br nm emission filter Fold
505 nm emission filter. Fold-increase of caspase activity was deter-mined by comparison of treated samples to untreated samples. For the Caspase-Glo assay, cells were plated in 96-well dishes at 4500 cells/well and allowed to incubate overnight. After transfection (see below) cells were treated with 4 mM MET, 1 mM SIM, and MET +SIM and incubated for 48 h. This caspase assay uses a luminogenic cas-pase 3/7 substrate which contains DEVD. Changes in caspase activity as indicated by luminescence were determined by comparison of treated samples to untreated samples. Each experiment was run with a mini-mum of three samples and/or repeated three times.
Fig. 1. Metformin (MET) and simvastatin (SIM) individually inhibit growth dose-dependently in all three cell lines, and are synergistic in combination (MET+SIM). MTS assays of cell proliferation inhibition at 72 h post-treatment yielded combination indices (CI) of 0.57–0.77 (CI b 1 indicates synergy) for MET+SIM. Data are presented as mean ± SEM. Each experiment was performed in quadruplicate and repeated at least twice. r> 2.6. Western immunoblotting
The effects of SIM, MET, and the combination on signaling targets within the PI3K/Akt/mTOR pathway and apoptosis were evaluated with western immunoblotting. Primary antibodies specific for phos-phorylated AMPKα (Thr172)(40H9), phosphorylated S6, Bax, Bcl-2, beclin-1 (D40C5), Bim (C34C5) and HRP-conjugated Methylpiperidino pyrazole (C-11) were used. Cells were plated in 60 mm dishes as described above, incubated for 24 h, and then treated with 1–8 μM SIM, 4 mM MET, or MET+SIM for 24 and 48 h. Prior to harvesting the cells for molecular analysis, brightfield images of the cells were captured at 10× and 20× using a Nikon inverted photo microscope. Cell lysates were prepared using RIPA buffer (Santa Cruz Biotechnology). Protein concentrations were determined by the DC Protein Assay Kit (Bio-Rad Laboratories). Thirty μg of protein per lysate were denatured at 95 °C, separated by SDS-PAGE gel electrophoresis, and transferred to a PVDF membrane. Mem-branes were blocked with 5% nonfat dry milk in TBST for one hour then probed with the primary antibody overnight at 4 °C, incubated with the appropriate HRP-conjugated secondary antibody, and proteins detected using either Luminol (Santa Cruz Biotechnology) or SuperSignal West Femto Chemiluminscent Substrate (ThermoFisher). All primary and secondary antibodies were purchased from Cell Signal-ing with the exception of actin (Santa Cruz). Protein bands were imaged and quantified by densitometry using LabWorks Image Acquisition and Analysis Software (UVP, Upland, CA).
Bim is a Bcl-2 family pro-apoptotic protein that interacts with and activates BAX . To verify apoptosis as a primary molecular pathway underlying reduced cell viability and increased caspase activity follow-ing exposure to treatments, Bim siRNA was used. RL95-2 cells were seeded at 4500 cells/well in a clear or white 96-well plate respectively and allowed to adhere for 72 h. Cells were transfected and treated as fol-lows. For each experiment, 40 nM of Bim siRNA (GE Dharmacon, Lafa-yette, CO) or 40 nM of ON-TARGET control pool siRNA (GE Dharmacon) was incubated in OptiMEM (ThermoFisher) for 5 min in separate tubes. During this incubation, 2 additional tubes of OptiMEM and DharmaFECT I transfection reagent (GE Dharmacon) were incu-bated at 25 °C. Next, the tubes containing the siRNA and its correspond-ing tubes of DharmFECT I were combined and incubated for 30 min at 25 °C. The reagents were then added to the cells in OptiMEM and transfected for 48 h. Following transfection, the cells were treated for 48 h with 1 μM SIM, 4 mM MET, or the combination. Viability was assessed by MTS assay and caspase-3 activity was assessed by Caspase 3/7 Glo Assay, both described above.
2.8. Statistical analysis
Results are presented as mean ± SEM and were compared statisti-cally with p b 0.05 considered significant. Multiple group comparisons