FIGURE 4.

Up-regulation of ATG5 and ATG7 is involved in K-Ras^V12-induced autophagic vacuole formation and malignant cell transformation. A, left, cellular mRNA was isolated 24, 48, and 72 h after infection with MFG or MFG-K-Ras^V12. RNA expression was examined by RT-PCR toward ATG5, ATG6 (Beclin-1), or ATG7. GAPDH served as an internal standard. The data represent a typical experiment conducted three times with similar results. Right, total cell lysates were isolated 24, 48, and 72 h after infection of MFG-control or MFG-K-Ras^V12 and were subjected to immunoblot analysis with anti-ATG5, -ATG6, or -ATG7 antibodies. β-Actin was used as a loading control. The data represent a typical experiment conducted three times with similar results. B, cells (5 × 10⁶ cells) were injected subcutaneously in the right flank of athymic nude mice. Tumors in subcutaneous mouse xenografts were subjected to immunoblot analysis with the indicated antibodies. β-Actin was used as a loading control. C, MCF10A cells were transfected with shRNA of ATG5, ATG7, or control and then infected with MFG-K-Ras^V12. After 72 h, punctate GFP-LC3 fluorescence was imaged by fluorescence microscopy. Cells (1 × 10⁵) were allowed to grow on soft agar, and colonies were monitored after 2 weeks. Bottom left, 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. (error bars). *, p < 0.01, significantly different from control. Bottom right, colonies greater than 50 mm in diameter were counted. Results from three independent experiments are presented as means ± S.E. *, p < 0.01, significantly different from control. D, as a rescue experiment, WT ATG5 or WT ATG7 was reintroduced into ATG5 or ATG7 knockdown cells, respectively, using a pcDNA vector carrying the ATG5 or ATG7 gene. Total cell lysates were subjected to immunoblot analysis with anti-ATG5 or -ATG7 antibodies. β-Actin was used as a loading control. Bottom left, After 72 h, 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. Bottom right, cells (1 × 10⁵) were allowed to grow on soft agar, and colonies were monitored after 2 weeks. Colonies greater than 50 mm in diameter were counted. Results from three independent experiments are presented as means ± S.E. *, p < 0.01, significantly different from control. E, MCF10A cells were transfected with shRNA of ATG5 or ATG7 and then infected with MFG-K-Ras^V12. Cells (5 × 10⁶ cells) were injected subcutaneous in the right flank of athymic nude mice. Tumor volumes were measured at 3-day intervals as described under “Experimental Procedures.”