Figure 3.

CDKIs trigger autophagy in an RB-dependent manner. A, immunoblot analysis of LC3 and p16. The cells were infected with AdRSV-p16 (300 pfu/cell) for 72 h. AdCMV-GFP was used as a control for viral infection and nonspecific protein expression. Actin was used as a loading control. B, green fluorescence punctation in U-87-EGFP-LC3. The cells were infected with AdRSV-p16 (300 pfu/cell); 72 h later, the cells were fixed and then examined by fluorescence microscopy. Top, representative images of the cells with the indicated treatments. Bottom, quantification of the cells with EGFP-LC3 dots (columns, mean; bars, SD). *, P = 0.004. Bar, 20 μm. C, top, immunoblot analysis of RB in U-87 MG cells transfected with shGFP or shRB-expressing plasmid. α-Tubulin was used as a loading control. Bottom, immunoblot analysis of LC3 and p16. The cells were infected with AdRSV-p16 (300 pfu/cell) for 72 h. AdCMV-β-gal was used as a control for viral infection and nonspecific protein expression. Actin was used as a loading control. D, immunoblot analysis of LC3. Cells were infected with AdMH4p27 at the indicated doses (pfu/cell). Seventy-two hours later, cell lysates were collected for immunoblot analysis. AdCMV-β-gal (100 pfu/cell) was added as a control for nonspecific protein expression. Actin was used as a loading control.