Differential regulation of autophagy by the two signalling pathways MEK/ERK and AKT/MTOR A. Western blot analysis of protein levels of pAKT (ser473), pS6R, pERKs, LC3 and tubulin after 24 h treatment with 1μM BRAFV600E inhibitor PLX4720 (lanes 2, 6 and 10), 1μM MEK inhibitor PD0325901 (lanes 3, 7 and 11) and 1μM of PI3K/MTOR inhibitor PI-103 (lanes 4, 8, 12), in the mutant BRAFV600E cell lines RKO, HT29 and Colo-205 respectively. The quantification of LC3 reflects the whole protein levels as compared to the untreated sample in each cell line (i) and the ratio of LC3II/LC3I in each sample separately (ii). B. The existence of autophagic vacuoles was analysed by 0,1 mM MDC staining under confocal microscope, after treatment of HT29 and Colo-205 cell lines with the kinase inhibitors, described in 3A. C. Cell viability of the mutant BRAFV600E colon cancer cell lines RKO, HT29 and Colo-205 after 24 (upper left panel), 48 (lower left panel) and 72 h (lower right panel) treatments with 1μM of each of the following BRAF/MEK/ERK and AKT/MTOR pathway inhibitors; PLX4720, PI-103, PD0325901 (PD), GDC0941 (GDC) (PI3K inhibitor), Rapamycin (MTOR inhibitor). Cells were also stained with Hoechst after 24h treatment in order to assess the number of apoptotic nuclei (upper right panel). D. Western blot analysis of LC3 and p62 protein levels in the mutant BRAFV600E colon cancer cell lines RKO, HT29 and Colo-205 respectively after 24 h treatment with 1μM of each of the following BRAF/MEK/ERK and AKT/MTOR pathway inhibitors: PLX4720 (2, 8, 14), PI-103 (3, 9, 15), PD0325901 (4, 10, 16), GDC0941 (5, 11, 17), Rapamycin (6, 12, 18). The quantification of LC3 reflects total protein levels as compared to the untreated sample in each cell line (i) and the ratio of LC3II/LC3I in each sample separately (ii). E. Confocal microscope images of two-dimensional culture in RKO cell line after treatment with 1μM of each of the following BRAF/MEK/ERK and PI3K/MTOR pathway inhibitors; PLX4720 (2 nd column), PI-103 (3 rd column), PD0325901(4 th column), GDC0941(5 th column), Rapamycin (6th column) as compared to control untreated cells (1st column). Cells were stained with MDC (upper row, light blue), phalloidin (middle row, red), and merged staining (lower row), in order to detect autophagic vacuoles (MDC) and cell number distribution. The number of cells stained with MDC (high-low) was recorded. The total number of cells by phalloidin staining and the number of MDC stained cells from five different confocal images for each sample was recorded. F. Western blot analysis of protein levels of pAKT(ser473), pS6R, LC3, p62 and tubulin after 24 h treatment with 1μM of PI-103 (lanes 2, 6, 10, 14), 1μM GDC0941 (lanes 3, 7, 11, 15) and 1μM Rapamycin (lanes 4, 8, 12, 16) in Caco-2, DLD-1, HCT116 and RKO cell lines. Protein levels were normalized against tubulin. The quantification of LC3 reflects the total protein levels as compared to the untreated sample in each cell line (i) and the ratio of LC3II/LC3I in each sample separately (ii). G. Confocal microscope images of two-dimensional culture in DLD-1 cell line after treatment with 1μM of each of the following PI3K/MTOR pathway inhibitors; PI-103 (2 nd column), GDC0941(3 rd column) and Rapamycin (4th column) as compared to control untreated cells (1st column). Cells were stained with Hoechst (upper row, blue), LC3 antibody (middle row, green) and merged (lower row). The total number of cells by Hoechst staining and the number of LC-3 stained cells from five different confocal images for each sample was recorded.
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