Journal: Cancer letters

Article Title: p-21 activated kinase 4 (PAK4) maintains stem cell-like phenotypes in pancreatic cancer cells through activation of STAT3 signaling

doi: 10.1016/j.canlet.2015.10.028

Figure Lengend Snippet: PAK4 induces transcriptional activity of STAT3 in human PC cells by promoting its nuclear translocation. The cells were co-transfected with Cignal STAT3 reporter (mixture of STAT3-responsive firefly luciferase reporter and Renilla luciferase plasmids) and negative control plasmids. (A) After 48 h, cells were harvested in passive lysis buffer and luciferase activity was assessed using a dual-luciferase assay system. Data are presented as normalized relative luciferase activity (mean ± SD; n = 3, *p < 0.05). Total, nuclear and cytoplasmic extracts were prepared and (B) cellular localization of STAT3 and (C) expression level of pSTAT3 or total were analyzed by immunoblot analysis. Lamin A (for nuclear fraction), α-tubulin (for cytoplasmic fraction) and β-actin (for total fraction) were used as loading controls.

Article Snippet: Antibodies targeting Lamin A, α-tubulin (mouse mono-clonal) and respective anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies were procured from Santa Cruz Biotechnology (Santa Cruz, CA). β-actin (mouse monoclonal) antibody was purchased from Sigma-Aldrich (St. Louis MO).

Techniques: Activity Assay, Translocation Assay, Transfection, Luciferase, Negative Control, Lysis, Expressing, Western Blot

Journal: Cancer letters

Article Title: p-21 activated kinase 4 (PAK4) maintains stem cell-like phenotypes in pancreatic cancer cells through activation of STAT3 signaling

doi: 10.1016/j.canlet.2015.10.028

Figure Lengend Snippet: Forced expression of STAT3 active mutant restores transcriptional activity and localization of STAT3 in PAK4-silenced PC cells. In this approach, PAK4 silenced cells were transfected with constitutively active STAT3 mutant (EF.STAT3C.Ubc.GFP) or empty vector (FUGW) and (A) after 48 h transfection, cells were again transfected with STAT3 luciferase promoter-reporter plasmid and transcriptional activity of STAT3 was examined. Data are presented as normalized relative luciferase activity (mean ± SD; n = 3, *p < 0.05). (B) Nuclear and cytoplasmic fractions were prepared after 24 h of transfection and expression level of STAT3 was examined by immunoblot analysis. Lamin A (for nuclear fraction), α-tubulin (for cytoplasmic fraction) and β-actin (for total fraction) were used as loading controls.

Article Snippet: Antibodies targeting Lamin A, α-tubulin (mouse mono-clonal) and respective anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies were procured from Santa Cruz Biotechnology (Santa Cruz, CA). β-actin (mouse monoclonal) antibody was purchased from Sigma-Aldrich (St. Louis MO).

Techniques: Expressing, Mutagenesis, Activity Assay, Transfection, Plasmid Preparation, Luciferase, Western Blot

Journal: Nutrients

Article Title: Metabolism of Caprine Milk Carbohydrates by Probiotic Bacteria and Caco-2:HT29–MTX Epithelial Co-Cultures and Their Impact on Intestinal Barrier Integrity

doi: 10.3390/nu10070949

Figure Lengend Snippet: Fold change of MUC4 , MUC2 , and MUC5AC mRNA from Caco-2:HT29–MTX (90:10) co-cultures after 3 h incubation with ( A ) probiotic bacteria or a carbohydrate fraction (CF) relative to untreated co-cultures; ( B ) bacteria/CF combinations compared to untreated monolayers; ( C ) bacteria/CF combinations compared to monolayers incubated with CF; and ( D ) bacteria/CF combinations relative to bacteria only controls. Data are expressed as the mean fold change (± SEM) of three replicates across three independent experiments; n = 3 A statistically significant difference in fold change at ±1.5 is indicated by * ( P < 0.05). LGG = L. rhamnosus GG; HN001 = L. rhamnosus HN001; Lp299v = L. plantarum 299v and LcS = L. casei Shirota.

Article Snippet: The abundance of mucin proteins in cell lysate (CL) and spent media (SM) was determined by indirect enzyme linked immunosorbent assay (indirect ELISA) using MUC2 mouse mono-clonal antibody (clone 4A4, 1:250; Creative Biomart, New York, NY, USA), MUC4 mouse mono-clonal antibody (clone 5B12, 1:500; Abnova, Taipei, Taiwan) or MUC5AC mouse mono-clonal antibody (clone 2H7, 1:250; Abnova) and horseradish peroxidase-rabbit anti-mouse immunoglobulin G conjugate (Abcam, Cambridge, UK; 1:5000 dilution for MUC2 and MUC5AC and 1:10,000 dilution for MUC4) as described previously [ ].

Techniques: Incubation, Bacteria

Journal: Nutrients

Article Title: Metabolism of Caprine Milk Carbohydrates by Probiotic Bacteria and Caco-2:HT29–MTX Epithelial Co-Cultures and Their Impact on Intestinal Barrier Integrity

doi: 10.3390/nu10070949

Figure Lengend Snippet: The abundance of MUC4, MUC2 and MUC5AC mucin protein from Caco-2:HT29–MTX (90:10) co-cultures after 3 h incubation with bacteria or a carbohydrate fraction (CF) from caprine milk either alone or in combination. Results are expressed as the mean abundance (±SEM); n = 3. * =significantly different ( P < 0.05) compared to untreated co-cultures, and z = significantly different ( P < 0.05) compared to co-cultures incubated with the respective bacteria only. UNT = untreated; LGG = L. rhamnosus GG; HN001 = L. rhamnosus HN001; Lp299v = L. plantarum 299v; and LcS = L. casei Shirota.

Article Snippet: The abundance of mucin proteins in cell lysate (CL) and spent media (SM) was determined by indirect enzyme linked immunosorbent assay (indirect ELISA) using MUC2 mouse mono-clonal antibody (clone 4A4, 1:250; Creative Biomart, New York, NY, USA), MUC4 mouse mono-clonal antibody (clone 5B12, 1:500; Abnova, Taipei, Taiwan) or MUC5AC mouse mono-clonal antibody (clone 2H7, 1:250; Abnova) and horseradish peroxidase-rabbit anti-mouse immunoglobulin G conjugate (Abcam, Cambridge, UK; 1:5000 dilution for MUC2 and MUC5AC and 1:10,000 dilution for MUC4) as described previously [ ].

Techniques: Incubation, Bacteria