Simian Virus 40 large T-antigen p53-inactivated human FTE cell lines FT190 and FT194 were obtained from Dr. Ronny Drapkin [15 (link)]. The FT190 and FT194 cell lines were cultured according to Karst et al [16 (link)] and treated with different concentrations of medroxyprogesterone acetate (Sigma; M1629), progesterone (Sigma; P8783), levonorgestrel (Sigma; N2260) and norethindrone (Sigma; N4128) for 72hrs on 6cm cell culture plates. At the end of treatment, 20–40μg of protein was used for standard western blotting analysis for cleaved caspase-3. To measure protein fold changes, densitometry values of cleaved caspase-3 normalized to actin protein bands were quantified using LabWorks software for both untreated and progestin-treated FTE cells. FTE cell viability, cytotoxicity and caspase activity were examined using ApoTox-Glo™ Triplex Assay (#G6321 Promega) according to the manufacturer’s instructions using 5000 cells/well on a 96-well white plate in phenol-free medium with different concentrations of MPA.
Protocol detail
Progestin-induced Apoptosis in FTE Cells
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Actin
Assay
Caspase
Caspase 3
Cell culture
Cell lines
Cell viability
Cells
Cytotoxicity
Densitometry
Human
Large t antigen
Levonorgestrel
Medroxyprogesterone acetate
Norethindrone
Phenol
Progesterone
Progestin
Promega
Protein
Protein changes
Simian virus 40
Western blotting analysis
White cells
Corresponding Organization : University of Chicago
Other organizations : Wake Forest University
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Variable analysis
independent variables
- Medroxyprogesterone acetate (Sigma; M1629)
- Progesterone (Sigma; P8783)
- Levonorgestrel (Sigma; N2260)
- Norethindrone (Sigma; N4128)
- Cleaved caspase-3 protein levels (measured by Western blotting)
- FTE cell viability
- FTE cell cytotoxicity
- FTE cell caspase activity (measured by ApoTox-Glo™ Triplex Assay)
- FTE cell lines FT190 and FT194
- Protein amount (20–40μg) used for Western blotting
- Cell density (5000 cells/well) used for ApoTox-Glo™ Triplex Assay
- Phenol-free medium used for ApoTox-Glo™ Triplex Assay
- Untreated FTE cells (negative control)
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