Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Biopanning of a phage library to identify mesothelin-binding peptides. (A) Schematic of biopanning. A phage library was incubated with parental HEK 293T cells; unbound phage was then incubated with HEK 293T cells transfected with a green fluorescent protein (GFP)-tagged mesothelin (MSLN) expression vector (MSLN-HEK 293T). Cell-bound phage was eluted and amplified for the next round. Created with BioRender.com . (B) MSLN-HEK 293T cells 48 h after transfection (green) and parental HEK 293T cells stained with an anti-MSLN antibody (red) and diamidino-2-phenylindole (DAPI) (blue). (C) Phage titer enrichment across screening rounds. pfu, plaque-forming units. (D) Phage-cell-binding ELISA showing binding of individual phage clones to parental versus MSLN-expressing HEK 293T cells. OD, optical density.

Article Snippet: HEK 293T (human embryonic kidney) cells and AsPC-1, MIA PaCa-2, PANC-1, and BxPC-3 (human pancreatic cancer) cells were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Binding Assay, Incubation, Transfection, Expressing, Plasmid Preparation, Amplification, Staining, Enzyme-linked Immunosorbent Assay, Clone Assay

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Binding of MSLNpep to MSLN-expressing cells. (A) Parental and GFP-tagged (green) MSLN-expressing HEK 293T cells were incubated with tetramethylrhodamine-5-maleimide (TAMRA)-labeled MSLNpep or a control peptide (red) at 4 °C for 1 h and DAPI (blue). (B) Immunoblotting of MSLN expression in AsPC-1 and MIA PaCa-2 pancreatic tumor cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) AsPC-1 and MIA PaCa-2 cells were incubated with fluorescein isothiocyanate (FITC)–MSLNpep (green) at 4 °C for 1 h and an anti-MSLN antibody (red). Nuclei were stained with DAPI (blue), and images were merged. (D and E) AsPC-1 cells were treated with small interfering RNA (siRNA) against MSLN (100 and 200 nM) to knock down MSLN expression or with control (Ctrl) siRNA (200 nM). Cells were incubated with an anti-MSLN antibody (D) or 25 μM FITC–MSLNpep (E) and analyzed by flow cytometry. *** P < 0.001 by one-way ANOVA. (F) AsPC-1 cells were treated with MSLN-siRNA or Ctrl-siRNA (200 nM). After silencing, cells were incubated with 25 μM FITC–MSLNpep (green) at 4 °C for 1 h and an anti-MSLN antibody (red) (1:1,000). Nuclei were stained with DAPI (blue), and images were merged. Scale bars, 30 μm.

Article Snippet: HEK 293T (human embryonic kidney) cells and AsPC-1, MIA PaCa-2, PANC-1, and BxPC-3 (human pancreatic cancer) cells were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Binding Assay, Expressing, Incubation, Labeling, Control, Western Blot, Staining, Small Interfering RNA, Knockdown, Flow Cytometry

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Binding of MSLNpep to MSLN protein. (A) HEK 293T and AsPC-1 cell lysates (20, 200, and 400 μg) were incubated with biotin-labeled MSLNpep or control peptide (Ctrlpep) and pulled down with streptavidin-coated magnetic beads. Immunoblotting of the precipitates was performed using an anti-MSLN antibody. (B) Quantification of band intensity (%) for each pull-down relative to input in (A) using ImageJ software. Data are mean ± standard error from 3 independent experiments. *** P < 0.001 by one-way analysis of variance (ANOVA). (C) HEK 293 and AsPC-1 cells were incubated at 4 °C for 1 h with FITC-labeled MSLNpep together with either soluble or mature MSLN. Nuclei were stained with DAPI (blue), and images were merged. Scale bars, 30 μm. (D) HEK 293 and AsPC-1 cells were pretreated with an anti-MSLN antibody at room temperature for 1 h and incubated with FITC–MSLNpep (green) at 4 °C for 1 h. Nuclei were stained with DAPI (blue), and images were merged. Scale bars, 30 μm. (E) ELISA quantifying MSLNpep binding to soluble and mature MSLN. Data are mean ± standard error from 3 independent experiments. ** P < 0.01 by unpaired t test. (F) In silico modeling of MSLNpep binding to MSLN using the PEPFOLD program. MSLN, green; MSLNpep, blue.

Article Snippet: HEK 293T (human embryonic kidney) cells and AsPC-1, MIA PaCa-2, PANC-1, and BxPC-3 (human pancreatic cancer) cells were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Binding Assay, Incubation, Labeling, Control, Magnetic Beads, Western Blot, Software, Staining, Enzyme-linked Immunosorbent Assay, In Silico

Journal: Biomaterials Research

Article Title: Mesothelin-Binding Peptide Inhibits Cell Migration and Enables Targeted Delivery of a Mitochondrial-Membrane-Damaging Peptide to Pancreatic Tumors

doi: 10.34133/bmr.0361

Figure Lengend Snippet: Cytotoxicity of an MSLN-targeted mitochondrial-membrane-damaging peptide. (A) AsPC-1 cells were incubated at 37 °C for 2 h for 1 h with 25 μM FITC-labeled control peptide (Ctrlpep) or MSLNpep (green). Nuclei were stained with DAPI (blue), and images were merged. Scale bars, 30 μm. (B) Diagram of MSLNpep-KLA (L-form) and MSLNpep-kla (D-form). (C to E) AsPC-1 (C), MIA PaCa-2 (D), and HEK 293T (E) cells were incubated with phosphate-buffered saline (PBS), Ctrlpep-KLA, Ctrlpep-kla, MSLNpep-KLA, or MSLNpep-kla for 24 h. After incubation, cell viability was measured using Cell Counting Kit reagents. The half-maximal inhibitory concentration (IC50) values are presented as mean ± standard error from 3 independent experiments. Created with BioRender.com .

Article Snippet: HEK 293T (human embryonic kidney) cells and AsPC-1, MIA PaCa-2, PANC-1, and BxPC-3 (human pancreatic cancer) cells were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Membrane, Incubation, Labeling, Control, Staining, Saline, Cell Counting, Concentration Assay