Figure 3.
The phosphorylation of ULK1 is required for autophagy activation in response to lipotoxicity. (A) ulk1 KO MEFs co-transfected with vectors encoding HA-WT ULK1 or the HA-ULK1S317A mutant were incubated with PA (500 μM) for 18 h and subjected to immunoblot analysis with antibodies against p-ULK1(S317), HA-ULK1, KEAP1, SQSTM1, LC3B, C.PARP, C.CASP3, and ACTB (loading control). (B) Densitometric analysis of KEAP1 and LC3B-II immunoblots. (C–E) Total mRNA isolation from cells treated as described in (A) and subjected to qRT-PCR analysis for relative mRNA expression of Gsta1 (C), Hmox1 (D), Nqo1 (E). (F) Confocal microscopy analysis of colocalization of Lysotracker and LC3B the cells treated as described in (A). The representative single optical sections and merge images are shown. Scale bar: 10 μm. (G) Quantitative analysis of colocalization. (H) TUNEL analysis of cells treated as in (A). Scale bar: 100 μm. (I) Quantification of TUNEL-positive cells. (J) Cell viability was estimated using a Cell titer-Glo assay kit. Live cell numbers were expressed as absorbance at luminescence. (K) Reactive oxygen species (ROS) were determined using CM-H2DCFH-DA. The representative images are shown. Scale bar: 100 μm. (L) Quantification of relative DCF fluorescence. Data are presented as the mean ± SD from 3 independent experiments. *p < 0.05, **p < 0.01