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. 2011 Apr 1;7(4):401–411. doi: 10.4161/auto.7.4.14397

Figure 3.

Figure 3

Autophagy regulates ATRA-induced cell differentiation. (A) HL-60 and NB4 cells were treated with ATRA (1 µM) with or without 3-methyladenine (“3-MA”, 10 mM) and rapamycin (100 nM) for 24–72 h, and CD11b expression was assayed by flow cytometric analysis (HL-60 cells: n = 3, *p = 0.005, 3-MA 48 h group versus ATRA 48 h group; p = 0.016, 3-MA 72 h group versus ATRA 72 h group; p < 0.001, rapamycin 48 h group versus ATRA 48 h group; p = 0.016, rapamycin 72 h group versus ATRA 72 h group; NB4 cells: n = 3, *p = 0.034, 3-MA 48 h group versus ATRA 48 h group; p = 0.019, 3-MA 72 h group versus ATRA 72 h group; p = 0.019, rapamycin 48 h group versus ATRA 48 h group; p = 0.016, rapamycin 72 h group versus ATRA 72 h group). (B) Morphological features of NB4 cells after 72 h of treatment with the drugs as indicated in (A). (C–E) After transfection with shRNA for 48 h, the indicated cells were treated with ATRA (1 µM) for 48 h, and then CD11b expression was assayed by flow cytometric analysis (C) (n = 3, *p = 0.003, ATG1 shRNA group versus control shRNA group; p = 0.007, ATG5 shRNA group versus control shRNA group; p = 0.005, PI3KC3 shRNA group versus control shRNA group). In parallel, cell differentiation was examined by NBT reduction (D) (HL-60 cells: n = 3, *p = 0.025, ATG1 shRNA group versus control shRNA group; p = 0.015, ATG5 shRNA group versus control shRNA group; p =0.014, PI3KC3 shRNA group versus control shRNA group. NB4 cells: n = 3, *p = 0.05, ATG1 shRNA group versus control shRNA group; p = 0.022, ATG5 shRNA group versus control shRNA group; p = 0.022, PI3KC3 shRNA group versus control shRNA group) or morphological assay (E). (F) HL-60 cells were treated as indicated with phorbol 12-myristate 13-acetate (“PMA”, 10 nM), arsenic trioxide (“As2O3”, 1 µM) and 1,25-vitamin D3 (“VitD3”, 20 nM) for 48 h, and then CD11b expression was assayed by flow cytometric analysis (n = 3, PMA: *p = 0.011, ATG5 shRNA group versus control shRNA group; As2O3: *p = 0.003, ATG5 shRNA group versus control shRNA group; VitD3: *p = 0.004, ATG5 shRNA group versus control shRNA group).