Journal: Cell Death & Disease

Article Title: Tissue-specific deregulation of selected HDACs characterizes ALS progression in mouse models: pharmacological characterization of SIRT1 and SIRT2 pathways

doi: 10.1038/cddis.2014.247

Figure Lengend Snippet: G93A-SOD1 toxicity is not mediated by p53 acetylation state or by IRS-2/Ras/ERK1/2 pathway in SH-SY5Y cells. ( a ) Western blot analysis of 20 μ M of total protein extract from cells infected with adenoviral vectors coding for Wt-SOD1 (Wt) and G93A-SOD1 (G93A) and treated with 3 μ M Ex527 or DMSO. Antibodies against SIRT1, Erk1/2, pErk1/2, p53, p53-Ac, tubulin and Ac-tubulin were used. β -Actin was used as loading control. One representative blot is shown from three independent experiments giving comparable results. ( b ) Densitometric analysis of results as in ( a ); data are expressed as acetylation ratio of p53 and tubulin. ( c ) Cells infected with adenoviral vectors coding for Wt-SOD1 (Wt) and G93A-SOD1 (G93A) were treated with 3 μ M Ex527 or with 3 μ M SL327 or both. Cell viability was assessed and reported for . Values significantly different from relative controls are indicated with * P <0.05 with respect to Ctrl and # P <0.05 with respect to DMSO. ( d ) Western blot analysis of 20 μ M of total protein extract from C2C12 cells differentiated for 5 days and expressing G93A-SOD1 (G93A) or untransfected (Ctrl). Antibodies against SIRT1, p53 and p53-Ac were used. β -Actin was used as loading control. One representative blot is shown from three independent experiments giving comparable results. ( e ) Densitometric analysis of data as in ( d ); data are expressed as acetylation ratio of p53

Article Snippet: AGK2, Ex527, Sirtinol, SRT1720 and SL327 were from Tocris Bioscience (Bristol, UK); all of them were resuspended in an appropriate volume of DMSO and kept stored at −80°C before use.

Techniques: Western Blot, Infection, Control, Expressing

Journal: Molecular cell

Article Title: An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells

doi: 10.1016/j.molcel.2019.05.017

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Biotinylated lectins (Vector Laboratories, Burlingame, CA) were incubated at different concentrations for 1 h, followed by washing and incubation with Streptavidin-Alexa Fluor488 (Invitrogen) for 1 h. Recombinant human Siglec Fc-chimera (R&D systems, Minneapolis, MN) were precomplexed with Alexa Fluor647 goat anti-human IgG antibody (Life Technologies) for 1 h followed by incubation with cells for 1 hr.

Techniques: Recombinant, Plasmid Preparation, Construct, Software

Journal: Cancer research

Article Title: Asporin Restricts Mesenchymal Stromal Cell Differentiation, Alters the Tumor Microenvironment, and Drives Metastatic Progression

doi: 10.1158/0008-5472.CAN-18-2931

Figure Lengend Snippet: ASPN binds to BMP-4 and restricts BMP-4-induced signaling. A, GSEA of TGF-β family pathway genes in Aspn+/+ and Aspn−/− fetal MSCs as determined from microarray (n=3). B, C, BMP-4-induced signaling in Aspn+/+ and Aspn−/− fetal MSCs (A and B represent independently derived MSCs) as measured by immunoblotting and quantified using ImageJ. Aspn+/+ and Aspn−/− fetal MSCs were serum-depleted and then incubated with recombinant mouse BMP-4 (5ng/mL) for 30 minutes. Statistical analyses performed using Student’s t-test (n≥3). D, E, Recombinant mouse ASPN restricts BMP-4-induced signaling in Aspn−/− fetal MSCs as measured by immunoblotting and quantified using ImageJ. Aspn+/+ and Aspn−/− fetal MSCs were serum-depleted and then incubated with recombinant mouse BMP-4 (5ng/mL) with vehicle or recombinant mouse ASPN (100ng/mL) for 30 minutes. Statistical analyses performed using Student’s t-test (n≥2). F, G, BMP-4-induced signaling in WPMY-1-Neo, WPMY-1-ASPN D13, and WPMY-1ASPN D14 (A and B represent independently derived clones) as measured by immunoblotting and quantified using ImageJ. WPMY-1-Neo, WPMY-1-ASPN D13, and WPMY-1ASPN D14 were serum-depleted and then incubated with recombinant human BMP-4 (5ng/mL) for 30 minutes. Statistical analyses performed using one-way ANOVA with Tukey multiple comparison (n≥3). H, Co-immunoprecipitation of ASPN and BMP-4. FLAG tagged mouse ASPN was transfected in HEK293T cells and then incubated with recombinant mouse BMP-4. ASPN was immunoprecipitated with anti-FLAG beads and then examined by immunoblotting for ASPN and BMP-4 (n=2). I, Aspn+/+ and Aspn−/− fetal MSCs were cultured in osteogenic-inducing media with either vehicle, 250nM LDN-193189, or recombinant mouse BMP4 (5ng/mL) and then stained for Alizarin Red. J, Aspn+/+ and Aspn−/− fetal MSCs were cultured in osteogenic-inducing media with either vehicle, 250nM LDN-193189, or recombinant mouse BMP4 (5ng/mL) and then examined for osteogenic-induced genes by qRT-PCR. Statistical analyses performed using one-way ANOVA with Tukey multiple comparison (n≥3). Graphs shown as mean±SEM, *P≤0.05, **P≤0.01, ***P≤0.001, and black bars=100μM.

Article Snippet: Co-immunoprecipitation assays HEK293T cells were transfected with a murine ASPN-Myc-FLAG tagged expression vector (Origene) or vehicle control using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Techniques: Microarray, Derivative Assay, Western Blot, Incubation, Recombinant, Clone Assay, Immunoprecipitation, Transfection, Cell Culture, Staining, Quantitative RT-PCR

Journal: Cancer research

Article Title: Asporin Restricts Mesenchymal Stromal Cell Differentiation, Alters the Tumor Microenvironment, and Drives Metastatic Progression

doi: 10.1158/0008-5472.CAN-18-2931

Figure Lengend Snippet: Key Resources

Article Snippet: Co-immunoprecipitation assays HEK293T cells were transfected with a murine ASPN-Myc-FLAG tagged expression vector (Origene) or vehicle control using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Techniques: Plasmid Preparation, Magnetic Beads, Recombinant, Transfection, Staining, Generated, Blocking Assay, Bicinchoninic Acid Protein Assay, Cell Migration Assay, Mutagenesis, Random Hexamer Labeling, Expressing, Software

Journal: Frontiers in Plant Science

Article Title: Macropinocytosis in Gracilariopsis lemaneiformis (Rhodophyta)

doi: 10.3389/fpls.2023.1225675

Figure Lengend Snippet: The genes encoding phosphatidylinositol kinase in Gracilariopsis lemaneiformis genome. (A) Phylogenetic analysis of phosphatidylinositol kinase members. The tree was constructed using a maximum likelihood method, and the bootstrap values (1,000 replicates) are shown on each node. Hs, Homo sapiens ; Dm, Drosophila melanogaster ; Dd, Dictyostelium discoideum ; Sc, Saccharomyces cerevisiae ; Cs, Coccomyxa subellipsoidea ; Pp, Physcomitrium patens ; At, Arabidopsis thaliana ; Gc, Gracilariopsis chorda ; Cr, Capsella rubella ; Sp, Schizosaccharomyces pombe ; Tp, Thalassiosira pseudonana ; Pm, Plasmodium malariae ; Ap, Aureobasidium pullulans ; RsJEL0117, Rhizoclosmatium sp. JEL0117. (B) Conserved domain of the proteins that blasted by phosphatidylinositol kinase from G. lemaneiformis protein database. (C) Statistical histogram of the number of endocytotic vesicles in non-epidermal cells under different concentrations of wortmannin. “*” indicate statistically significant differences (p < 0.05) between the experimental group (10 μM or 20 μM) and control group (0 μM) by Student’s t-test, n=34.

Article Snippet: For determining the inhibition of endocytosis, different inhibitors [5-(N-ethyl-N-isopropyl)] amiloride (EIPA) (MedChemExpress, Cat. HY-101840), lacunculin B (Aladdin, Cat. L275457), and wortmannin (MedChemExpress, Cat. HY-10197) were incubated with slices for 1 h, and then, the slices were incubated with the mixture of inhibitors and FITC-dextran for 50 min. After fixing in 4% paraformaldehyde (Solarbio, Cat. P1110) at 4°C for 2 h, the slices were observed using a laser confocal microscope (Nikon A1R HD25, Japan).

Techniques: Construct, Control