Journal: Molecular Biology of the Cell

Article Title: Posttranslational Modifications of Tubulin and the Polarized Transport of Kinesin-1 in Neurons

doi: 10.1091/mbc.E09-01-0044

Figure Lengend Snippet: Inhibition of GSK3β results in increased levels of tubulin PTMs and disrupts the polarized sorting of CA-Kinesin-1 and JIP1 in polarized stage 3 and unpolarized stage 2 cells. (A–C) Effects of GSK3β inhibition in polarized stage 3 cells. (A) Stage 3 cortical neurons were treated for 8 h with 10 μM SB216763. Cell lysates were analyzed by Western blotting with antibodies to acetylated α-tubulin, detyrosinated α-tubulin, polyglutamylated tubulin, or total β-tubulin. (B) Stage 3 hippocampal neurons were treated for 2–4 h with DMSO or 5–10 μM SB216763. The cells were then transfected with CA-Kinesin-mCherry and soluble YFP, allowed to express the exogenous proteins under additional treatment for 4–5 h, and then fixed and imaged. (C) Quantification of the percentage of stage 3 minor neurites or stage 5 dendrites that have accumulated CA-Kinesin-mCherry after treatment described in B. (D–F) Effects of GSK3β inhibition in unpolarized stage 2 cells. (D) Stage 2 hippocampal neurons expressing CA-Kinesin-1-mCit since the time of plating were treated for 8 h with DMSO or 10 μM SB216763 and then fixed and stained for total β-tubulin. (E) Stage 2 hippocampal neurons were treated for 8 h with DMSO or 10 μM SB216763 and then fixed and stained with antibodies to JIP1 and acetylated α-tubulin. (F) Quantification of the percentage of stage 2 neurites with CA-Kinesin-1 (D) or JIP1 (E) accumulated at the tips after treatment with DMSO or SB216763. Bars, 20 μm. Error bars, SEM. t test: *p < 0.01 compared with control DMSO-treated cells.

Article Snippet: Cells were treated for the indicated times and concentrations with Taxol (Sigma-Aldrich), the tubulin deacetylase inhibitors trichostatin A (TSA; Sigma-Aldrich) or a close structural analog of tubacin, MAZ1370, that is more potent in cell-based assays and referred to in the text as tubacin for simplicity ( Reed et al. , 2006 ), or the glycogen synthase kinase (GSK)3β inhibitor SB216763 (Sigma-Aldrich).

Techniques: Inhibition, Western Blot, Transfection, Expressing, Staining

Journal: Cancer cell

Article Title: Small Molecule MYC Inhibitors Suppress Tumor Growth and Enhance Immunotherapy

doi: 10.1016/j.ccell.2019.10.001

Figure Lengend Snippet: Key Resources Table

Article Snippet: GSK-3β (3D10) , Cell Signaling Technology , 9832S.

Techniques: Control, Virus, Staining, Recombinant, SYBR Green Assay, Magnetic Beads, Transfection, Protease Inhibitor, Luciferase, Proliferation Assay, Enzyme-linked Immunosorbent Assay, In Situ, Derivative Assay, Construct, Expressing, Software

Journal: iScience

Article Title: Type 2 diabetes sex-specific effects associated with E167K coding variant in TM6SF2

doi: 10.1016/j.isci.2021.103196

Figure Lengend Snippet:

Article Snippet: Phospho-GSK-3β (Ser9) (D85E12) Rabbit mAb , Cell Signaling , #5558; RRID: AB_10013750.

Techniques: Virus, Recombinant, Protease Inhibitor, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Microarray, Software

Journal: iScience

Article Title: Type 2 diabetes sex-specific effects associated with E167K coding variant in TM6SF2

doi: 10.1016/j.isci.2021.103196

Figure Lengend Snippet:

Article Snippet: GSK-3β (D5C5Z) Rabbit mAb , Cell Signaling , #12456; RRID: AB_2636978.

Techniques: Virus, Recombinant, Protease Inhibitor, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Microarray, Software

Journal: Cellular and Molecular Immunology

Article Title: MCP-1-induced ERK/GSK-3β/Snail signaling facilitates the epithelial–mesenchymal transition and promotes the migration of MCF-7 human breast carcinoma cells

doi: 10.1038/cmi.2015.106

Figure Lengend Snippet: MCP-1 treatment enhances ERK and GSK-3β (Ser9) phosphorylation in MCF-7 cells. (a) MCP-1 induces ERK activation in a time-dependent manner. MCF-7 cells were serum-starved overnight and treated with 50 ng/ml of MCP-1 for the indicated times. Total protein was subjected to western blotting using anti-p-ERK and Akt antibodies. β-actin was used as a loading control. (b) MCP-1 leads to the inactivation of GSK-3β. MCF-7 cells were serum-starved overnight and treated with 50 ng/ml of MCP-1 or RS for 1 h, and total protein was subjected to immunoblotting with anti-p-ERK, ERK, p-GSK-3β, GSK-3β and Snail antibodies. (c) Inhibition of the MCP-1 receptor CCR2 by RS reverses the expression of EMT biomarkers. The expression levels of E-cad, FN, Vim and β-actin were detected by western blotting. CCR2, cysteine–cysteine chemokine receptor 2; EMT, epithelial–mesenchymal transition; FN, fibrinogen; GSK-3β, glycogen synthase kinase-3β MCP-1, monocyte chemoattractant protein-1.

Article Snippet: After culture in 12- or 24-well plates overnight, the cells were incubated for a period of time as indicated with one of the following treatments: 50 ng/ml of MCP-1 (PeproTech, Rocky Hill, CT, USA); 20 μM of the CCR2 receptor antagonist RS102895 (Santa Cruz Biotech, Santa Cruz, CA, USA); 30 μM of the MEK1/2 inhibitor U0126 (Sigma, Carlsbad, CA, USA); 40 μM of the GSK-3β inhibitor lithium chloride (LiCl; Sigma); or 10 μM of the PI3K inhibitor LY294002 (Sigma).

Techniques: Activation Assay, Western Blot, Inhibition, Expressing

Journal: Cellular and Molecular Immunology

Article Title: MCP-1-induced ERK/GSK-3β/Snail signaling facilitates the epithelial–mesenchymal transition and promotes the migration of MCF-7 human breast carcinoma cells

doi: 10.1038/cmi.2015.106

Figure Lengend Snippet: Inhibition of ERK abolishes the MCP-1-induced changes in GSK-3β, Snail, EMT biomarker expression, and cell migration and invasion. MCF-7 cells were incubated with or without U0126 for 1 h before MCP-1 treatment. (a, b) Cell lysates were collected and the protein levels of p-ERK, p-GSK-3β (Ser9), Snail, FN, Vim and E-cad were measured by western blotting. (c) The morphological changes were observed under phase contrast microscopy. (d, e) MCF-7 cells were treated as described above and analyzed for changes in migration by a wound closure assay (d) and a transwell assay (e). *P<0.05 MCP-1 vs control; #P<0.05 U1026 or U1026+MCP-1 vs MCP-1. GSK-3β, glycogen synthase kinase-3β MCP-1, monocyte chemoattractant protein-1.

Article Snippet: After culture in 12- or 24-well plates overnight, the cells were incubated for a period of time as indicated with one of the following treatments: 50 ng/ml of MCP-1 (PeproTech, Rocky Hill, CT, USA); 20 μM of the CCR2 receptor antagonist RS102895 (Santa Cruz Biotech, Santa Cruz, CA, USA); 30 μM of the MEK1/2 inhibitor U0126 (Sigma, Carlsbad, CA, USA); 40 μM of the GSK-3β inhibitor lithium chloride (LiCl; Sigma); or 10 μM of the PI3K inhibitor LY294002 (Sigma).

Techniques: Inhibition, Biomarker Assay, Expressing, Migration, Incubation, Western Blot, Microscopy, Wound Closure Assay, Transwell Assay

Journal: Cellular and Molecular Immunology

Article Title: MCP-1-induced ERK/GSK-3β/Snail signaling facilitates the epithelial–mesenchymal transition and promotes the migration of MCF-7 human breast carcinoma cells

doi: 10.1038/cmi.2015.106

Figure Lengend Snippet: Pretreatment of MCF-7 cells with LiCl (a GSK-3β inhibitor) abrogates the MCP-1-induced changes in Snail, EMT biomarker expression, and cell migration and invasion. MCF-7 cells were incubated with or without LiCl for 1 h before MCP-1 treatment. (a, b) Cell lysates were collected and protein levels of p-GSK-3β (Ser9), Snail, FN, Vim and E-cad were measured by western blotting. (c) The morphological changes were observed under phase contrast microscopy. (d, e) MCF-7 cells were treated as described above and analyzed for changes in migration by a wound closure assay (d) and a transwell assay (e). *P<0.05 MCP-1 vs control; #P<0.05 U1026 or U1026+MCP-1 vs MCP-1.

Article Snippet: After culture in 12- or 24-well plates overnight, the cells were incubated for a period of time as indicated with one of the following treatments: 50 ng/ml of MCP-1 (PeproTech, Rocky Hill, CT, USA); 20 μM of the CCR2 receptor antagonist RS102895 (Santa Cruz Biotech, Santa Cruz, CA, USA); 30 μM of the MEK1/2 inhibitor U0126 (Sigma, Carlsbad, CA, USA); 40 μM of the GSK-3β inhibitor lithium chloride (LiCl; Sigma); or 10 μM of the PI3K inhibitor LY294002 (Sigma).

Techniques: Biomarker Assay, Expressing, Migration, Incubation, Western Blot, Microscopy, Wound Closure Assay, Transwell Assay

Journal: Cellular and Molecular Immunology

Article Title: MCP-1-induced ERK/GSK-3β/Snail signaling facilitates the epithelial–mesenchymal transition and promotes the migration of MCF-7 human breast carcinoma cells

doi: 10.1038/cmi.2015.106

Figure Lengend Snippet: The MCP-1-induced EMT of MCF-7 cells is partially dependent on the PI3K/Akt signaling pathway. (a) The cells were incubated with or without 20 nM LY294002 (LY) for 1 h before MCP-1 treatment. The protein levels of p-Akt, Akt, p-GSK-3β (Ser9), GSK-3β, p-ERK and ERK were determined by western blotting. (b) EMT phenotypic changes were observed under phase contrast microscopy. All the data are representative of three independent experiments.

Article Snippet: After culture in 12- or 24-well plates overnight, the cells were incubated for a period of time as indicated with one of the following treatments: 50 ng/ml of MCP-1 (PeproTech, Rocky Hill, CT, USA); 20 μM of the CCR2 receptor antagonist RS102895 (Santa Cruz Biotech, Santa Cruz, CA, USA); 30 μM of the MEK1/2 inhibitor U0126 (Sigma, Carlsbad, CA, USA); 40 μM of the GSK-3β inhibitor lithium chloride (LiCl; Sigma); or 10 μM of the PI3K inhibitor LY294002 (Sigma).

Techniques: Incubation, Western Blot, Microscopy

Journal: Cellular and Molecular Immunology

Article Title: MCP-1-induced ERK/GSK-3β/Snail signaling facilitates the epithelial–mesenchymal transition and promotes the migration of MCF-7 human breast carcinoma cells

doi: 10.1038/cmi.2015.106

Figure Lengend Snippet: Schematic representation of the proposed mechanism for MCP-1-induced EMT in MCF-7 cells. MCP-1 treatment leads to the activation of the MEK/ERK or PI3K/Akt/ERK signaling pathways. Activated ERK leads to the inactivation of GSK-3β by phosphorylation of the Ser9 active site, which leads to the disassociation of Snail from GSK-3β. Snail then translocates to the nucleus where it functions as a transcription factor to regulate FN, Vim and E-cad to initiate EMT or VEGF, MMP-2 and MMP-9 to promote cell migration and invasion.

Article Snippet: After culture in 12- or 24-well plates overnight, the cells were incubated for a period of time as indicated with one of the following treatments: 50 ng/ml of MCP-1 (PeproTech, Rocky Hill, CT, USA); 20 μM of the CCR2 receptor antagonist RS102895 (Santa Cruz Biotech, Santa Cruz, CA, USA); 30 μM of the MEK1/2 inhibitor U0126 (Sigma, Carlsbad, CA, USA); 40 μM of the GSK-3β inhibitor lithium chloride (LiCl; Sigma); or 10 μM of the PI3K inhibitor LY294002 (Sigma).

Techniques: Activation Assay, Migration

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