Research Paper Volume 9, Issue 11 pp 2352—2375

Figure 6. Senescent ERas cells fail to induce cytoprotective autophagy upon MEK/ERK inhibition (A) Senescent cells with suppressed MEK/ERK retain high mTORC1 activity and phosphorylate AMPK and Ulk1 Ser555. Cells were treated with NaBut+PD or NaBut and then lysed and processed to Western-blotting in 10 and 12% gels. Numbers below present densitometry of bands. (B) Immunofluorescence images, demonstrating that senescent cells with suppressed MEK/ERK have no LC3 and LAMP1 colocalization. Cells were treated with inhibitors or left untreated, fixed and stained with antibodies against pan-LC3, LAMP1 and p62/SQSTM1 (p62). Confocal images are shown. Upper row: pan-LC3 (green), LAMP1 (red); bottom row: pan-LC3 (green), p62/SQSTM1 (red). Nuclei stained with DAPI (blue). Scale bars: 25 µm (upper panel), 10 µm (lower panel). (C) Western-blot analysis of LC3I to LC3II conversion and p62/SQSTM1 degradation. Cells were processed to Western-blotting in 15% gel. Numbers below present densitometry of bands. (D) Images demonstrating low lysosomal β-galactosidase activity in senescent cells with MEK/ERK suppression. Cells were treated with inhibitors for 72 h and then fixed and stained with β-galactosidase substrate in pH 4.0 buffer and visualized using Pascal LSM5 microscope. Decrease of viability of senescent cells treated with autophagy inhibitor Bafilomycin A1 (E) or AMPK mimetic AICAR (F), analyzed by MTT-test. Cells were treated with NaBut and Bafilomycin A1 or NaBut and AICAR. Cells were processed with MTT reagent, amount of formazan was measured at 570 nm wavelength. Data are presented as mean ±S.E.M. of three independent experiments (n=3).