Journal: Theranostics

Article Title: Vitamin C kills thyroid cancer cells through ROS-dependent inhibition of MAPK/ERK and PI3K/AKT pathways via distinct mechanisms

doi: 10.7150/thno.35219

Figure Lengend Snippet: Induction of AKT ubiquitination and proteasome degradation by vitamin C via a ROS-dependent pathway. Western blot analysis of total AKT in 8305C ( A ) and C643 ( B ) cells with vitamin C (VC) and cycloheximide (CHX) treatment alone or in combination for the indicated time points with β-Actin as a loading control. ( C ) 8305C and C643 cells was treated with 2 mM VC for 2 h after pretreatment of 25 μM MG132 for the indicated time points, and western blot analysis was then used to evaluate AKT expression. β-Actin was used as a loading control. ( D ) 8305C and C643 cells was pretreated with 25 μM MG132 for 2 h, and VC was then added for another 2 h before harvesting. Lysates were incubated with anti-AKT antibody for 4 h and then conjugated with agarose. Bounding proteins were analyzed by immunoblot with anti-ubiquitin (Ub) antibody. Input samples were taken prior to immunoprecipitation and immunoblotted with the indicated antibodies. Data were presented as mean ± SD. **, P <0.01; ***, P <0.001.

Article Snippet: To assess the effect of vitamin C on AKT protein stability, cells were incubated with 200 µg/mL CHX (MP Biomedicals, Santa Ana, CA) for 2 h to inhibit de novo protein synthesis, and then treated with 2 mM vitamin C. At the indicated time points, cell lysates were harvested and subjected to Western blot analysis using the indicated antibodies.

Techniques: Western Blot, Expressing, Incubation, Immunoprecipitation