Supplementary MaterialsS1 Data: In independent linens, the excel spreadsheet contains the numerical data and statistical analysis for Figs 1C, 1E, 6B, 6C, 6D, 6E, 7A, 7C, 7D, 7E, 7F, 7G, 7H and 7I; S12A, S12B, S12D, S12E, S12F, S13A, S13B, S14A, S14C, S14E, S15A, S15C, S15E, S16A, S16B, S17A, S17B, S17C, S17D, S17E, S17F and S18B Figs. and nocodazole for western blot analysis. (C) Abiraterone inhibitor ULK1 phosphorylation in mitosis interferes with ULK1 antibody acknowledgement. The ULK1 antibody (Cell Signaling Technology, #8054) could not identify the upshifted band for mitotic ULK1 but could identify when the PVDF membrane was treated with lambda phosphatase for 1 hour. PVDF, polyvinylidene fluoride; ULK1, unc-51-like autophagy activating kinase 1.(TIF) pbio.3000288.s003.tif (457K) GUID:?C7A61C0B-9C55-4256-B8D3-97B32BB9E174 S2 Fig: Aurora A is less likely to be responsible for ULK1 band shift in mitosis. (A-B) HeLa cells synchronized and treated as Fig 3A were subjected to western blot analysis. However, we found that the additional 2 Aurora A inhibitors, MLN8054 and Aurora A inhibitor I, did not impact ULK1 band shift (A). In addition, MLN8237 treatment for any shorter time (1 hour or 0.5 hours) did not cause ULK1 band shift switch as 1.5-hour treatment (B). MLN8237, MLN8054, and Aurora A inhibitor I were Aurora A inhibitor. CDK, cyclin-dependent kinase; ULK1, unc-51-like autophagy activating kinase 1.(TIF) pbio.3000288.s004.tif (534K) GUID:?3D85BAA7-3240-48EC-B03B-129CBDF7BA69 S3 Abiraterone inhibitor Fig: CDK1 regulates ULK1 phosphorylation independent of ULK1 kinase activity. (A-B) ULK1-KO cells were founded. HeLa and 293T cells transiently transfected with the CRISPR/Cas9 plasmid subcloned gRNA for human being ULK1 were screened by western blot analysis and the ULK1-KO clones were identified. The reddish circles indicate the ULK1-KO clones for the following assay. (C-D) K46I kinase-dead ULK1 also underwent significant electrophoretic mobility shift and phosphorylation in mitosis as WT ULK1. HeLa ULK1-KO cells reconstituted with FLAG-tagged mULK1-K46I were treated as Figs ?Figs1C1C and ?and3B3B (the lower panel) and then analyzed by european blot analysis and immunoprecipitation, respectively. (E) In vitro kinase assay indicated that purified CDK1/cyclin B could induce K46I kinase-dead ULK1 to undergo significant electrophoretic mobility shift and phosphorylation. CDK, Abiraterone inhibitor cyclin-dependent kinase; gRNA, guideline RNA; KO, knockout; mULK1, mouse ULK1; ULK1, unc-51-like autophagy activating kinase 1; WT, crazy type.(TIF) pbio.3000288.s005.tif (1014K) GUID:?FB52F8DC-899C-48BD-8B5A-0B6BCB2ABDDC S4 Fig: ULK2 is also phosphorylated and upshifted in mitosis. The 293T cells transiently transfected with Abiraterone inhibitor FLAG-tagged mULK2 treated as Fig 3A were analyzed by western blot analysis and immunoprecipitation. mULK2, mouse ULK2; ULK2, unc-51-like autophagy activating kinase 2.(TIF) pbio.3000288.s006.tif (213K) GUID:?6E99F6FC-4537-41CD-8532-381EBA00949B S5 Fig: ATG13 is upshifted in mitosis. (A) HeLa cells were treated as Fig 4C for ATG13 mobility shift analysis. (B) ATG13 mobility shift in mitosis is definitely decreased by CDK1 inhibitor RO-3306, similarly to ULK1, although to a lesser extent. The 293T cells overexpressing FLAG-tagged mULK1 were synchronized by single-thymidine in the presence or absence of nocodazole, treated with 10 M RO-3306 for 5 or 30 minutes. The coimmunoprecipitate by FLAG antibody was subjected to immunoblotting with ATG13, FIP200 antibodies. (C-D) ULK1 manifestation level does not affect ATG13 mobility shift in mitosis. HeLa ULK1-KO cells with or without FLAG-tagged mULK1 manifestation synchronized into mitosis with thymidine and nocodazole (C) or in asynchronous condition (D) were treated with 10 M RO-3306 for 30 minutes for western blot analysis. ATG, autophagy-related; CDK, cyclin-dependent kinase; FIP200, FAK family-interacting protein of 200 kDa; KO, knockout; mULK1, mouse ULK1; ULK1, unc-51-like autophagy activating kinase 1.(TIF) pbio.3000288.s007.tif (1.0M) GUID:?5B955E83-4BEE-4C2E-AD48-6DED0AC73197 S6 Fig: Identification of ULK2 phosphorylation sites in mitosis. (A-B) The initial recognition of ULK2 phosphorylation sites in mitosis. The immunoprecipitate with FLAG antibody in asynchronous or mitotic 293T cells transfected with mULK2-3FLAG was subjected to SDS-PAGE and Coomassie amazing blue staining (A) and mass spectrometry analysis of phosphorylation sites for mitotic mULK2 compared with asynchronous mULK2 (B). (C) The contribution of the potential residues to mitotic ULK2 band shift. HeLa cells were transfected with the mutant mULK2-3FLAG plasmid in indicated sites and analyzed by cell cycle synchronization and western blot. mULK2, mouse ULK2; ULK2, unc-51-like autophagy activating IL2RA kinase 2.(TIF) pbio.3000288.s008.tif (806K) GUID:?24EF06EB-7727-4A08-B7E6-C5BA01936BFB S7 Fig: ATG13-KO cells establishment. HeLa cells were transiently transfected with the CRISPR/Cas9 plasmid that subcloned gRNA for human being ATG13. The ATG13-KO clones were screened by western blot and recognized. The red circle shows the ATG13-KO clones for the following assay. ATG, autophagy-related; gRNA, guideline RNA; KO, knockout.(TIF) pbio.3000288.s009.tif (113K) GUID:?913014AB-EBC4-4817-B8DD-CD79E00DCFBC S8 Fig: The phosphorylation sites we recognized in both ULK1 and ATG13 are conserved between different isoforms. (A) The difference between the construct we used in this paper (“type”:”entrez-protein”,”attrs”:”text”:”Q6PB82″,”term_id”:”81911376″,”term_text”:”Q6PB82″Q6PB82 in Uniprot, “type”:”entrez-nucleotide”,”attrs”:”text”:”BC059835″,”term_id”:”37590579″,”term_text”:”BC059835″BC059835 in GenBank) with the ULK1 used in some other studies (“type”:”entrez-protein”,”attrs”:”text”:”O70405″,”term_id”:”6136125″,”term_text”:”O70405″O70405 in Uniprot) is definitely that there are 6 additional amino.