Journal: Cells

Article Title: miR-338-3p Is Regulated by Estrogens through GPER in Breast Cancer Cells and Cancer-Associated Fibroblasts (CAFs)

doi: 10.3390/cells7110203

Figure Lengend Snippet: c-Fos and miR-338-3p are involved in Cyclin D1 regulation in SkBr3 cancer cells and CAFs. Luciferase activity of Cyclin D1 reporter gene in SkBr3 cancer cells ( A ) and CAFs ( B ) transfected for 8 h with a vector or a dominant-negative c-Fos construct (DN-Fos) before treatment with 100 nM of E2 and 100 nM G-1 for 18 h. Luciferase activity of Cyclin D1 reporter gene in SkBr3 cancer cells ( C ) and CAFs ( D ) transfected for 24 h with 25 nM miR-Ctrl or miR-338-3p mimic (miR-338-3p m) in combination or not with 50 nM miR-338-3p inhibitor (miR-338-3p i) before treatment for 18 h with 100 nM E2 or 100 nM G-1. The luciferase activity was normalized to the internal transfection control, values of cells receiving vehicle (-) were set as 1-fold induction upon which the activity obtained upon the indicated treatments was calculated. mRNA expression of Cyclin D1 in SkBr3 cells ( E ) and CAFs ( F ) transfected for 48 h with 25 nM miR-Ctrl or miR-338-3p mimic (miR-338-3p m) in combination or not with 50 nM miR-338-3p inhibitor (miR-338-3p i) before treatment for 8 h with 100 nM E2 or 100 nM G-1. Each column represents the mean ± SD of three independent experiments performed in triplicate. (*) indicates p < 0.05 for cells receiving treatments vs cells treated with vehicle (-).

Article Snippet: Equal amounts of protein extract were resolved on SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (Amersham Biosciences, Italy), probed overnight at 4 °C with antibodies against: c-Fos (E-8, sc-166940) and β-Actin (AC-15, sc-69879) (Santa Cruz Biotechnology, DBA, Italy), GPER (AB137479) (Abcam, Euroclone, Milan, Italy) and Cyclin D1 (Origene, DBA, Milan, Italy).

Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Dominant Negative Mutation, Construct, Expressing

Journal: Cells

Article Title: miR-338-3p Is Regulated by Estrogens through GPER in Breast Cancer Cells and Cancer-Associated Fibroblasts (CAFs)

doi: 10.3390/cells7110203

Figure Lengend Snippet: miR-338-3p prevents Cyclin D1 protein induction by E2 and G1 in SkBr3 cancer cells and CAFs. Cyclin D1 protein expression in SkBr3 cancer cells ( A , B ) and CAFs ( C , D ) transfected for 48 h with 25 nM miR-Ctrl or miR-338-3p mimic (miR-338-3p m) in combination or not with 50 nM miR-338-3p inhibitor (miR-338-3p i) before treatment for 12h with 100 nM E2 or 100 nM G-1. Side panels show densitometry analysis of the blots normalized to the loading control β-actin. (*) indicates p < 0.05 for cells receiving treatments vs cells treated with vehicle (-).

Article Snippet: Equal amounts of protein extract were resolved on SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (Amersham Biosciences, Italy), probed overnight at 4 °C with antibodies against: c-Fos (E-8, sc-166940) and β-Actin (AC-15, sc-69879) (Santa Cruz Biotechnology, DBA, Italy), GPER (AB137479) (Abcam, Euroclone, Milan, Italy) and Cyclin D1 (Origene, DBA, Milan, Italy).

Techniques: Expressing, Transfection

Journal: Molecular Cancer

Article Title: Krüppel-like factor 5 is a crucial mediator of intestinal tumorigenesis in mice harboring combined Apc Min and KRAS V 12 mutations

doi: 10.1186/1476-4598-9-63

Figure Lengend Snippet: Immunohistochemical and Western blot analyses of cyclin D1 in the normal-appearing small intestinal tissues of wild type and mutant mice . (A-D) Immunohistochemical staining of cyclin D1 in the normal-appearing small intestines of wild type (WT) and mutant mice. A small focus of adenomatous tissue is demarcated by the red broken lines in panel D. (E) Quantification of cyclin D1 staining intensities in all fours sections using the Metamorph image analysis software. N = 10; **, P < 0.01. (F) Western blot analyses of Klf5, β-catenin, and cyclin D1 in the small intestines of wild type and mutant mice. Actin serves as a loading control.

Article Snippet: Rabbit monoclonal cyclin D1 antibodies were purchased from Biocare Medical (Concord, CA) and used at 1:200 dilutions in immunohistochemical analyses and 1:2,500 dilutions for Western blot analysis.

Techniques: Immunohistochemical staining, Western Blot, Mutagenesis, Staining, Software, Control

Journal: Molecular Cancer

Article Title: Krüppel-like factor 5 is a crucial mediator of intestinal tumorigenesis in mice harboring combined Apc Min and KRAS V 12 mutations

doi: 10.1186/1476-4598-9-63

Figure Lengend Snippet: Immunohistochemical analyses of Ki67 in the normal-appearing small intestinal tissues of wild type and mutant mice . (A-D) Immunohistochemical staining of Ki67 in the normal-appearing small intestines of wild type (WT) and mutant mice. (E) Quantification of Ki67 cyclin D1 staining intensities in all fours sections using the Metamorph image analysis software. N = 10; **, P < 0.01.

Article Snippet: Rabbit monoclonal cyclin D1 antibodies were purchased from Biocare Medical (Concord, CA) and used at 1:200 dilutions in immunohistochemical analyses and 1:2,500 dilutions for Western blot analysis.

Techniques: Immunohistochemical staining, Mutagenesis, Staining, Software