Journal: Scientific Reports

Article Title: Casein Kinase 1 Epsilon Regulates Glioblastoma Cell Survival

doi: 10.1038/s41598-018-31864-x

Figure Lengend Snippet: CK1ε is highly expressed in GBM. ( A ) mRNA levels of CK1 genes in four GBM cell lines. Data was retrieved from the CellMiner database. The arbitrary copy numbers are shown. Error bars represent standard deviations from four different sets of data. ( B ) mRNA levels of CK1 genes in normal and GBM tissues. Data was retrieved from the Oncomine database. Fold changes of mRNAs in GBM tissues over mRNAs in normal brain tissues are shown. P values determine the statistical significance of mRNA difference between GBM and normal brain tissues. N/A: not available. ( C ) Immunofluorescence analysis of CK1ε in U251 cells. Green: CK1ε; Blue: nuclei. Data were from The Human Protein Atlas. ( D ) Immunohistochemical analyses of CK1ε in normal brain tissues and specimens of high-grade glioma. Data were from the Human Protein Atlas. N.D.: not detected.

Article Snippet: Immunofluorescence images of CK1ε in U251 cells and images of immunohistochemical analyses of CK1ε in cerebral cortex and high-grade glioma were obtained from the Human Protein Atlas.

Techniques: Immunofluorescence, Immunohistochemical staining

Journal: Scientific Reports

Article Title: Casein Kinase 1 Epsilon Regulates Glioblastoma Cell Survival

doi: 10.1038/s41598-018-31864-x

Figure Lengend Snippet: CK1ε is important for GBM cell survival. ( A ) Knockdown of CK1ε in nine GBM cell lines. GBM cells were treated with non-silencing (NS) or CK1ε shRNA. Protein levels of CK1ε in U87MG cells are shown in the left panel. ACTB (β-actin) is the loading control. Band intensities were quantified using Image J. The viability of GBM cell lines was determined by the MTS viability assay and is shown in the right panel. ( B ) Knockdown of MELK in nine GBM cell lines. Left panel: immunoblotting of MELK in U87MG cells. Right panel: cell viability. ( C ) Viability of primary GBM cells upon depletion of CK1ε or MELK. ( D ) Knockdown of CK1ε in astrocytes. Top panel: cell viability; bottom panel: immunoblotting of CK1ε. Full length blots were presented in supplemental materials. * P < 0.05; # P > 0.05.

Article Snippet: Immunofluorescence images of CK1ε in U251 cells and images of immunohistochemical analyses of CK1ε in cerebral cortex and high-grade glioma were obtained from the Human Protein Atlas.

Techniques: Knockdown, shRNA, Control, Viability Assay, Western Blot

Journal: Scientific Reports

Article Title: Casein Kinase 1 Epsilon Regulates Glioblastoma Cell Survival

doi: 10.1038/s41598-018-31864-x

Figure Lengend Snippet: Cell responses to CK1ε depletion do not correlate with levels of CK1ε. ( A ) Immunoblotting of CK1ε in GBM cell lines. ACTB (β-actin) is the loading control. Band intensities were quantified using Image J. ( B ) Correlation of CK1ε protein levels and viability of CK1ε-deficient GBM cell lines. A linear regression model was used to determine the correlation between CK1ε levels and GBM cell viability upon CK1ε depletion (Fig. , right panel). R square is the coefficient of determination. ( C ) Immunoblotting of MELK in GBM cell lines. ( D ) Correlation of MELK protein levels and viability of GBM cell lines. Full length blots were presented in supplemental materials.

Article Snippet: Immunofluorescence images of CK1ε in U251 cells and images of immunohistochemical analyses of CK1ε in cerebral cortex and high-grade glioma were obtained from the Human Protein Atlas.

Techniques: Western Blot, Control

Journal: Scientific Reports

Article Title: Casein Kinase 1 Epsilon Regulates Glioblastoma Cell Survival

doi: 10.1038/s41598-018-31864-x

Figure Lengend Snippet: CK1ε depletion induces apoptosis and growth inhibition through activating β-catenin. ( A ) β-catenin signaling in CK1ε-deficient U87MG cells. Phosphorylated, active, and total β-catenin was assessed using immunoblotting. ACTB (β-actin) is the loading control. Band intensities were quantified using Image J. ( B ) β-catenin signaling in CK1ε-deficient A172 cells. ( C ) Luciferase reporter assay. The TOPFlash plasmid harbors TCF/LEF binding sites and responds to β-catenin activation. FOPFlash contains mutated TCF/LEF binding sites and does not respond to β-catenin activation. ( D ) Immunoblotting of cleaved caspase 3 (c-CASP3, an apoptosis marker) and LC3B (an autophagy marker). ( E ) Caspase 3/7 activity assay. ( F ) Knockdown of β-catenin. U87MG cells were transduced with viruses of NS shRNA or β-catenin shRNA. ( G ) Viability of U87MG cells treated with CK1ε shRNA and/or β-catenin shRNA. ( H ) Immunoblotting of c-CASP3 in U87MG cells upon depletion of CK1ε and/or β-catenin. Error bars represent standard deviations from three independent experiments. Full length blots were presented in supplemental materials. * P < 0.05; # P > 0.05. N.D.: not detected.

Article Snippet: Immunofluorescence images of CK1ε in U251 cells and images of immunohistochemical analyses of CK1ε in cerebral cortex and high-grade glioma were obtained from the Human Protein Atlas.

Techniques: Inhibition, Western Blot, Control, Luciferase, Reporter Assay, Plasmid Preparation, Binding Assay, Activation Assay, Marker, Activity Assay, Knockdown, Transduction, shRNA

Journal: Scientific Reports

Article Title: Casein Kinase 1 Epsilon Regulates Glioblastoma Cell Survival

doi: 10.1038/s41598-018-31864-x

Figure Lengend Snippet: CK1ε regulates β-catenin activity and self-renewal of GSCs. ( A ) Viability of GS9-6/NOTCH1 GSCs upon depletion of survival kinase genes. GS9-6/NOTCH1 cells were transduced with viruses of NS shRNA or shRNAs of individual kinase genes from our previous RNA interference screen (ref. ). ( B ) Viability of LN-18/GSC, LN229/GSCs, and U251/GSC upon depletion of CK1ε or MELK. ( C ) Images of GS9-6/NOTCH1 spheres treated with NS, CK1ε, or MELK shRNA. Scale bar: 25 μm. ( D ) Self-renewal assay of VTC-001/GSC, GS9-6/NOTCH1, and LN-18/GSC treated with NS, CK1ε, or MELK shRNA. Percentages of wells with spheres represent capabilities of GSCs to self-renew. ( E ) Differentiation of GS9-6/NOTCH1 cells. Cells were treated with fetal bovine serum (FBS) to induce differentiation, which was indicated by elevated levels of GFAP (an astrocyte marker). ( F ) Immunoblotting of active β-catenin, β-catenin, c-CASP3, NOTCH1, and GFAP in CK1ε-deficient GS9-6/NOTCH1 cells. ( G ) Immunoblotting of active β-catenin, β-catenin, or c-CASP3 in CK1ε-deficient LN229/GSCs. Full length blots were presented in supplemental materials. * P < 0.05; # P > 0.05.

Article Snippet: Immunofluorescence images of CK1ε in U251 cells and images of immunohistochemical analyses of CK1ε in cerebral cortex and high-grade glioma were obtained from the Human Protein Atlas.

Techniques: Activity Assay, Transduction, shRNA, Marker, Western Blot

Journal: Scientific Reports

Article Title: Casein Kinase 1 Epsilon Regulates Glioblastoma Cell Survival

doi: 10.1038/s41598-018-31864-x

Figure Lengend Snippet: The CK1ε inhibitor IC261 blocks GBM cell growth with no effect on astrocytes. ( A ) IC50 of IC261 in U87MG cells. U87MG cells were treated with IC261 at various doses. Viability was determined using the MTS assay and IC50 was calculated using Prism. ( B ) IC50 of PF-4800567 in U87MG cells. ( C ) Cytotoxicity of IC261 in astrocytes and nine GBM cell lines. ( D ) Immunoblotting of CK1ε/β-catenin signaling in U87MG cells treated with IC261 or PF-4800567. Full length blots were presented in supplemental materials. * P < 0.05; # P > 0.05.

Article Snippet: Immunofluorescence images of CK1ε in U251 cells and images of immunohistochemical analyses of CK1ε in cerebral cortex and high-grade glioma were obtained from the Human Protein Atlas.

Techniques: MTS Assay, Western Blot

Journal: Scientific Reports

Article Title: Casein Kinase 1 Epsilon Regulates Glioblastoma Cell Survival

doi: 10.1038/s41598-018-31864-x

Figure Lengend Snippet: The CK1ε inhibitor IC261 blocks the growth GSCs in vitro and in vivo . ( A ) IC50 of IC261 in LN229/GSCs. ( B ) Viability of GS9-6/NOTCH1 treated with IC261. ( C ) Tumor growth in mice. LN229/GSCs were injected subcutaneously into immune-deficient mice followed by treatment of DMSO or IC261 (30 mg/kg). ( D ) Images of xenograft tumors at the end point. ( E ) Histological analysis of tumors.

Article Snippet: Immunofluorescence images of CK1ε in U251 cells and images of immunohistochemical analyses of CK1ε in cerebral cortex and high-grade glioma were obtained from the Human Protein Atlas.

Techniques: In Vitro, In Vivo, Injection

Journal: Scientific Reports

Article Title: Casein Kinase 1 Epsilon Regulates Glioblastoma Cell Survival

doi: 10.1038/s41598-018-31864-x

Figure Lengend Snippet: A noncanonical CK1ε/β-catenin signaling in GBM cell survival.

Article Snippet: Immunofluorescence images of CK1ε in U251 cells and images of immunohistochemical analyses of CK1ε in cerebral cortex and high-grade glioma were obtained from the Human Protein Atlas.

Techniques:

Journal: Frontiers in Neuroanatomy

Article Title: 3D Digital Anatomic Angioarchitecture of the Rat Spinal Cord: A Synchrotron Radiation Micro-CT Study

doi: 10.3389/fnana.2020.00041

Figure Lengend Snippet: Overview and quantification of the 3D morphology of the rat spinal cord microvasculature. (A–C) The raw image of the spinal cord microvasculature among cervical, thoracic, and lumbar regions. (D–F) Representative images of the segmented vasculature and the color-coded labeled vessels of the spinal cord among the three regions. The major arteries and veins including the posterior spinal artery (PSA), central sulcus artery (CSA), anterior spinal artery (ASA), and the posterior spinal vein (PSV). (G–I) The pseudocolored image of the spinal cord microvasculature among three regions. The color bar on the left of the panel indicates the associated vessel diameters. The smallest capillaries are depicted in red and the largest vessels in blue. (J–N) The mean vessel volume fraction, vessel thickness, segment density, bifurcation density, and segment length were relatively lower in the thoracic region than the cervical and lumbar spinal cord; * p < 0.05. (O) Vessel distribution histogram revealed the vessel thickness in the thoracic region with a diameter of less than 25 μm was slightly lower than that of the cervical and lumbar regions. C, cervical spinal cord; T, thoracic spinal cord; L, lumbar spinal cord. * p < 0.05. Scale bar = 200 μm.

Article Snippet: After the vasculature was segmented from the spinal cord parenchyma with Image Pro Analyser 3D software (Version 7.0, Media Cybernetics Inc., USA) based on the iterative gray-level threshold algorithm (Reichold et al., ), we performed the following protocol for the calculation of the 3D vascular morphological parameters (Kim et al., ). (1) Vectorization, skeletonization based on the voxel erosion (Lang et al., ) plugin in Image Pro Analyser 3D software, was used to automatically extract the centerline of the vessel tree ( ). (2) Once the centerline was extracted, numerous vascular quantification parameters such as vessel segment numbers, vessel segment density, vessel segment length, etc could be systematically analyzed and robustly quantified for complex vessel structure. defined the schema of vessel tree parameters.

Techniques: Labeling

Journal: Frontiers in Neuroanatomy

Article Title: 3D Digital Anatomic Angioarchitecture of the Rat Spinal Cord: A Synchrotron Radiation Micro-CT Study

doi: 10.3389/fnana.2020.00041

Figure Lengend Snippet: Representative 3D printed model of the rat spinal cord microvasculature. (A,C) The spinal cord vasculature template obtained from SRμCT for different perspectives. (B,D) The corresponding 3D printed spinal cord vasculature model shows the complex interweaving of vessel branches. ASA, anterior spinal arteries; CSA, central sulcus arteries. (A,C) Scale bar = 200 μm; (B,D) scale bar = 5 cm.

Article Snippet: After the vasculature was segmented from the spinal cord parenchyma with Image Pro Analyser 3D software (Version 7.0, Media Cybernetics Inc., USA) based on the iterative gray-level threshold algorithm (Reichold et al., ), we performed the following protocol for the calculation of the 3D vascular morphological parameters (Kim et al., ). (1) Vectorization, skeletonization based on the voxel erosion (Lang et al., ) plugin in Image Pro Analyser 3D software, was used to automatically extract the centerline of the vessel tree ( ). (2) Once the centerline was extracted, numerous vascular quantification parameters such as vessel segment numbers, vessel segment density, vessel segment length, etc could be systematically analyzed and robustly quantified for complex vessel structure. defined the schema of vessel tree parameters.

Techniques:

Journal: Cells

Article Title: Proteins and Molecular Pathways Relevant for the Malignant Properties of Tumor-Initiating Pancreatic Cancer Cells

doi: 10.3390/cells9061397

Figure Lengend Snippet: Inhibition of S100A8, S100A9, and LGALS3BP expression impairs migration of pancreatic cancer cell line L3.6sl. ( A ) Representative images of the specific knockdown cells (shS100A8, shS100A9, and shLGALS3BP) compared to non-target control shRNA cells (sh ctrl) shown are after 0.0, 6.0, 12.0, and 24.0 h. Black lines enclose areas free of migratory cancer cells. Quantification of migration ability over time ( B ): 0.0 h control shRNA area was set to 0%; all other values were normalized accordingly. Measurements were done with the ProgRes Software. Error bars represent standard deviations of six measurements. Statistical significances were assessed by a two-way ANOVA followed by a Dunnett’s post-hoc test (* for p ≤ 0.05, ** for p ≤ 0.01. and *** for p ≤ 0.001, n = 6).

Article Snippet: Cell movement was assessed after 0.0, 6.0, 12.0, and 24.0 h and documented using a transmission light microscope with ProgRes capture system (Jenoptik GmbH, Jena, Germany).

Techniques: Inhibition, Expressing, Migration, Knockdown, Control, shRNA, Software