Fig 1: Trametinib enhances CMA activity in AML12 cells. (A) Representative fluorescence microscopy images of AML12 cells expressing the KFERQ-Dendra2 CMA reporter, following treatment for 4 h with DMSO (solvent control), Tram, or Buparlisib. Drug concentrations are shown in figure. (B) Quantification of results shown in (A). p values represent the results of a Kolmogorov-Smirnov non-parametric test. (C) Representative western blots and quantification of a macroautophagic flux assay in AML12 cells treated with Tram for 4 h. n = 6 for each condition. PINT is the interaction term from a 2-factor ANOVA. There is no detectable effect on macroautophagy after 4 h of treatment. (D) Representative western blots of lysates from AML12 cell treated for 48 h with Tram or Tram + Leupeptin and Ammonium Chloride (to block lysosomal proteolysis). LAMP2A is a control for effective leupeptin administration. ERK and pERK are controls for effective Tram treatment. EEF1B2, EEF1G, EEF2, ACLY, ACACA, and FASN are CMA substrates. (E) Quantifications of blots shown in (D). n = 8 for each condition. P values on the graphs are the results of unpaired t tests, to evaluate if Leupeptin and Ammonium Chloride “rescued” the effects of Trametinib treatment. Levels of histone H3 were measured in each experiment and used to normalize across replicate gels.