, -22, -25, -27, -32, -35 and -39). (b) Pearson’s correlation test in 16 ALK þ ALCL. The percentage of tumoral cells expressing VEGF was graded as negative (0); 1: o50%, 2: 50–75% and 3: 475%.

Article Snippet: VEGF and HIF1a immunohistochemistry Sections (5mM) were cut from each tissue microarray, deparaffinized, subjected to heat antigen retrieval and stained with rabbit polyclonal anti-VEGF antibody (A20, 1/40 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-HIF-1a antibody (clone H1a67, 1/100, Novus Biologicals, Cambridge, UK).

Techniques: Expressing, Immunohistochemistry, Immunoperoxidase Staining, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: ADP-ribosylation factor 6 expression increase in oesophageal adenocarcinoma suggests a potential biomarker role for it

doi: 10.1371/journal.pone.0263845

Figure Lengend Snippet: (A) Alignment of ARF6 primer sequence with ARF6 gene sequence to estimate the ARF6 amplicon size. B) PCR products showing the specificity of ARF6 primers. (B) Agarose gel electrophoresis of PCR products obtained using the ARF6 primers with either ARF6/pEGFP-N1 or Arf1/pEGFP-N1 plasmid as a template (ladder: 1000 bp; 800 bp; 600 bp; 400 bp; 200bp). Cycle threshold values were obtained by performing ARF6 RT-qPCR using a dilution series of ARF6 encoding plasmid DNA (C) or MDM-MB-231 cDNA (D).

Article Snippet: The tissue microarray (Biomax, US; Cat. No. ES8011a), containing 35 cases of EAC and 5 healthy tissues in duplicate, were incubated in CC1 retrieval buffer (pH8-8.5) for 40 minutes, previously validated anti-ARF6 3A-1 mouse monoclonal antibody (Santa Cruz Biotech., US) at a dilution of 1:150 was applied and incubated at 36°C for 40min [ , – ].

Techniques: Sequencing, Amplification, Agarose Gel Electrophoresis, Plasmid Preparation, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: ADP-ribosylation factor 6 expression increase in oesophageal adenocarcinoma suggests a potential biomarker role for it

doi: 10.1371/journal.pone.0263845

Figure Lengend Snippet: The expression of ARF6 mRNA in healthy tissues relative to that in the adrenal gland (the lowest expressing tissue). Data is shown using a box plot with whiskers showing + SD.

Article Snippet: The tissue microarray (Biomax, US; Cat. No. ES8011a), containing 35 cases of EAC and 5 healthy tissues in duplicate, were incubated in CC1 retrieval buffer (pH8-8.5) for 40 minutes, previously validated anti-ARF6 3A-1 mouse monoclonal antibody (Santa Cruz Biotech., US) at a dilution of 1:150 was applied and incubated at 36°C for 40min [ , – ].

Techniques: Expressing

Journal: PLoS ONE

Article Title: ADP-ribosylation factor 6 expression increase in oesophageal adenocarcinoma suggests a potential biomarker role for it

doi: 10.1371/journal.pone.0263845

Figure Lengend Snippet: Relative folds change in expression of ARF6 mRNA in cancerous tissues when compared to that in healthy corresponding tissues. Data is shown using a box plot with whiskers showing + SD.

Article Snippet: The tissue microarray (Biomax, US; Cat. No. ES8011a), containing 35 cases of EAC and 5 healthy tissues in duplicate, were incubated in CC1 retrieval buffer (pH8-8.5) for 40 minutes, previously validated anti-ARF6 3A-1 mouse monoclonal antibody (Santa Cruz Biotech., US) at a dilution of 1:150 was applied and incubated at 36°C for 40min [ , – ].

Techniques: Expressing

Journal: PLoS ONE

Article Title: ADP-ribosylation factor 6 expression increase in oesophageal adenocarcinoma suggests a potential biomarker role for it

doi: 10.1371/journal.pone.0263845

Figure Lengend Snippet: (A) Cancer stage-dependent expression of ARF6 mRNA in the oesophagus. (B) Immunostaining of EAC TMA was performed in a benchmark Ultra IHC staining module with an anti-ARF6 antibody. (C) The cores are displayed as 10x magnification and the insets were enlarged to 40x magnification. (D) Pooled ARF6 expression score between healthy and cancer specimens. (D) The cancer specimens were then separated into grades. Each core was scored based on the total sum of proportional of epithelial cells stained (score of 0 to +5) and the intensity of the staining (0 = none, +1 = weak, +2 = moderate and 3+ = strong). Scale bar 100μm.

Article Snippet: The tissue microarray (Biomax, US; Cat. No. ES8011a), containing 35 cases of EAC and 5 healthy tissues in duplicate, were incubated in CC1 retrieval buffer (pH8-8.5) for 40 minutes, previously validated anti-ARF6 3A-1 mouse monoclonal antibody (Santa Cruz Biotech., US) at a dilution of 1:150 was applied and incubated at 36°C for 40min [ , – ].

Techniques: Expressing, Immunostaining, Immunohistochemistry, Staining