Fig. 2. Arzanol drives LC3-II accumulation independent of the AKT/mTOR-pathway.
A Structure of arzanol. B–F Immunoblot analysis of mTOR/ULK1-pathway proteins during serum- and amino acid starvation. Wild-type HeLa cells were starved in serum- and amino acid-free medium for 6 h while being incubated with baf A1 or different concentrations of self-isolated arzanol. Data show quantified means of biological triplicates normalized to β-actin (ACTB) ± SEM. Representative immunoblots are additionally shown. Nonadjacent lanes (i.e., full medium control lane) are indicated by vertical black lines. Statistical analysis was performed using two-way ANOVA with Dunnett’s multiple comparison test comparing treated to untreated samples, ***P < 0.001, ****P < 0.0001. G Simplified pathway of key proteins of the mTOR/ULK1-axis in starvation-induced autophagy. H, I Immunoblot analysis of LC3-II and p62/SQSTM1 during serum-starvation. Wild-type HeLa cells were starved in serum-free (dark bars) or serum- and amino acid-free (light bars) medium for 6 h while being incubated with baf A1 or different concentrations of commercially available arzanol. Data show quantified means of biological triplicates normalized to ACTB ± SEM. J HeLa cells stably expressing GFP-LC3-RFP-LC3ΔG were starved in serum- and amino acid-free medium for 6 h while incubated with different concentrations of commercially available arzanol. Data show fold increase of GFP-LC3 fluorescence relative to mock-treated control as means ± SEM of three biological replicates.