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: Oncology Reports

Article Title: Combination of gemcitabine and erlotinib inhibits recurrent pancreatic cancer growth in mice via the JAK-STAT pathway

doi: 10.3892/or.2018.6198

Figure Lengend Snippet: The gemcitabine-erlotinib (E+G) combination group inhibits the activity of the JAK-STAT pathway, as well as the expression of downstream HIF-1α, cyclin D1 and p53. (A and B) The protein levels of phosphorylated STAT3 Tyr 705 (p-STAT3 Tyr 705 ), HIF-1α, p53, cyclin D1 in BxPC-3 and PANC-1 cells were analyzed by western blotting, respectively. (C and D) The protein expression of p-STAT3 Tyr 705 , HIF-1α, p53, cyclin D1 in BxPC-3 and PANC-1 cells was calculated using one-way ANOVA. *P<0.05 denotes the controls vs. the treatment groups. # P<0.05 denotes the monotherapy (E or G) vs. the combination (E+G) groups. (E) Phosphorylation levels of JAK1 (Tyr 1022 ), JAK2 (Tyr 221 ), JAK3(Tyr 981 ) and STAT3 (Tyr 701 ) in BxPC-3 and PANC-1 cells were analyzed by western blotting, respectively. (F and G) The protein intensity of phospho-JAK1 (p-JAK1), p-JAK2 and p-JAK3 in BxPC-3 and PANC-1 cells was calculated using one-way ANOVA. # P<0.01; *P<0.05. (H) The protein intensity of p-STAT1 in BxPC-3 and PANC-1 cells was assessed using one-way ANOVA. # P<0.01, *P<0.05. E, erlotinib; G, gemcitabine.

Article Snippet: The following primary antibodies were used: anti-JAK1 (rabbit polyclonal; cat. no. AF5012), phospho-JAK1 (Tyr 1022 ) (rabbit polyclonal; cat. no. AF2012), anti-JAK2 (rabbit polyclonal; cat. no. AF6022), phospho-JAK2 (Tyr 221 ) (rabbit polyclonal; cat. no. AF3023), anti-JAK3 (mouse monoclonal; cat. no. BF0256), phospho-JAK3 (Tyr 981 ) (rabbit polyclonal; cat. no. AF8160), phospho-STAT1 (Tyr 701 ) (rabbit polyclonal; cat. no. AF3300) and anti-STAT1 (rabbit polyclonal; cat. no. AF6300) were all purchased from Affinity Biosciences (Cambridge, UK); anti-STAT3 (rabbit monoclonal; cat. no. ab76315), and anti-pSTAT3 Try 705 (rabbit monoclonal; cat. no. ab68153) were obtained from Abcam (Cambridge, MA, USA); anti-HIF-1α (rabbit polyclonal; cat. no. BS3514) and anti-cyclin D1 (rabbit polyclonal; cat. no. BS6532) were purchased from Bioworld Technology Co., Ltd. (Nanjing, China); anti-p53 (rabbit, monoclonal; cat. no. 2527) was acquired from Cell Signaling Technology, Inc. (Danvers, MA, USA); while GAPDH was purchased from EarthOx Life Sciences (Millbrae, CA, USA).

Techniques: Activity Assay, Expressing, Western Blot

Journal: PLoS ONE

Article Title: The POZ-ZF Transcription Factor Kaiso (ZBTB33) Induces Inflammation and Progenitor Cell Differentiation in the Murine Intestine

doi: 10.1371/journal.pone.0074160

Figure Lengend Snippet: Cell proliferation was evaluated by Ki67 ( A ) and Cyclin D1 ( B ) staining. Both markers exhibited reduced staining in Kaiso Tg /+ mice compared to their Non-Tg siblings. Reduced CyclinD1 expression was also confirmed by immunoblot analysis of 3 different mice intestines ( C ). ** represents significance.

Article Snippet: Antibodies used were as follows: anti-Kaiso rabbit polyclonal antibody at a 1∶30,000 dilution, anti-Cyclin D1 rabbit polyclonal antibody (US Biological) at a 1∶5,000 dilution, anti-β-actin mouse monoclonal antibody (Sigma Aldrich) at a 1∶30,000 dilution.

Techniques: Staining, Expressing, Western Blot