Figure 4.

Knockdown of JMJD2B suppresses autophagy and sensitizes cells to Nutlin. A, A549 cells were transfected with control siRNA or JMJD2B siRNA and then treated 24 h with Nutlin (10 μm); lysates were immunoblotted for the indicated proteins. B and D, A549 and U2OS cells were transfected with control siRNA or JMJD2B siRNA and then treated 24 h with Nutlin (10 μm) and/or BafA1 (10 nm). Whole cell lysates were immunoblotted for the indicated proteins. Note: A and B are from the same experiment. In B, the first four lanes of the JMJD2B blot are reused from A to illustrate differences in JMJD2B expression in the absence and presence of BafA1. C, left panel, A549 cells were transfected with control siRNA or JMJD2B siRNA and then treated 24 h with vehicle or Nutlin (10 μm). MDC sequestration was determined and average relative MDC (triplicate) is presented with S.D. indicated (left panel). The asterisk indicates there is a significant difference between vehicle-treated and Nutlin-treated conditions in control siRNA (p < 0.01) and as well as Nutlin-treated control siRNA and JMJD2B siRNA (p < 0.01) cells. C, right panel, cells were treated similarly for 72 h and then analyzed for sub-G₁. % sub-G₁ cell were presented with S.D. indicated. There is a significant difference between Nutlin-treated control siRNA and JMJD2B siRNA (p < 0.01) cells. E, left panel, U2OS cells were transfected with control siRNA or JMJD2B siRNA and then treated 24 h with vehicle or Nutlin (10 μm). MDC sequestration was determined and average relative MDC (triplicate) is presented with S.D. indicated. The asterisk indicates there is a significant difference between vehicle-treated and Nutlin-treated conditions in control siRNA (p < 0.05) as well as Nutlin-treated control siRNA and JMJD2B siRNA (p < 0.05) cells. E, right panel, cells were treated similarly for 72 h and analyzed for sub-G₁. % sub-G₁ cells were presented with S.D. indicated. There is a significant difference between Nutlin-treated control siRNA and JMJD2B siRNA (p < 0.01) cells.