Journal: FEBS letters

Article Title: MUC1 Negatively Regulates GalNAc Transferase 5 Expression in Pancreatic Cancer

doi: 10.1002/1873-3468.13532

Figure Lengend Snippet: MUC1 regulates glycosyltransfereases in PC cells. A. Schematic representation of MUC1 constructs. Full length MUC1-F construct including FLAG tag, Tandem Repeat domain, Transmembrane domain and cytoplasmic tail (top) and MUC1-CT construct excluding tandem repeat domain (bottom). B. Real-time RT PCR analysis of GALNT5, GALNT6, GALNT8 and ST6GalNAc-1 in S2–013 Neo and S2–013 MUC1-F cells. C. Real-time RT PCR analysis of GALNT5, GALNT6, GALNT8 and ST6GalNAc-1 in FG Neo and FG MUC1 KD cells. All values have been normalized to 18S rRNA and relative to respective control cells (S2–013 MUC1-F / S2–013 Neo and FG MUC1-KD / FG Neo). The values are expressed as average of three reactions ± s.e. mean (*, p<0.05; **, p<0.005; ns, non-significant). D. Immunoblot analysis of MUC1-CT and GalNAc-T5 expression in S2–013 Neo, S2–013 MUC1-CT, S2–013 MUC1-F, FG-Neo and FG-MUC1 KD cells. β-actin was used as a loading control. E. Immunoflourescence analysis of protein expression of MUC1 (red color) and GalNAc-T5 (green color) in S2–013 MUC1-F and S2–013 Neo cells. The images were taken at the magnification of 20X.

Article Snippet: The following primary antibodies were used to probe the membrane for the protein expressions: rabbit anti-GalNAc-T5 (Abgent, San Diego, CA, USA, 1:1000), hamster anti-MUC1-CT (CT2, Abcam, USA, 1:1000), and mouse anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA, 1:5000).

Techniques: Construct, FLAG-tag, Quantitative RT-PCR, Western Blot, Expressing

Journal: FEBS letters

Article Title: MUC1 Negatively Regulates GalNAc Transferase 5 Expression in Pancreatic Cancer

doi: 10.1002/1873-3468.13532

Figure Lengend Snippet: MUC1 suppresses GalNAc-T5 expression through complex with p53 and c-Jun. A. Immunoprecipitation of MUC1-CT with anti-MUC1-CT antibody (CT2) in S2–013 MUC1-CT cell lysate and probed with p53, c-Jun and MUC1-CT specific antibodies (lane 1: input, lane 2: IP with Armenian Hamster IgG, lane 3: IP with MUC1-CT). Membrane re-probed with anti-Armenian Hamster IgG-HRP was served as loading control. B. Protein expressions of GalNAc-T5 and MUC1-CT2 in S2–013 MUC1-CT cells treated with varying concentrations of Pifithrin-α hydrobromide (1, 2.5 and 5μM) for 24 hours. C. Immunohistochemical analysis of MUC1 (AR20.5 and CT2) and GalNAc-T5 expression in human normal (n=3) and primary pancreatic tumor tissues (n=25). Original magnification x 20. A p value of less than 0.05 was considered as stastically significant. D. Proposed mechanism of MUC1-CT mediated transcriptional down regulation of GALNT5 gene expression through complex with p53 and c-Jun and displacement of Sp1.

Article Snippet: The following primary antibodies were used to probe the membrane for the protein expressions: rabbit anti-GalNAc-T5 (Abgent, San Diego, CA, USA, 1:1000), hamster anti-MUC1-CT (CT2, Abcam, USA, 1:1000), and mouse anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA, 1:5000).

Techniques: Expressing, Immunoprecipitation, Immunohistochemical staining

Journal: International Journal of Nanomedicine

Article Title: An Isolation System to Collect High Quality and Purity Extracellular Vesicles from Serum

doi: 10.2147/IJN.S328325

Figure Lengend Snippet: Procedures and characteristics of SEC for separating EVs from serum. ( A ) Schematic overview of the experimental workflow. ( B ) Concentrations of particles and proteins in the SEC fractions were determined with nanoparticle tracking analysis (gray) and BCA (red), respectively. Data shows all 24 fractions. ( C ) SDS-PAGE was used to determine to directly visualize the relative presence of proteins in the all collected fractions, 15 μL of each fraction was mixed with 5 μL 4-fold concentrated reducing sample buffer, boiled for 5 minutes, and loaded on a 10% gradient gel. ( D ) The presence of the vesicle marker CD9, CD81, and the serum contaminant protein albumin and apolipoprotein marker apoA were determined in pooled EVs concentrates of fractions 8–13 and pooled Non-EVs concentrates fractions 14–24 with Western blot. ( E ) Droplets of fractions 9–11 and 20 were loaded onto grids, negative stained, and evaluated with transmission electron microscopy (TEM). Examples of EV-like structures (cup-shaped) are indicated by white arrows. Scale bars are 200 nm.

Article Snippet: CD9 was detected using rabbit anti-mouse CD9 (220642, Zen Bio, 1:1000); CD63 with rabbit anti-mouse CD63 (510953, Zen Bio, 1:1000); CD81 with rabbit anti-mouse CD81 (381296, Zen Bio, 1:1000); apoA1 with rabbit anti-mouse apoA1 (381145, Zen Bio, 1:1000); Albumin with rabbit anti-mouse Albumin (ab207327, Abcam, 1:1000).

Techniques: SDS Page, Marker, Western Blot, Staining, Transmission Assay, Electron Microscopy

Journal: International Journal of Nanomedicine

Article Title: An Isolation System to Collect High Quality and Purity Extracellular Vesicles from Serum

doi: 10.2147/IJN.S328325

Figure Lengend Snippet: Systematically compare the separation efficiency of SEC, UC, and TEI. ( A ) Schematic overview of the experimental workflow. ( B ) total protein was determined by BCA (expressed as mg/mL originating serum; mean ± SD, n = 3). ( C ) the concentration of particles as detected by NTA (particles/mL originating serum; mean ± SD, n = 3). ( D ) the ratio of particle to protein for SEC, UC, and TEI. ( E ) size distribution of particles detected in C (representative for n = 3). The particle size at peak optimum is indicated. ( F ) samples were analyzed by SDS-PAGE followed by Bio-Safe Coomassie G-250. ( G ) the same samples were analyzed by Western blotting for the presence of the EV markers CD9, CD81, CD63, and the serum contaminant protein albumin and apolipoprotein marker apoA. The experiment shown is representative of 3 independent experiments. ( F and G ) SEC, TEI, and UC samples are all separated from equivalent volumes of 1mL originating serum, and the final volume is 100 μL. UCx3 samples were separated from 3mL originating serum, and the final volume is 100 μL. All samples were loaded on equal volumes for electrophoresis. ( H ) The whole amount of TEM images of particles collected by SEC, UC, or TEI. Examples of EV-like structures (cup-shaped) are indicated by white arrows. Scale bars are 500 nm. Data were used for comparative analysis and presented as columns with bars representing means ± SD. Marks: *p≤0.05; **p≤0.01; ***p≤0.001 and non-significant differences were indicated using ns symbol.

Article Snippet: CD9 was detected using rabbit anti-mouse CD9 (220642, Zen Bio, 1:1000); CD63 with rabbit anti-mouse CD63 (510953, Zen Bio, 1:1000); CD81 with rabbit anti-mouse CD81 (381296, Zen Bio, 1:1000); apoA1 with rabbit anti-mouse apoA1 (381145, Zen Bio, 1:1000); Albumin with rabbit anti-mouse Albumin (ab207327, Abcam, 1:1000).

Techniques: Concentration Assay, SDS Page, Western Blot, Marker, Electrophoresis

Journal: International Journal of Nanomedicine

Article Title: An Isolation System to Collect High Quality and Purity Extracellular Vesicles from Serum

doi: 10.2147/IJN.S328325

Figure Lengend Snippet: Comparison of two different combination methods and SEC. ( A ) Schematic overview of the experimental workflow. ( B ) total protein was determined by BCA (expressed as mg/mL originating serum; mean ± SD, n = 3). ( C ) the concentration of particles as detected by NTA (particles/mL originating serum; mean ± SD, n = 3). ( D ) the ratio of particle to protein for SEC, SEC+UC, and SEC+TEI. ( E ) size distribution of particles detected in C (representative for n = 3). The particle size at peak optimum is indicated. ( F ) samples were analyzed by SDS-PAGE followed by Bio-Safe Coomassie G-250. ( G ) the same samples were analyzed by Western blotting for the presence of the EV markers CD9, CD81, CD63, and the serum contaminant protein albumin and apolipoprotein marker apoA. The experiment shown is representative of 3 independent experiments. ( F and G ) SEC, SEC+UC, and SEC+TEI samples are all separated from equivalent volumes of 1mL originating serum, and the final volume is 100 μL. SEC+UCx3 samples were separated from 3mL originating serum, and the final volume is 100μL. All samples were loaded on equal volumes for electrophoresis. ( H ) The whole amount of TEM images of particles collected by SEC, SEC+UC, or SEC+TEI. Examples of EV-like structures (cup-shaped) are indicated by white arrows. Scale bars are 500 nm. Data were used for comparative analysis and presented as columns with bars representing means ± SD. Marks: *p≤0.05; **p≤0.01; ***p ≤ 0.001 and non-significant differences were indicated using ns symbol.

Article Snippet: CD9 was detected using rabbit anti-mouse CD9 (220642, Zen Bio, 1:1000); CD63 with rabbit anti-mouse CD63 (510953, Zen Bio, 1:1000); CD81 with rabbit anti-mouse CD81 (381296, Zen Bio, 1:1000); apoA1 with rabbit anti-mouse apoA1 (381145, Zen Bio, 1:1000); Albumin with rabbit anti-mouse Albumin (ab207327, Abcam, 1:1000).

Techniques: Comparison, Concentration Assay, SDS Page, Western Blot, Marker, Electrophoresis