Journal: Antioxidants

Article Title: CHK2 Promotes Metabolic Stress-Induced Autophagy through ULK1 Phosphorylation

doi: 10.3390/antiox11061166

Figure Lengend Snippet: ULK1 is a physiological substrate of CHK2. ( A , B ) Immunoprecipitation assays testing the endogenous interaction between CHK2 and ULK1 in HCT116 cells. Lysates were immunoprecipitated with ULK1 ( A ) or CHK2 ( B ) antibody. The immunoprecipitates and lysates were analyzed by Western blot. ( C ) CHK2 binds with ULK1 in vitro. GST pull-down assays were performed by incubating purified GST or GST-ULK1 with invitro-translated flag-tagged CHK2. Arrows indicate GST and GST-ULK1 bands. ( D ) Lysates from HCT116 cells treated with EBSS were immunoprecipitated with CHK2 antibody or rabbit IgG. The immunoprecipitates and lysates were analyzed by Western blot. ( E ) Lysates from HCT116 cells treated with H 2 O 2 (500 μM) were immunoprecipitated with CHK2 antibody or rabbit IgG. The immunoprecipitates and lysates were analyzed by Western blot. ( F ) Lysates from HCT116 cells pretreated with NAC (SIGMA, A7250, 2 mM) for 3 h and then cultured for 30 min in EBSS starvation were immunoprecipitated with CHK2 antibody. The expression of p-CHK2 Thr68, CHK2, and ULK1 was detected by immunoblotting. ( G ) Lysates from HCT116 cells pretreated with CHK2 inhibitor (SIGMA, C3742, 20 μM) for 4 h and then treated with EBSS for 30 min were immunoprecipitated with CHK2 antibody. The expression of p-CHK2 Thr68, CHK2, and ULK1 was detected by immunoblotting. ( H ) Lysates from HCT116 cells expressing theindicated plasmids, treated or untreated with EBSS, were immunoprecipitated with FLAG antibody. The immunoprecipitates and lysates were analyzed by Western blot with the indicated antibodies. Quantitative analysis for the binding intensity of ULK1 are shown.

Article Snippet: CHK2 (Addgene, no. 41901, Watertown, MA, USA) was subcloned into pcDNA3.1 with an amino-terminal 3× Flag tag.

Techniques: Immunoprecipitation, Western Blot, In Vitro, Purification, Cell Culture, Expressing, Binding Assay

Journal: Antioxidants

Article Title: CHK2 Promotes Metabolic Stress-Induced Autophagy through ULK1 Phosphorylation

doi: 10.3390/antiox11061166

Figure Lengend Snippet: CHK2 phosphorylates ULK1 at Ser556. ( A ) Clustal alignment of the conserved sites in ULK1 matching the optimal CHK2 substrate motif. ( B ) In vitro CHK2 kinase assay using CHK2 recombinant human protein with FLAG-ULK1WT and FLAG-ULK1S556A as substrates, followed by immunoblotting analysis. ( C , D ) Western blot analysis with the indicated antibodies in H1299 cells with CHK2 knockdown. Cells were treated with EBSS ( C ) or H 2 O 2 (500 μM) ( D ). ( E ) Western blot analysis with indicated antibodies in H1299 cells pretreated with CHK2 inhibitor for 3 h and treated with EBSS for 1 h. ( F , G ) Western blot analysis with indicated antibodies in H1299 cells with ATM knockdown. Cells were treated with EBSS ( F ) or H 2 O 2 (500 μM) ( G ). ( H ) Western blot analysis with the indicated antibodies in H1299 cells pretreated with ATM inhibitor for 3 h and treated with EBSS for 1 h. ( I ) Western blot analysis with the indicated antibodies in H1299 cells pretreated with NAC for 4 h and treated with EBSS for 1 h. The results from three independent experiments are presented as mean ± SEM. ** p < 0.05, *** p < 0.001.

Article Snippet: CHK2 (Addgene, no. 41901, Watertown, MA, USA) was subcloned into pcDNA3.1 with an amino-terminal 3× Flag tag.

Techniques: In Vitro, Kinase Assay, Recombinant, Western Blot, Knockdown

Journal: Antioxidants

Article Title: CHK2 Promotes Metabolic Stress-Induced Autophagy through ULK1 Phosphorylation

doi: 10.3390/antiox11061166

Figure Lengend Snippet: CHK2-mediated ULK1 phosphorylation promotes autophagy. ( A ) Western blot analysis with reconstituted expression of the indicated proteins in H1299 cells. ( B , C ) Western blot analysis of p62and LC3 in H1299 cells with reconstituted expression of ULK1 WT, S556A mutant, or S556D mutant treated with EBSS for 3 h with ( B ) or without ( C ) CHK2. ( D , E ) Western blot analysis of p62 and LC3 in H1299 cells with reconstituted expression of ULK1 WT, S556A mutant, or S556D mutant treated with H 2 O 2 (500 μM) for 3 h with ( D ) or without ( E ) CHK2. ( F ) The red puncta is shown by representative confocal microscopic images in 293 cells expressing the indicated plasmids treated with EBSS for 2 h. ( G ) Data are presented as mean ± SEM. * ULK1 SA mutant treated with EBSS compared to ULK1 WT treated with EBSS, p < 0.001; # ULK1 SD mutant compared to ULK1 WT, p < 0.001 and & ULK1 SD mutant treated with EBSS without CHK2 compared to ULK1 WT treated with EBSS without CHK2, p < 0.001. Scale bar, 10 μm.

Article Snippet: CHK2 (Addgene, no. 41901, Watertown, MA, USA) was subcloned into pcDNA3.1 with an amino-terminal 3× Flag tag.

Techniques: Phospho-proteomics, Western Blot, Expressing, Mutagenesis

Journal: Antioxidants

Article Title: CHK2 Promotes Metabolic Stress-Induced Autophagy through ULK1 Phosphorylation

doi: 10.3390/antiox11061166

Figure Lengend Snippet: CHK2-ULK1 mediated autophagy protects cells against metabolic stress-induced cell death. ( A , B ) Flow cytometry analysis of apoptosis was performed in H1299 cells expressing the indicated plasmids treated with EBSS for 8 h with ( A ) or without ( B ) CHK2. The results from three independent experiments are presented as mean ± SEM. *** p < 0.001 compared to ULK1 S556A treated with EBSS. ( C , D ) Flow cytometry analysis of apoptosis was performed in H1299 cells expressing the indicated plasmids treated with H 2 O 2 (500 μM) for 8 h with ( C ) or without ( D ) CHK2. The results from three independent experiments are presented as mean ± SEM. *** p < 0.001 compared to ULK1 S556A treated with H 2 O 2 (500 μM) stimulation.

Article Snippet: CHK2 (Addgene, no. 41901, Watertown, MA, USA) was subcloned into pcDNA3.1 with an amino-terminal 3× Flag tag.

Techniques: Flow Cytometry, Expressing