Journal: PLoS ONE

Article Title: MicroRNA-200c Modulates the Expression of MUC4 and MUC16 by Directly Targeting Their Coding Sequences in Human Pancreatic Cancer

doi: 10.1371/journal.pone.0073356

Figure Lengend Snippet: (A) S2.028 and (B) T3M-4-miR200c and respective vector control transfected cell lysates were separated by SDS-Agarose gel electrophoresis and subjected to western blot using an anti-MUC16 antibody (left panels). Band intensity was quantified by densitometry and analyzed using the imageJ program (right panels). Significant reductions of both high and low molecular weight MUC16 protein isoforms were observed in miR-200c expressing S2.028 (A) and T3M-4 cells (B) than compared to vector control cells. α-tubulin was used as a loading control.

Article Snippet: The membranes were incubated with the following primary antibodies: MUC1 (AR20.5-Mouse IgG, Quest PharmaTech Inc, Edmonton, Alberta, CA), MUC4 (8G7- Mouse IgG, kind gift from Dr. Surinder K Batra, Department of Biochemistry and Molecular biology, UNMC), MUC16 (AR9.6R333 Mouse IgG, Quest PharmaTech Inc, Edmonton, Alberta, CA) and α-tubulin (Mouse IgG, Developmental studies hybridoma bank, Iowa, USA) (1:1000 dilutions with 5% non-fat dry milk powder in TBS-T), overnight at room temperature.

Techniques: Plasmid Preparation, Control, Transfection, Agarose Gel Electrophoresis, Western Blot, Molecular Weight, Expressing

Journal: PLoS ONE

Article Title: MicroRNA-200c Modulates the Expression of MUC4 and MUC16 by Directly Targeting Their Coding Sequences in Human Pancreatic Cancer

doi: 10.1371/journal.pone.0073356

Figure Lengend Snippet: qRT-PCR analysis of MUC4 (A and B) and MUC16 (C and D) were carried out in vector control and miR-200c transfected S2.028 (A and C) and T3M-4 cells (B and D). The relative expression of MUC4 and MUC16 was evaluated by the 2 -ΔΔCt method using GAPDH as an internal control. The miR-200c expressing cells (S2.028 and T3M-4) showed significantly reduced levels of MUC4 mRNAs compared to vector control cells (A and B). Expression of MUC16 transcript was reduced in miR-200c expressing S2.028 and T3M-4 cells (C and D). All measurements were carried out in triplicate. The fold increase in transcript levels over vector control were expressed as Mean ± S.D. A p-value less than 0.05 was considered to be statistically significant.

Article Snippet: The membranes were incubated with the following primary antibodies: MUC1 (AR20.5-Mouse IgG, Quest PharmaTech Inc, Edmonton, Alberta, CA), MUC4 (8G7- Mouse IgG, kind gift from Dr. Surinder K Batra, Department of Biochemistry and Molecular biology, UNMC), MUC16 (AR9.6R333 Mouse IgG, Quest PharmaTech Inc, Edmonton, Alberta, CA) and α-tubulin (Mouse IgG, Developmental studies hybridoma bank, Iowa, USA) (1:1000 dilutions with 5% non-fat dry milk powder in TBS-T), overnight at room temperature.

Techniques: Quantitative RT-PCR, Plasmid Preparation, Control, Transfection, Expressing

Journal: PLoS ONE

Article Title: MicroRNA-200c Modulates the Expression of MUC4 and MUC16 by Directly Targeting Their Coding Sequences in Human Pancreatic Cancer

doi: 10.1371/journal.pone.0073356

Figure Lengend Snippet: Possible miR-200c targeting regions in MUC4 and MUC16 were identified by using the RegRNA MicroRNA target prediction web server ( http://regrna.mbc.nctu.edu.tw/index1.php ). A, RegRNA miRNA target prediction shows that miR-200c binds between base pairs 820-842 in the first exon of MUC4. B, In MUC16 mRNA, the miR-200c is predicted to bind nine different exons including E1, E3, E19, E39, E44, E49, E54, E64 and E73. The numbers indicate the region of mRNAs that interact with miR-200c.

Article Snippet: The membranes were incubated with the following primary antibodies: MUC1 (AR20.5-Mouse IgG, Quest PharmaTech Inc, Edmonton, Alberta, CA), MUC4 (8G7- Mouse IgG, kind gift from Dr. Surinder K Batra, Department of Biochemistry and Molecular biology, UNMC), MUC16 (AR9.6R333 Mouse IgG, Quest PharmaTech Inc, Edmonton, Alberta, CA) and α-tubulin (Mouse IgG, Developmental studies hybridoma bank, Iowa, USA) (1:1000 dilutions with 5% non-fat dry milk powder in TBS-T), overnight at room temperature.

Techniques:

Journal: PLoS ONE

Article Title: MicroRNA-200c Modulates the Expression of MUC4 and MUC16 by Directly Targeting Their Coding Sequences in Human Pancreatic Cancer

doi: 10.1371/journal.pone.0073356

Figure Lengend Snippet: (A) Wild type and mutated sequences of MUC4 and MUC16 were cloned into the pMIR-Luciferase vector (pMIR-MUC4 wt and MUC16 wt) and (pMIR-MUC4 mt and MUC16 mt) respectively. Vectors expressing pMIR-Luc, pMIR-MUC4 wt, pMIR-MUC16 wt, pMIR-MUC4 mt and pMIR-MUC16 mt were transfected into S2.028miR-200c (B and D) and T3M-4miR-200c cells (C and E) and luciferase activity was quantified 48 h after transfection. The results were expressed as relative luciferase activity (Mean ± SEM of three independent determinations). The p value was determined using Student’s t-test ( *** p<0.0005).

Article Snippet: The membranes were incubated with the following primary antibodies: MUC1 (AR20.5-Mouse IgG, Quest PharmaTech Inc, Edmonton, Alberta, CA), MUC4 (8G7- Mouse IgG, kind gift from Dr. Surinder K Batra, Department of Biochemistry and Molecular biology, UNMC), MUC16 (AR9.6R333 Mouse IgG, Quest PharmaTech Inc, Edmonton, Alberta, CA) and α-tubulin (Mouse IgG, Developmental studies hybridoma bank, Iowa, USA) (1:1000 dilutions with 5% non-fat dry milk powder in TBS-T), overnight at room temperature.

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

Journal: Biochemistry and Biophysics Reports

Article Title: Identification of ligand-selective peptidic ActRIIB-antagonists using phage display technology

doi: 10.1016/j.bbrep.2017.06.001

Figure Lengend Snippet: Binding activities and ligand selectivities of peptides as determined by SPR. Sensorgrams (left) and equilibrium plots (right) for the interaction of (A) AR2mini, (B) AR9, and (C) AR8 with immobilized ActRIIB-Fc. The top concentration was 100 μM. Serial 2-fold dilutions were performed.

Article Snippet: AR2mini, AR2mut, AR8, and AR9 were synthesized internally by standard 9-fluorenylmethyloxycarbonyl (F-moc)-based solid-phase peptide synthesis (SPPS) using the Syro Wave microwave-assisted peptide synthesizer (Biotage, Uppsala, Sweden) with disulfide bond formation after cleavage from the solid support, followed by purification by reversed-phase high-performance liquid chromatography (RP-HPLC).

Techniques: Binding Assay, Concentration Assay

Journal: Biochemistry and Biophysics Reports

Article Title: Identification of ligand-selective peptidic ActRIIB-antagonists using phage display technology

doi: 10.1016/j.bbrep.2017.06.001

Figure Lengend Snippet: IC 50 and K D values of peptides.

Article Snippet: AR2mini, AR2mut, AR8, and AR9 were synthesized internally by standard 9-fluorenylmethyloxycarbonyl (F-moc)-based solid-phase peptide synthesis (SPPS) using the Syro Wave microwave-assisted peptide synthesizer (Biotage, Uppsala, Sweden) with disulfide bond formation after cleavage from the solid support, followed by purification by reversed-phase high-performance liquid chromatography (RP-HPLC).

Techniques: Sequencing, Activity Assay

Journal: Biochemistry and Biophysics Reports

Article Title: Identification of ligand-selective peptidic ActRIIB-antagonists using phage display technology

doi: 10.1016/j.bbrep.2017.06.001

Figure Lengend Snippet: Binding competition assay. The binding activity of a biotinylated AR2mini-derivative peptide (2 µM) to ActRIIB-Fc was evaluated in the presence of non-labeled peptides (AR2mini-derivative, AR9, or AR8) at a concentration of 100 µM. Percent inhibition was calculated as described in the “Materials and methods” section.

Article Snippet: AR2mini, AR2mut, AR8, and AR9 were synthesized internally by standard 9-fluorenylmethyloxycarbonyl (F-moc)-based solid-phase peptide synthesis (SPPS) using the Syro Wave microwave-assisted peptide synthesizer (Biotage, Uppsala, Sweden) with disulfide bond formation after cleavage from the solid support, followed by purification by reversed-phase high-performance liquid chromatography (RP-HPLC).

Techniques: Binding Assay, Competitive Binding Assay, Activity Assay, Labeling, Concentration Assay, Inhibition