Figure 3.

Synergistic cytotoxic effect of MJ and PI3K/Akt pathway inhibitors toward MCA-105 and SaOS-2 cells. (A) MCA-105 (left panels) and SaOS-2 (right panels) cells were seeded into 96-well plates at a concentration of 3 x 10³ cells per well and were allowed to adhere. After incubation, vehicles (ethanol and DMSO), wortmannin (40 µM), and MJ (0.5, 1 mM, 1 hour later) were added for 16 hours. The cytotoxic effect was measured using the XTT Cell Proliferation Kit as described in the Materials and Methods section. Values are means ± SD of at least three experiments. Statistical analysis: * Indicates that the experimentally measured combined cytotoxic effect is greater than the expected additive effect as calculated using the Bliss additivism model. (B) MCA-105 (left panels) and SaOS-2 (right panels) cells were seeded into six-well plates at a concentration of 0.7 x 10⁶ cells per well and were allowed to adhere. After the overnight incubations, cells were incubated for 16 additional hours in the presence of vehicle (DMSO) or wortmannin (40 µM). Total cell lysates were subjected to SDS-PAGE followed by immunoblot analysis using anti-pAkt and anti-Akt antibodies. (C) MCA-105 (left panels) and SaOS-2 (right panels) cells were seeded into 96-well plates at a concentration of 3 x 10³ cells per well and were allowed to adhere overnight. After the overnight incubations, cells were incubated for 16 additional hours in the presence of vehicles (ethanol and DMSO), Akt inhibitor (25 µM), and MJ (0.5 mM, added 1 hour later). The cytotoxic effect was measured using the XTT Cell Proliferation Kit as described in the Materials and Methods section. * Indicates that the experimentally measured combined cytotoxic effect is greater than the expected additive effect as calculated using the Bliss additivism model. (D) MCA-105 cells were seeded into six-well plates at a concentration of 0.7 x 10⁶ cells per well and were serum-starved (0.5% FCS) for the duration of the experiment. After the overnight incubations, cells were incubated for 16 additional hours in the presence of vehicles (ethanol and DMSO), Akt inhibitor (50 µM), and MJ (1 mM, added 1 hour later). Total cell lysates were subjected to SDS-PAGE followed by immunoblot analysis using anti-pAkt, anti-pGSK-3α/β, and anti-Akt antibodies.