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: Methods in molecular biology (Clifton, N.J.)

Article Title: Measuring Autophagy in Stressed Cells

doi: 10.1007/978-1-4939-2522-3_10

Figure Lengend Snippet: Summary of western blot methods for key autophagy proteins

Article Snippet: Tandem mCherry/GFP-LC3 fusion expression plasmid (Addgene #22418). shRNAs to Atg5 (Sigma-Aldrich custom, see text below), Atg7 (Sigma-Aldrich TRCN0000375444), Beclin1 (sigma), Ulk1 (Addgene #27633), Ulk2 (Addgene #27634), p62 (Sigma-Aldrich TRCN0000098619). siRNAs to Atg5 (Dharmacon L-004374-00-0005), Atg12 (Thermo Scientific siGenome SMARTpool M-050953-01), Ulk1 (Thermo Scientific siGenome SMARTpool M040155-00).

Techniques: Western Blot, Extraction, Membrane, Blocking 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: KEY RESOURCES TABLE

Article Snippet: pCMV6-DDK-human ULK1 , Origene , RC215643.

Techniques: Recombinant, Western Blot, Software