Journal: Life Science Alliance

Article Title: Disruption of stromal hedgehog signaling initiates RNF5-mediated proteasomal degradation of PTEN and accelerates pancreatic tumor growth

doi: 10.26508/lsa.201800190

Figure Lengend Snippet: (A) Western blots and quantification for total and phosphorylated GSK3β Tyr-216 Smo KO or Smo KO fibroblasts. Graph represents quantitation of three individual Western blots relative to Smo WT . N = 3, bars represent means ± SD. (B) Western blots of total and phosphorylated PTEN Thr-366 upon treatment with vehicle or MG132 in Smo KO fibroblasts. N = 3. (C, D) Western blots and quantification for total and phosphorylated PTEN Thr-366 and phosphorylated glycogen synthase (GS) Ser-614 in Smo KO or Smo KO fibroblasts at the indicated time points. Graph represents quantitation of three individual Western blots relative to Smo WT . N = 3, dots represent means ± SD.

Article Snippet: Three GSK3β inhibitors were used: SB-216763 (#sc-200646; Santa Cruz), CT99021 (#CHIR99021; Sigma-Aldrich), and AR-A014418 (#ALX-270-468-M001; Enzo Lifesciences).

Techniques: Western Blot, Quantitation Assay

Journal: Life Science Alliance

Article Title: Disruption of stromal hedgehog signaling initiates RNF5-mediated proteasomal degradation of PTEN and accelerates pancreatic tumor growth

doi: 10.26508/lsa.201800190

Figure Lengend Snippet: Genetic deletion of Smo or pharmacologic inhibition of SMO in pancreatic CAFs activates GSK3β, leading to enhanced PTEN phosphorylation and proteasomal degradation via the E3 ubiquitin ligase enzyme RNF5. Subsequent AKT activation leads to enhanced GLI2 binding and activation of the Tgfa promoter. TGF-α production by SMO-null fibroblasts cross-talks with the adjacent tumor cells, leading to activation of epidermal growth factor receptor (EGFR) and accelerated growth of the tumor epithelium.

Article Snippet: Three GSK3β inhibitors were used: SB-216763 (#sc-200646; Santa Cruz), CT99021 (#CHIR99021; Sigma-Aldrich), and AR-A014418 (#ALX-270-468-M001; Enzo Lifesciences).

Techniques: Inhibition, Phospho-proteomics, Ubiquitin Proteomics, Activation Assay, Binding Assay

Journal: PLoS ONE

Article Title: miR-27a-3p regulates expression of intercellular junctions at the brain endothelium and controls the endothelial barrier permeability

doi: 10.1371/journal.pone.0262152

Figure Lengend Snippet: (A) Schematic representation of the GSK3ß 3’-UTR with the miR-27a-3p binding site. Complementary sequences are represented in blue. (B) Effect of miR-27a-3p mimic on GSK3ß 3’UTR luciferase reporters (wild type and mutant). Results are expressed as the fold change of the ratio (firefly to Renilla luciferase activity). Experiments were carried out three times with tests performed in triplicates for each experiment. (C, D) Relative mRNA expression of GSK3ß (C) and ß-catenin (D) measured by real-time PCR in hCMEC/D3 cells transfected with miR-27a-3p mimic or control for 72h. GAPDH was used as an internal standard. Data are represented as (2 ^-ΔCt ). Experiments were carried out three times with PCR performed in duplicates for each experiment. (E, F) Protein expression of the GSK3ß (E) and nuclear ß-catenin (F) measured by western-blot in hCMEC/D3. Optical densities of three independent images were analyzed with Image Lab 6.0.1 software(Bio-Rad) and normalized to GAPDH. Results are represented as normalized optical densities. Experiments were carried out three to six times with each preparation representing pooled protein lysates from monolayer cultures performed in triplicates. Data represent mean ± SD from the independent experiments (biological replicates). *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 .

Article Snippet: For GSK3ß inhibition, cells were transfected with 20 nM of GSK3B siRNA (cat # sc-35527, Santa Cruz), and for co-transfection of GSK3ß-siRNA/miR-27a-3p miRNA inhibitor, cells were co-transfected with 20 nM of GSK3B siRNA and 30 nM of miR-27a-3p inhibitor or AllStars negative control for 72h.

Techniques: Binding Assay, Luciferase, Mutagenesis, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Control, Western Blot, Software

Journal: PLoS ONE

Article Title: miR-27a-3p regulates expression of intercellular junctions at the brain endothelium and controls the endothelial barrier permeability

doi: 10.1371/journal.pone.0262152

Figure Lengend Snippet: hCMEC/D3 cells were transfected with miR-27a-3p inhibitor and/or GSK3B siRNA or negative control for 72h. (A) Protein expression of GSK3ß, nuclear ß-catenin, claudin-5 and occludin measured by western-blot in hCMEC/D3. Optical densities of three independent images were analyzed with Image Lab 6.0.1 software(Bio-Rad) and normalized to GAPDH. Results are represented as normalized optical densities. Experiments were carried out three to four times with each preparation representing pooled protein lysates from monolayer cultures performed in triplicates. (B) Transendothelial electrical resistance (TEER) of hCMEC/D3 cells treated with miR-27a-3p inhibitor and/or GSK3B siRNA or negative control after 72 hours of transfection. Experiments were carried out six times with monolayer cultures performed in triplicates. (C, D) The permeability coefficient (P e , cm/s) of the endothelial monolayer assessed by the 4 (C) and 70 kDa FITX-dextran flux assay (D). Experiments were carried out five times with monolayer cultures performed in triplicates. Data represent mean ± SD from the independent experiments (biological replicates). *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 .

Article Snippet: For GSK3ß inhibition, cells were transfected with 20 nM of GSK3B siRNA (cat # sc-35527, Santa Cruz), and for co-transfection of GSK3ß-siRNA/miR-27a-3p miRNA inhibitor, cells were co-transfected with 20 nM of GSK3B siRNA and 30 nM of miR-27a-3p inhibitor or AllStars negative control for 72h.

Techniques: Transfection, Negative Control, Expressing, Western Blot, Software, Permeability, Flux Assay

Journal: Cell Systems

Article Title: Genome-Scale Oscillations in DNA Methylation during Exit from Pluripotency

doi: 10.1016/j.cels.2018.06.012

Figure Lengend Snippet:

Article Snippet: Gsk-3β inhibitor CHIR9902 , Wellcome – MRC Cambridge Stem Cell Institute , https://www.stemcells.cam.ac.uk/research/facilities/tissueculture.

Techniques: Recombinant, Methylation, Sequencing, Amplification, Software