Research Paper Volume 9, Issue 11 pp 2352—2375

Figure 2. MEK/ERK suppression in control ERas cells leads to activation of AMPK-mediated autophagy which helps to overcome cell death. (A) MEK/ERK suppression does not decrease mTORC1 activity and activates AMPK and Ulk1 Ser555 phosphorylation. Western-blot analysis of 4E-BP1, S6, AMPK, Ulk1 (Ser757, Ser555) phosphorylation after MEK/ERK suppression. Cells were treated with PD and then lysed and processed to Western-blotting in 10 and 12% gels. Numbers below present densitometry of bands. (B) Immunofluorescence images, demonstrating LC3 and LAMP1 colocalization and p62/SQSTM1 degradation. Cells were cultivated with PD, 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: 25µm (upper panel), 10 µm (lower panel). (C) Western-blot analysis of LC3I to LC3II conversion and p62/SQSTM1 degradation upon MEK/ERK suppression. Cells were exposed to PD and processed to Western-blotting in 15% gel. Numbers below present densitometry of bands. Decrease of viability of cells with inhibited MEK/ERK pathway after suppression of autophagy with Bafilomycin A1 (D) and after inhibition of AMPK with Compound C (E) as analyzed by MTT-test. Cells were treated with PD together with Bafilomycin A1 or with Compound C. In the indicated time intervals 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).