FIGURE 6.

Activation of JNK plays a role in K-Ras^V12-induced autophagy and malignant cell transformation. A, MCF10A cells were infected with MFG-control or MFG-K-Ras^V12. At the indicated times, total cell lysates were subjected to immunoblot analysis with the indicated antibodies. β-actin was used as a loading control. B, cells were transfected with siRNA of ERK2, p38 MAPK, or JNK1 or were pretreated with PD98059 (25 μmol/liter), SB203580 (10 μmol/liter), or SP600125 (10 μmol/liter) and then infected with MFG-control or MFG-K-Ras^V12. After 72 h, total cell lysates were subjected to immunoblot analysis with anti-ATG5 or -ATG7 antibodies. β-Actin was used as a loading control. C, MCF10A cells were transfected with siRNA of ERK2, p38 MAPK, or JNK1 and then infected with MFG-K-Ras^V12. Left, after 72 h, punctate GFP-LC3 fluorescence was imaged by fluorescence microscopy. The percentage of autophagic cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP positive cells. Results from three independent experiments are presented as means ± S.E. *, p < 0.01, significantly different from control. Middle, cells (1 × 10⁵) were allowed to grow on soft agar, and colonies were monitored after 2 weeks. Results from three independent experiments are presented as means ± S.E. (error bars). *, p < 0.01, significantly different from control. Right, after 72 h, cells were loaded with DCFH-DA for 15 min. The amount of retained DCF was measured using flow cytometry as described under “Experimental Procedures.” Results from three independent experiments are presented as means ± S.E. D, MCF10A cells were pretreated with NAC and then infected with MFG-control or MFG-K-Ras^V12. After 72 h, total cell lysates were subjected to immunoblot analysis with the indicated antibodies. β-Actin was used as a loading control.