Figure 2. Modulation of autophagy by α-ketoglutarate precursors. (A) Inhibition of starvation-induced autophagy by DMKG, TFMKG and O-KG. U2OS cells stably expressing the autophagic markers GFP-LC3 were incubated in HBSS medium (NF) and left untreated or incubated with α-ketoglutarate precursors for 4h. Co-treatment with bafilomycin A1 (BafA1) was used to assess autophagic flux. Representative pictures (in presence of BafA1) (right panel) and quantification (left panel) are shown. Data represent mean ± S.D. (one representative experiment, n=3). *** p < 0.001 (compared to Control); ### p < 0.001 (compared to NF) (unpaired t test). Scale bar 10 μm. (B) Induction of autophagy by TFMKG and O-KG, but not DMKG, in complete medium. *** p < 0.001 (compared to Control); (unpaired t test). Scale bar 10 μm. (C, D) Immunoblotting showing the conversion of LC3I to LC3II in U2OS cells treated with α-ketoglutarate precursors in NF (C) or complete medium (D) in presence or absence of BafA1 to monitor autophagic flux (one representative experiment, n=3).