FIGURE 4.

Beclin 1 and Atg12, but not p27Kip1 are required for dasatinib-induced autophagy. Dasatinib-induced growth inhibition partially depends upon autophagy induction. (A), Effects of p27Kip1, Beclin 1 and Atg12 siRNAs on dasatinib (Das)-induced autophagy determined by AO staining. HEY cells were transfected with negative control (Neg siR), p27Kip1#1 (p27 siR1), Beclin 1 (BECN1 siR), or Atg12 (Atg siR) siRNAs (50 nM final concentration). Cells were treated either DMSO or Das for 24 hrs. AO staining was analyzed by flow cytometry. *, P < 0.05 compared to Neg siR/Das-treated group; **, P < 0.01 compared to Neg siR/Das-treated group. Data shown was averaged from 4 independent experiments (p27 siR data from 6 independent experiments). (B–D), Effect of p27Kip1, Beclin 1 and Atg12 siRNAs on their respective protein expression by Western blotting. Mock was the transfection reagent-treated cells. (E), Beclin 1 levels in stable subclones #46 and #69 that express Beclin 1 shRNA (shBECN1) detected by Western blotting. (F), Effects of shBECN1 on dasatinib-induced growth inhibition. HEY stable shBECN1 subclones #46 and #69 and their control piMARK cells were treated with Das (150 nM) for 72 hr. Cell viability was measured with crystal violet staining. DMSO-treated control (CTRL) was set as 1 for each subclone of piMARK, shBECN1#46 and shBECN1#69. Das-treated groups in these three subclones were expressed as ratio of respective CTRL. **, P < 0.01 compared to piMARK CTRL group. (G), Effects of shBECN1 on dasatinib-induced autophagy. shBECN1 subclones #46 and #69 and control piMARK cells were treated with Das for 24 hr and AO staining was performed.