ATRA and ATO induce autophagy through the mTOR pathway. (A) Lysates from NB4 cells treated with or without ATO (1μM) or ATRA (1μM) were analyzed by IB by the use of antibodies against P-p70S6K. Tubulin was used as a loading control. Data from 3 independent experiments were quantified. Data are expressed as the mean ± SEM. (B) Rapamycin inhibits mTOR and induces autophagy and PML/RARA degradation. Lysates from control or rapamycin-treated (200nM for 4 hours) NB4 cells were examined by Western blotting by the use of anti-P-p70S6K, anti-RARA, anti-LC3, or anti-tubulin antibodies. (C-F) Measurement of CD11b by was performed with fluorescence-activated cell sorting. NB4 cells were incubated or not with ATRA (1μM) for 24 hours, then incubated for another 24 hours with 3MA (5mM) or BafA1 (50nM) (C-D) or rapamycin (100nM; E-F) in the presence or absence of ATRA. The cells were then incubated with FITC-conjugated anti-CD11b antibody and analyzed by flow cytometry. (C-D) Representative flow diagrams are shown. The graph represents mean of 4 independent experiments done in duplicate ± SEM. (E-F) Representative flow diagrams are shown. The graph represents mean of 2 independent experiments done in duplicates ± SEM.