Journal: Cell Death & Disease

Article Title: IMP2/p62 induces genomic instability and an aggressive hepatocellular carcinoma phenotype

doi: 10.1038/cddis.2015.241

Figure Lengend Snippet: p62 promotes DLK1-RAC1-driven ROS generation. ( a and b ) Levels of RAC1 mRNA presented as mean±S.E.M. ( a ) and activated RAC1 protein levels determined by pull-down assay ( b ) in HepG2 cells after treatment with 1 μ g/ml DLK1 protein ( n =3 in duplicate). ( b ) Representative pull-down assay with activated RAC1 (aRAC1) and total RAC1 (tRAC1) is shown. X-fold signal intensities of 5-min treatment with DLK1 were normalized to untreated control (co). ( c ) ROS levels: representative experiment (quintuplicates) of HepG2 cells treated with 0.5 or 1 μ g/ml DLK1 or H 2 O 2 as positive control for 0–30 min (upper part). Data are normalized to untreated HepG2 cells. ROS levels in HepG2 cells treated with either DLK1 or RAC1 inhibitor NSC23766 alone or in combination (lower part). Untreated HepG2 cells served as control. H 2 O 2 -induced ROS formation was set to 100% ( n =2, quintuplicate). Data are presented as mean±S.E.M. ( d ) RAC1 expression in HepG2 cells overexpressing (left) p62 sense plasmid (p62) compared with antisense-plasmid (co-v), untreated control (co), and siRNA knockdown (right) of p62 (si p62) compared with random siRNA (si co; n =3 triplicate/quadruplicate). Data show mean±S.E.M. Western blot knockdown/overexpression control was densitometrically quantified ( n =4 triplicate/quadruplicate; upper part). ( e ) RAC1 expression in human HCC (GSE14520) normalized to the mean of normal samples. ( f ) Overview of p62-promoted DLK1-RAC1-induced genomic instability. DLK1-overexpressing cells with stem-cell-like features secrete DLK1 protein, which activates RAC1 in a paracrine manner, in turn leading to ROS generation via NADPH oxidase. Elevated ROS levels finally result in genomic instability

Article Snippet: Knockdown and overexpression experiments for p62 in HepG2 were performed as previously described. p62 sense and antisense constructs are available at Addgene (#42174 and #42175, Cambridge, MA, USA).

Techniques: Pull Down Assay, Control, Positive Control, Expressing, Plasmid Preparation, Knockdown, Western Blot, Over Expression

Journal: Cell reports

Article Title: Pancreatic islet protection at the expense of secretory function involves serine-linked mitochondrial one-carbon metabolism

doi: 10.1016/j.celrep.2023.112615

Figure Lengend Snippet: (A) Scheme of adenoviral GFP (Ad- GFP ) and human ATF4 (Ad- ATF4 ) plasmid transduction into dispersed mouse islet cells and subsequent generation of pseudo-islets. Representative images of pseudo-islets infected with Ad- GFP . Scale bar, 100 μm. (B–H) Relative mRNA expression of (B) human ATF4 , (C) Phgdh , (D) Psat1 , (E) Psph , (F) Shmt2 , (G) Mthfd2 , and (H) Aldh1l2 in pseudo-islets infected with Ad- GFP or Ad- ATF4 . Reproduced in one (C and E) and three (D and F–H) independent experiments. (I) Immunoblot of Aldh1l2, Phgdh, ATF4, Beta-actin, and GFP in pseudo-islets infected with Ad- GFP as control or Ad- ATF4 . Relative protein expression was normalized to GFP expression levels. Reproduced in two independent experiments. (J) Insulin secretion of Ad- GFP and Ad- ATF4 infected pseudo-islets, after low (2 mM) and high (20 mM) glucose stimulation. Reproduced in two independent experiments. (K) Insulin content of infected pseudo-islets. Reproduced in two independent experiments. (L) Flow cytometry analysis of cell viability using FVS660. Histograms of Ad- GFP or Ad- ATF4 infected mouse islet cells, demonstrating live (FL4-H negative) and dead (FL4-H positive) cell populations after treatment with 2.5 mM STZ for 24 h. Reproduced in two independent experiments. (M) Bright-field images of infected pseudo-islets after 24 h of 2.5 mM STZ treatment. Scale bar, 100 μm. Please note that each dot in the insulin secretion and content panels represents an independent well containing seven or eight islets each, and data are presented as mean ± SEM with corresponding p values and quantifications shown as percentage of Ad- GFP control. Statistical significance was evaluated by two-tailed unpaired Student’s t test (B–H and K) or two-way ANOVA followed by Tukey’s multiple comparison test (J).

Article Snippet: For efficient overexpression of ATF4 in pancreatic islets, dispersed islet cells were infected for 6 h in a humidified incubator at 37°C and 5% CO 2 with a multiplicity of infection (MOI) of 200 with adenoviruses containing either a human ATF4 overexpression plasmid (Vector Biolabs, # ADV-201618, RefSeq: BC011994, further designated as Ad- ATF4 ) or a GFP control plasmid (ViraQuest Inc., VQAd CMV eGFP, further designated as Ad- GFP ), followed by the formation of pseudo-islets by hanging drop technique.

Techniques: Plasmid Preparation, Transduction, Infection, Expressing, Western Blot, Flow Cytometry, Two Tailed Test, Comparison

Journal: Cell reports

Article Title: Pancreatic islet protection at the expense of secretory function involves serine-linked mitochondrial one-carbon metabolism

doi: 10.1016/j.celrep.2023.112615

Figure Lengend Snippet:

Article Snippet: For efficient overexpression of ATF4 in pancreatic islets, dispersed islet cells were infected for 6 h in a humidified incubator at 37°C and 5% CO 2 with a multiplicity of infection (MOI) of 200 with adenoviruses containing either a human ATF4 overexpression plasmid (Vector Biolabs, # ADV-201618, RefSeq: BC011994, further designated as Ad- ATF4 ) or a GFP control plasmid (ViraQuest Inc., VQAd CMV eGFP, further designated as Ad- GFP ), followed by the formation of pseudo-islets by hanging drop technique.

Techniques: Virus, Plasmid Preparation, Recombinant, Fractionation, Enzyme-linked Immunosorbent Assay, Software, Staining