Figure 4.

Administration of ethanolamine induces autophagy and reduces chronological senescence in mammalian cells. (a) HPLC-ELSD-assisted measurement of U2OS total cellular PE levels with and without 10 mM ethanolamine (Etn) treatment. (b, c) Effect of ethanolamine administration on autophagy induction. Wild-type human osteosarcoma U2OS cells (b) and their GFP-LC3 expressing counterparts (c) were either left untreated or treated with 10 mM ethanolamine. Thereafter, cells were processed for the immunochemical detection of LC3 lipidation (b) and for fluorescence microscopic quantification of GFP-LC3 positive dots (c). Administration of ethanolamine resulted in increased autophagy flux, as measured by LC3 lipidation in the absence or in the presence of bafilomycin A1 (bafA1). GAPDH levels were detected to ensure equal loading. Representative immunoblots are depicted in (b). Autophagy induction by ethanolamine treatment was confirmed by microscopic detection of GFP-LC3⁺ puncta in U2OS cells stably expressing GFP-LC3, in the absence or in the presence of bafilomycin A1 (c). Nutrient deprivation (EBSS) was used as a positive control of autophagy stimulation. (d) Ethanolamine reduces yeast-like chronological senescence and increases replicative viability in mammalian cells. Highly confluent U2OS cells were left untreated or stimulated with 10 mM ethanolamine or 1 μM rapamycin (Rapa, positive control) for one week