Journal: Cell Death & Disease

Article Title: Identification of KW-2449 as a dual inhibitor of ferroptosis and necroptosis reveals that autophagy is a targetable pathway for necroptosis inhibitors to prevent ferroptosis

doi: 10.1038/s41419-024-07157-9

Figure Lengend Snippet: A , B MEF cells were pre-treated with DMSO or KW-2449 for 30 min followed by treatment with RSL3 for the indicated time. Cells were lysed and immunoblotted with indicated antibodies. C HEK293T cells were transfected with empty vector or Flag-tagged human ULK1. After 16 h, cells were treated with KW-2449 at indicated concentrations for 8 h. Cells were then lysed and immunoblotted with indicated antibodies. D Quantification of ADP-Glo kinase assays performed with recombinant human ULK1 in the presence of increasing concentrations of KW-2449. E Overall binding model of ULK1 with KW-2449. KW-2449 is shown in blue. α - helix is shown in orange. β-sheet is shown in yellow. F Closeup view of KW-2449 bound to ULK1. The π−alkyl interactions were indicated by the black line. Key residues that contact KW-2449 are labeled. G Paired WT and ULK1-deficient MEF cells were pre-treated with DMSO or KW-2449 for 30 min. Then, WT MEF cells were treated with RSL3 for 6 h and ULK1-deficient MEF cells were treated with RSL3 for 24 h, respectively. Cell viability was analyzed by CellTiter-Lumi assay. H ULK1-deficient MEF cells were infected with the lentivirus containing the pLVX with Flag-tagged mouse ULK1 to reconstitute the expression of ULK1. The expression of ULK1 was examined by Western blotting with ULK1 antibody. I ULK1-deficient and ULK1-deficient with Flag-ULK1 expressed MEF cells were pre-treated with DMSO or KW-2449 for 30 min followed by treatment with RSL3 for 6 h. Cell viability was analyzed by CellTiter-Lumi assay. Bar graphs represent the mean ± SD from three independent experiments. All Western data are representative of three independent experiments. Statistical analysis was performed using a two-sided student’s t-test. The levels of significance were indicated by ***P < 0.001; ns not significant.

Article Snippet: The mammalian expression plasmids of Flag-tagged human ULK1 (Cat#HG12031-NF) were purchased from SinoBiological (Beijing, China).

Techniques: Transfection, Plasmid Preparation, Recombinant, Binding Assay, Labeling, Infection, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: MiR-26 enhances chemosensitivity and promotes apoptosis of hepatocellular carcinoma cells through inhibiting autophagy

doi: 10.1038/cddis.2016.461

Figure Lengend Snippet: MiR-26a/b directly targets ULK1 and inhibits autophagy at the initial stage in HCC cells. ( a ) A schematic diagram of ULK1 3′-UTR as a putative target for miR-26a/b. The seed-recognizing sites in the ULK1 3′-UTR by miR-26a/b are indicated in red. ( b ) Relative luciferase activity in 293T that were transfected with firefly luciferase reporters containing WT or mutant 3′-UTRs of ULK1, pre-miR-26a/b or random pre-miR-NC. ( c ) Huh-7 and HepG2 cells were transfected with pre-miR-NC, pre-miR-26a/b or anti-miR-NC, anti-miR-26a/b. The ULK1 expression level were detected using immunoblotting. ( d ) HepG2 cells were transfected with pre-miR-NC, pre-miR-26 or anti-miR-26 for 24 h, cells were treated with/without 3-MA, CQ or rapamycin for 24 h. The expression levels of LC3, ULK1, Beclin-1 and ATG7 were detected. ( e ) Quantitative analysis of ULK1 protein levels. ( f ) Quantitative analysis of LC3-II/LC3-I protein levels. ( g ) HepG2 cells were transfected with GFP-LC3 plasmid, pre-miRNA-NC, miR-26a/b mimics, anti-miR-NC or anti-miR-26; after 24-h transfection, cells were treated with 3-MA, CQ or rapamycin for a further 24 h before observation to count GFP-LC3 puncta under confocal microscopy. Blue indicates DAPI-stained nuclei. Green indicates GFP-LC3. One of 10 representative micrographs is shown. ( h ) The relative number of GFP-LC3 punctae in cells treated with 3-MA, CQ or rapamycin was calculated from 10 random fields. The data are presented as the means±S.E. obtained from three independent experiments. * P <0.05; ** P <0.01; and *** P <0.001

Article Snippet: For the ULK1 overexpression assay, a pcDNA3.1 vector was designed to specifically express the open reading frame of human ULK1 containing full-length 3′-UTR and purchased from GeneCopoeia (Germantown, MD, USA).

Techniques: Luciferase, Activity Assay, Transfection, Mutagenesis, Expressing, Western Blot, Plasmid Preparation, Confocal Microscopy, Staining

Journal: Cell Death & Disease

Article Title: MiR-26 enhances chemosensitivity and promotes apoptosis of hepatocellular carcinoma cells through inhibiting autophagy

doi: 10.1038/cddis.2016.461

Figure Lengend Snippet: MiR-26a/b regulates autophagy through targeting ULK1. ( a ) Protein levels of ULK1 and LC3 were determined in HepG2 cells overexpressed with pre-miR-NC, pre-miR-26, ULK1-vector or pre-miR-26 plus ULK1-expressing plasmids. GAPDH was served as internal control. The right histograms represent quantitative analysis of ULK1 and LC3-II/LC3-I protein level. ( b ) Representative photographs of HepG2 cells transfected with GFP-LC3-expressing plasmids plus pre-miR-NC, pre-miR-26, ULK1-vector or pre-miR-26+ULK1-vector. ( c ) Protein levels of ULK1 and LC3 were detected in HepG2 cells overexpressed with anti-miR-NC, anti-miR-26, Si-ULK1 or anti-miR-26 plus Si-ULK1. ( d ) Representative photographs of HepG2 cells transfected with GFP-LC3-expressing plasmids plus anti-miR-NC, anti-miR-26, Si-ULK1 or anti-miR-26+Si-ULK1. The right histogram represents quantitative analysis of GFP-LC3 punctate from 10 micrographs. * P <0.05; ** P <0.01; and *** P <0.001

Article Snippet: For the ULK1 overexpression assay, a pcDNA3.1 vector was designed to specifically express the open reading frame of human ULK1 containing full-length 3′-UTR and purchased from GeneCopoeia (Germantown, MD, USA).

Techniques: Plasmid Preparation, Expressing, Control, Transfection

Journal: Cell Death & Disease

Article Title: MiR-26 enhances chemosensitivity and promotes apoptosis of hepatocellular carcinoma cells through inhibiting autophagy

doi: 10.1038/cddis.2016.461

Figure Lengend Snippet: MiR-26a/b is inversely correlated with increased autophagy in tumor tissues of patients with HCC. ( a – c ) Relative levels of miR-26a/b (expressed as the miRNA/U6 ratio) in tumor tissues (T) and the corresponding background livers (N) were determined using RT-qPCR and in situ hybridization assays. Data are shown as the means±S.E.M.; n =30 in each group. ( d ) Protein levels of ULK1, Beclin-1 and ATG7 were determined using western blotting in 30 pairs of samples. GAPDH was used as an internal control. ( e – h ) Quantitative analyses of the protein and mRNA levels of ULK1, Beclin-1 and ATG7 in 30 pairs of HCC samples. ( i – l ) Pearson's correlation scatter plot of the fold changes of miR-26a/b and ULK1 protein, mRNA in HCC tissues. * P <0.05; ** P <0.01; and *** P <0.001

Article Snippet: For the ULK1 overexpression assay, a pcDNA3.1 vector was designed to specifically express the open reading frame of human ULK1 containing full-length 3′-UTR and purchased from GeneCopoeia (Germantown, MD, USA).

Techniques: Quantitative RT-PCR, In Situ Hybridization, Western Blot, Control

Journal: Cell Death & Disease

Article Title: MiR-26 enhances chemosensitivity and promotes apoptosis of hepatocellular carcinoma cells through inhibiting autophagy

doi: 10.1038/cddis.2016.461

Figure Lengend Snippet: MiR-26a/b enhances the sensitivity of HCC cells to chemotherapeutic drugs and promotes HCC apoptosis by inhibiting autophagy in vitro . ( a ) Representative western blotting analyses of ULK1 in HepG2 cells after treatment with Dox over time. ( b ) Different expression levels of ULK1 in HepG2 and HepG2/Dox cells. ( c ) ULK1 levels in HepG2/Dox cells transfected with/without miR-26 mimics under different treatments. ( d ) HepG2 cells transfected with pre-miR-NC, pre-miR-26, ULK1-vector or pre-miR-26 plus ULK1-vector, treated with/without Dox. Protein levels of ULK1 and LC3 were determined. ( e ) Representative photographs of HepG2 cells transfected with GFP-LC3-expressing plasmids plus pre-miR-NC, pre-miR-26, ULK1-vector or pre-miR-26+ULK1-vector, treated with/without Dox. The right-hand histogram represents a quantitative analysis of GFP-LC3 punctae from 10 micrographs. ( f and g ) Cell viabilities of HepG2 cells under different treatments were determined using a CCK-8 assay at various time points. ( h ) Cell apoptosis of HepG2 cells under various treatments was analyzed using flow cytometry. ( i ) The sensitivities of HepG2 cells under different transfections with Dox were determined using a CCK-8 assay. * P <0.05; ** P <0.01; and *** P <0.001

Article Snippet: For the ULK1 overexpression assay, a pcDNA3.1 vector was designed to specifically express the open reading frame of human ULK1 containing full-length 3′-UTR and purchased from GeneCopoeia (Germantown, MD, USA).

Techniques: In Vitro, Western Blot, Expressing, Transfection, Plasmid Preparation, CCK-8 Assay, Flow Cytometry

Journal: Cell Death & Disease

Article Title: MiR-26 enhances chemosensitivity and promotes apoptosis of hepatocellular carcinoma cells through inhibiting autophagy

doi: 10.1038/cddis.2016.461

Figure Lengend Snippet: Intravenous injections of miR-26a/b-expressing lentivirus enhance the efficiency of chemotherapeutic drugs by inhibiting autophagy in vivo . ( a and b ) Quantitative analysis of miR-26a/b levels in tumors and livers of four mouse groups. ( c ) Western blotting analyses of ULK1, Beclin-1 and ATG7 proteins in the tumors and livers of mice that were treated with PBS, Lenti-miR-26, Dox or Dox plus Lenti-miR-26. ( d ) Representative micrographs of the immunofluorescence staining of LC3 puncta in tumor and liver tissues. Red indicates LC3; blue indicates nuclei. ( e ) Schematic illustrating how miR-26 induces chemosensitivity to drugs. MiR-26 inhibits autophagy and sensitize tumor cells to chemotherapy by suppressing ULK1 and downstream events. Pointed arrows and blunted arrows indicate activation and repression, respectively. * P <0.05; ** P <0.01; and *** P <0.001

Article Snippet: For the ULK1 overexpression assay, a pcDNA3.1 vector was designed to specifically express the open reading frame of human ULK1 containing full-length 3′-UTR and purchased from GeneCopoeia (Germantown, MD, USA).

Techniques: Expressing, In Vivo, Western Blot, Immunofluorescence, Staining, Activation Assay

Journal: Molecular cell

Article Title: ULK1/2 Regulates Stress Granule Disassembly Through Phosphorylation and Activation of VCP/p97

doi: 10.1016/j.molcel.2019.03.027

Figure Lengend Snippet: (A) Integrated ULK1 interactome from in-house–generated proteomic data and public databases (STRING, BIOPLEX, InWeb, and BIOGRID). Underlined proteins indicate genes that are mutated in patients with IBM. Pathway analysis for the ULK1 interactome was performed using ENRICHR and Fisher’s exact test.

Article Snippet: Plasmid Constructs The pCMV6-DDK-human ULK1 (RC215643) and pCMV6-DDK-human ULK2 (RC206010) were purchased from Origene.

Techniques: Generated

Journal: Molecular cell

Article Title: ULK1/2 Regulates Stress Granule Disassembly Through Phosphorylation and Activation of VCP/p97

doi: 10.1016/j.molcel.2019.03.027

Figure Lengend Snippet: (A) Cross-sections of quadriceps from control (Ulk1+/−Ulk2−/flox) and Ulk1/2 hypomorph (Ulk1−/− Ulk2−/flox) mice at 24 weeks were stained with H&E and modified Gomori trichrome staining. Arrow indicates a crescentic inclusion and arrowhead indicates a vacuole. Scale bars, 25 μm.

Article Snippet: Plasmid Constructs The pCMV6-DDK-human ULK1 (RC215643) and pCMV6-DDK-human ULK2 (RC206010) were purchased from Origene.

Techniques: Staining, Modification

Journal: Molecular cell

Article Title: ULK1/2 Regulates Stress Granule Disassembly Through Phosphorylation and Activation of VCP/p97

doi: 10.1016/j.molcel.2019.03.027

Figure Lengend Snippet: (A) Body weight of control, Ulk1/2Ckmm-Cre cDKO, and Atg7Ckmm-Cre cKO mice at 8 weeks of age.

Article Snippet: Plasmid Constructs The pCMV6-DDK-human ULK1 (RC215643) and pCMV6-DDK-human ULK2 (RC206010) were purchased from Origene.

Techniques:

Journal: Molecular cell

Article Title: ULK1/2 Regulates Stress Granule Disassembly Through Phosphorylation and Activation of VCP/p97

doi: 10.1016/j.molcel.2019.03.027

Figure Lengend Snippet: (A) HEK293T cells were transfected with ULK1-DDK or ULK2-DDK and subjected to heat shock at 43°C for 1 h. Immediately following heat shock, cells were lysed and immunoprecipitated for DDK, followed by immunoblot against the indicated proteins.

Article Snippet: Plasmid Constructs The pCMV6-DDK-human ULK1 (RC215643) and pCMV6-DDK-human ULK2 (RC206010) were purchased from Origene.

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: Molecular cell

Article Title: ULK1/2 Regulates Stress Granule Disassembly Through Phosphorylation and Activation of VCP/p97

doi: 10.1016/j.molcel.2019.03.027

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Plasmid Constructs The pCMV6-DDK-human ULK1 (RC215643) and pCMV6-DDK-human ULK2 (RC206010) were purchased from Origene.

Techniques: Recombinant, Western Blot, Software

Journal: iScience

Article Title: Treatment of calcific arterial disease via enhancement of autophagy using GSK343

doi: 10.1016/j.isci.2023.108360

Figure Lengend Snippet: Impaired autophagy is a determinant of VSMC calcification (A) RNA sequencing differential gene expression analysis for calcified versus healthy human VSMCs. Calcified VSMCs were grown in osteogenic media for 3 days. (B) Analysis of canonical pathways impacted by global transcriptomic differential gene expression in calcified VSMCs. (C) Heat map of mRNA expression of autophagy-related genes in healthy versus calcified VSMCs. (D) Quantitative western blot analysis of calcification (RUNX2), autophagy initiation proteins (ULK1, ULK2, ATG13 and p62), and VSMC contractile (CNN1, SM22α) markers along with alizarin red stain for calcium. (E) Quantitative western blot analysis of autophagy flux markers in calcified versus healthy VSMCs in the presence or absence of chloroquine (CQ). (F) Alizarin red staining of human VSMCs under osteogenic conditions indicates calcification is inhibited in cells treated with rapamycin (autophagy activator) and is increased in cells treated with a ULK1 inhibitor (SBI-0206965; autophagy inhibition). (G) (Left panel) Ex vivo treatment of mouse aortas with ULK1 inhibitor shows increased calcification (OsteoSense, red) and inflammation as measured by MMP activity (MMPsense, green). Right panel shows increased calcium deposition in aortas treated with ULK1 inhibitor (alizarin red staining) under osteogenic conditions and increased nuclear Runx2 signal (red) as assessed by immunofluorescence. (H) Alizarin red staining and in situ hybridization for ULK1 mRNA (green) in fixed human tissue from aortic calcification and calciphylaxis patients were compared with healthy tissue, demonstrating reduced ULK1 mRNA levels in the setting of large and small vessel calcification. Significance was calculated using 1-way ANOVA with a Tukey’s multi-comparisons test. Scale bars = 50 um.

Article Snippet: Stellaris® FISH Probes recognizing human ULK1 (SMF-1082-5) and labeled with Quasar® 570-labeled oligos (Biosearch Technologies, Inc., Petaluma, CA) were hybridized to tissue samples, following the manufacturer’s instructions available online at www.biosearchtech.com/stellarisprotocols .

Techniques: RNA Sequencing, Gene Expression, Expressing, Western Blot, Staining, Inhibition, Ex Vivo, Activity Assay, Immunofluorescence, In Situ Hybridization

Journal: iScience

Article Title: Treatment of calcific arterial disease via enhancement of autophagy using GSK343

doi: 10.1016/j.isci.2023.108360

Figure Lengend Snippet: Differential expression of autophagy initiation genes is associated with human coronary artery disease, and therapeutic activation of autophagy inhibits vascular calcification (A) Volcano plot of differentially expressed genes in normal (n = 24) versus ischemic (n = 36) human coronary artery tissues as detected using DESeq2. Significant differentially expressed genes were considered using FDR adjusted p value <0.05, as indicated by the green points. Autophagy genes are labeled with human gene symbols. (B) Violin plots of differentially regulated autophagy genes in normal versus ischemic coronary artery tissues. Values represent log 10 normalized gene expression levels quantified using a collapsed gene model and RNA-SeQC. p values shown are derived from DESeq2 analysis in normal (n = 24) versus ischemic (n = 36) human coronary artery samples. (C) Immunoblot analysis of RUNX2 from total lysates of human VSMCs under normal or osteogenic conditions with or without treatment of autophagy activator (either GSK343 or rapamycin). (D) Treatment with either GSK343 (10 μM) or rapamycin (100 nM), activators of autophagy, inhibited the calcification of human VSMCs grown in calcification media for 10 days, as evidenced by alizarin red stain. (E) Collagen gel contraction assay was performed demonstrating that autophagy activation with either GSK343 or rapamycin restores the contractility of VSMCs that is disrupted when grown in osteogenic media and decreases cellular calcium content (mg/dL) by colorimetric cresolphthalein method. (F) Heat map of the fold change in mRNA levels of autophagy-related genes from human VSMCs grown under osteogenic media treated without or with GSK343 at low (5 μM) or high (10 μM) concentrations compared with VSMCs in normal media. (G) Immunoblot analysis of ULK1, ULK2, p62, and LC3BI/II from total lysates of human VSMCs under normal or osteogenic conditions and treated with GSK343 (10 μM with three replicates). (H) Quantitative western blot analysis of autophagy flux markers in calcified versus healthy VSMCs shows decreased p62 protein in GSK343-treated cells. (I) Pie chart showing the genomic distribution of the differentially accessible chromatin regions from human healthy VSMCs (top panel, left chart), calcified VSMCs (middle chart), and calcified VSMCs treated with GSK343 (right chart). (J) Overlapping ATAC-seq tracks of the promoter regions of the autophagy-initiation genes (ULK1, ATG13A, BECN1), the contractile gene (CNN1), the calcification marker (RUNX2), and the lysosome marker (LAMP1). ATAC-seq peaks for healthy VSMCs are presented in green and for calcified VSMCs in red. The ATAC-seq data have been normalized to take depth into account, and the scale on the y axis was chosen for optimal visualization of peaks. Also, representative ATAC-seq tracks of the autophagy-initiation genes (ULK1 and ATG13A) from VSMCs under osteogenic conditions treated with DMSO (red peaks) or GSK343 (yellow peaks) are presented. Shown are a portion of the promoter regions (1-2Kb). Significance was calculated using 1-way ANOVA with a Tukey’s multi-comparisons test. Scale bars = 50 um.

Article Snippet: Stellaris® FISH Probes recognizing human ULK1 (SMF-1082-5) and labeled with Quasar® 570-labeled oligos (Biosearch Technologies, Inc., Petaluma, CA) were hybridized to tissue samples, following the manufacturer’s instructions available online at www.biosearchtech.com/stellarisprotocols .

Techniques: Quantitative Proteomics, Activation Assay, Labeling, Gene Expression, Derivative Assay, Western Blot, Staining, Collagen Gel Contraction Assay, Marker

Journal: iScience

Article Title: Treatment of calcific arterial disease via enhancement of autophagy using GSK343

doi: 10.1016/j.isci.2023.108360

Figure Lengend Snippet: Therapeutic activation of autophagy by GSK343 inhibits calcification in ex vivo calcification (A) Immunofluorescence staining showing reduced ULK1 (green) and CNN1 (magenta) levels in fresh-fixed tissue of human calcified aortas (left panel) and dermal biopsies from calciphylaxis (right panel) compared with healthy controls. (B) Ex vivo calcification assays were performed with fresh surgical biopsies from patients with either aortic calcification (left panel) or (C) calciphylaxis (right panel). Alizarin red staining shows the calcification levels of surgical biopsies in normal or osteogenic media treated for 21 days with DMSO or GSK343 (20 μM). Treatment with GSK343 inhibited the ex vivo calcification of aortic and calciphylaxis tissues (middle panels) and was associated with increased ULK1 and CNN1 expression (lower panel). (D) In our summative model of autophagy in VSMC calcification, defects in the autophagy initiation machinery at the transcriptional level impair autophagy flux and exacerbate calcification of VSMCs. GSK343 pharmacologically activates autophagy by relaxing chromatin and restoring expression of autophagy initiation genes (e.g., ULK1, BECN1, ATG13) to favor the formation of autolysosomes, which associates with inhibition of VSMC calcification. p values determined as compared with the corresponding control, by one-sample t test. Scale bars = 50 um.

Article Snippet: Stellaris® FISH Probes recognizing human ULK1 (SMF-1082-5) and labeled with Quasar® 570-labeled oligos (Biosearch Technologies, Inc., Petaluma, CA) were hybridized to tissue samples, following the manufacturer’s instructions available online at www.biosearchtech.com/stellarisprotocols .

Techniques: Activation Assay, Ex Vivo, Immunofluorescence, Staining, Expressing, Inhibition, Control

Journal: iScience

Article Title: Treatment of calcific arterial disease via enhancement of autophagy using GSK343

doi: 10.1016/j.isci.2023.108360

Figure Lengend Snippet:

Article Snippet: Stellaris® FISH Probes recognizing human ULK1 (SMF-1082-5) and labeled with Quasar® 570-labeled oligos (Biosearch Technologies, Inc., Petaluma, CA) were hybridized to tissue samples, following the manufacturer’s instructions available online at www.biosearchtech.com/stellarisprotocols .

Techniques: Recombinant, Staining, Contraction Assay, Calcium Colorimetric Assay, Generated