Figure 4
Normal levels of hVPS34 are not required for AICAR-mediated differentiation of U937 cells. (a) Cells were transfected with siRNA against hVPS34 and respective non-targeting siRNA was used as a negative control. AICAR (0.5 mM), ATRA (1 μM), metformin (15 mM) or PMA (50 nM) were added 24 h after transfection. Total cell lysates were isolated 3 or 48 h after addition of agents and analyzed by western blot. The expression of differentiation markers was analyzed by flow cytometry 48 or 72 h after addition of agents. Western blot analyses and data obtained by flow cytometry are shown for each of the three independent experiments (Exp 1–3). (b, c) Cells were transfected with siRNA against hVPS34 or respective non-targeting siRNA. AICAR (0.5 mM) or metformin (15 mM) were added 48 h after transfection. Total cell lysates were isolated 3 or 48 h after addition of agents and analyzed by western blot for the expression of LC3B-II (b) or Beclin 1 (c). Representative images of at least three independent experiments are shown. (d) Cells were transfected with siRNA against Beclin 1 or respective non-targeting siRNA. AICAR (0.5 mM) was added 48 h after transfection. Total cell lysates were isolated 48 h after addition of AICAR and analyzed by western blot. Representative images of at least three independent experiments are shown.
