Fig 2: Changes in the transcriptional level of additional autophagy-related markers in vivo.Expression of ULK1, Bcl-2, Atg5, Atg9a, Atg12, Atg14, DRAM1, ZFYVE1, and VMP1 mRNA in the diaphragms of T. spiralis-infected mice on 0, 7, 14, 21, and 28 dpi. The data represent at least 3 independent experiments and are expressed as mean ± SD. p<0.05(*), p<0.01(**), p<0.001(***).

Fig 3: In silico high-throughput screening of candidate small-molecule compounds targeting ULK1. (A) Schematic drawing of the activation mechanism of ULK1 agonist. (B) Candidate binding pockets in the structure of the kinase domain of ULK1. The inhibitor pocket is shown in red and our virtual screening pocket is shown in green. (C) Virtual screening protocol for the identification of the novel ULK1 agonist. (D) The top twenty candidate small-molecule compounds targeting ULK1 from Drugbank.

Fig 4: The ULK1 S405 and S415 residues are critical for ULK1 activation and autophagy induction.a Prediction of putative GSK3B phosphorylation sites in the Serine/Proline-Rich domain of ULK1. b Conservation of five candidate GSK3B phosphorylation sites in human and rodent ULK1. c Measurement of cell death after the expression of wild-type (WT) and phosphorylation-defective mutants of ULK1. Cell death was analyzed after insulin withdrawal for 48 h (n = 3). d Measurement of cell death after the expression of ULK1 with phosphorylation-defective or phosphorylation-mimicking mutations at the S405 and S415 residues in sgUlk1 HCN cells. Cell death was analyzed after insulin withdrawal for 24 h (n = 3). e Western blotting analysis of ATG13 and BECN1 phosphorylation in sgUlk1 HCN cells transfected with the indicated ULK1 constructs after insulin withdrawal for 6 h. The blots shown are representative of at least 3 experiments with similar results. f GFP-ZFYVE1 puncta assay in sgUlk1 HCN cells cotransfected with GFP-ZFYVE1 and the indicated mCherry-Ulk1 constructs after insulin withdrawal for 6 h. Scale bar, 10 μm. The images were taken using an LSM780 confocal microscope (Carl Zeiss). The graph shows the quantification of puncta from three experiments (n = 30 cells per condition). *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 5: Neuron starvation(A) Hippocampal neurons in culture stained with an antibody against the axonal protein Tau1. Left image are 3 DIV neurons in culture with control media. Right image are 3 DIV neurons which have been starved for the last 24 h in culture by diluting media five times with HBSS. Tau1 signal is in gray scale and DAPI staining is in blue. Here we show a maximal projection image of a 3D image acquisition using a TLSM Leica SP8 confocal microscope. To generate this image, several fields of view were stitched together with the confocal’s proprietary software. Arrows indicate uni-polar neurons. Arrowheads indicate multi-polar neurons. Green dashed lines are examples of quantified axons and ramifications that equal to the total axonal length. Scale bars represent 100 micrometers.(B) Western blot of samples from cortical neurons starved for 4 h with diluted N2 culture media. Different membranes were incubated with either anti ULK1 or anti phospho ULK1.(C) Western blot of samples from neurons starved for 24 h or treated with staurosporine to induce apoptosis as a positive control. The membrane was incubated with anti cleaved caspase 3 antibody.