The human pancreatic cancer cell lines MiaPaPc-2, BxPC-3 and PANC-1 were purchased from the American Type Culture Collection. The S2VP10 cells, derived from the Suit-2 cells as reported30 (link), were originally provided by Dr. M. Hollingsworth (University of Nebraska). Cell line authentication was determined by short tandem repeat analysis (STR) performed by the American Type Culture Collection. OGT overexpression MiaPaPc-2 and BxPC-3 cells were generated by transfection of pCMV-flag-OGT (OGT, Addgene, #29760); and OGT knockdown S2VP10 and PANC-1 cells were generated by infected with lentiviruses containing a short hairpin RNA (shRNA) specific for OGT (shOGT, Addgene, #10878). PANC-1 cells with FADD knockdown were established as we previously described by shRNA targeting FADD (Open Biosystems)31 (link). All stable clones were selected by 2 μg/ml puromycin as we previously reported31 (link). Sequencing analysis was performed to confirm the correct sequence, and protein expression was validated by Western blot analysis. Apoptosis was induced by TRA-8, a DR5 agonist antibody that was generated as previously described32 (link); and determined by Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining kit (BD Biosciences, San Jose, CA) and analyzed by flow cytometry as we previously reported11 (link).
Protocol detail
Modulation of Pancreatic Cancer Cell Apoptosis
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Annexin v
Antibody
Apoptosis
Cell line authentication
Cell lines
Cells
Clones
Fadd
Flow cytometry
Fluorescein
Human
Isothiocyanate
Lentiviruses
Pancreatic cancer
Propidium iodide
Protein
Puromycin
Sequencing analysis
Short tandem repeat
Shrna
Tra 8
Transfection
Western blot analysis
Corresponding Organization : Birmingham VA Medical Center
Other organizations : Union Hospital, Huazhong University of Science and Technology
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Variable analysis
independent variables
- OGT overexpression in MiaPaPc-2 and BxPC-3 cells
- OGT knockdown in S2VP10 and PANC-1 cells
- FADD knockdown in PANC-1 cells
- Apoptosis induced by TRA-8 (DR5 agonist antibody)
- Cell lines used: MiaPaPc-2, BxPC-3, PANC-1, S2VP10
- Cell line authentication by short tandem repeat (STR) analysis
- Stable clones selected by 2 μg/ml puromycin
- Sequence analysis and protein expression validation by Western blot
- TRA-8, a DR5 agonist antibody, to induce apoptosis
- Not explicitly mentioned
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