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

Figure 2.

Figure 2

Atg1, Atg5 and PI3KC3 are required for ATRA-induced autophagy. (A) Western blot analysis of Atg1, Atg5 and PI3KC3 expression in HL-60 or NB4 cells after treatment with ATRA (1 µM) for 48 h. (B) Western blot analysis of Atg1, Atg5 and PI3KC3 expression in HL-60 or NB4 cells after transfection with specific target shRNA for 48 h. (C) The indicated cells were treated with ATRA (1 µM) for 48 h, then autophagy was assayed by quantification of the average number of LC3 puncta per cell (HL-60 cells: n = 3, *p = 0.008, ATG1 shRNA group versus control shRNA group; p = 0.005, ATG5 shRNA group versus control shRNA group; p = 0.007, PI3KC3 shRNA group versus control shRNA group. NB4 cells: n = 3, *p = 0.01, ATG1 shRNA group versus control shRNA group; p = 0.005, ATG5 shRNA group versus control shRNA group; p = 0.008, PI3KC3 shRNA group versus control shRNA group). Representative images in HL-60 cells are shown in the right part. Bar = 20 µm. (D) HL-60 cells were treated as indicated with ATRA (1 µM) for 48 h and LC3-I/II protein expression was assayed by western blot. (E) Cells were treated as indicated with ATRA (1 µM) for 48 h, and then apoptosis was assayed by quantification of Annexin V-positive cells (HL-60 cells: n = 3, *p = 0.002, ATG1 shRNA group versus control shRNA group; p < 0.001, ATG5 shRNA group versus control shRNA group; p = 0.001, PI3KC3 shRNA group versus control shRNA group. NB4 cells: n = 3, *p < 0.001, ATG1 shRNA group versus control shRNA group; p = 0.003, ATG5 shRNA group versus control shRNA group; p = 0.001, PI3KC3 shRNA group versus control shRNA group).