Figure 2

ROS are involved in oridonin-induced RARα stabilization. (A) NB4 cells were treated with the indicated concentrations of oridonin for 12 h (left panel) or with 10 μM oridonin for the indicated times (right panel), followed by detection of ROS levels by flow cytometry. The symbols * and # represent P values less than 0.05 and 0.01, respectively. (B) After pretreatment with or without 2 mM NAC for 1 h, NB4 cells were incubated with 10 μM oridonin for 12 h, followed by detection of ROS levels by flow cytometry (left panel) and western blot detection for RARα protein with β-actin as loading control (right panel). The symbol # represents a P value less than 0.01. (C) Primary AML cells were treated as NB4 cells in the panel B, and the levels of RARα protein were measured. (D, E) NB4 cells were treated with the indicated concentrations of H₂O₂ for 2 h (D) or with 5 μM H₂O₂ for the indicated times (E), then the level of RARα protein was assessed. (F) NB4 cells were treated with 5 μM H₂O₂ for the indicated times, and RARα mRNA levels were evaluated by real-time RT-PCR. (G) NB4 cells were incubated with 5 μg/mL CHX alone or in combination with 5 μM H₂O₂, followed by western blot detection of RARα protein with β-actin as loading control. (H) NB4 cells were infected with pSIREN-RetroQ-derived retroviruses carrying shRNA specifically against catalase (sh-CAT) or non-specific scrambled shRNA as a control (NC). Infected cells were assayed for ROS production (left panel) and western blotted for the indicated proteins. The symbol # represents P values less than 0.01, respectively. All experiments were repeated three times and gave consistent results.