Journal: Life

Article Title: Casomorphine-10 (CM-10) Peptide Orchestrates Circadian and Neurodevelopmental Gene Clusters via δ-Opioid Receptor Signaling: Insights from Transcriptome Analysis with δ-Opioid Receptor-Expressing HEK293 Cells

doi: 10.3390/life15101636

Figure Lengend Snippet: δ-opioid receptor (DOR)-expressing HEK293 cells and HEK293 cells stained with anti-DOR antibody and followed with FITC-conjugated anti-mouse IgG ( A ). Volcano plot of identified differentially expressed genes (DEGs) in DOR-HEK293 cells after 1 h treatment with CM-10 ( B ). Red boxes show changed gene expression over 1.4-time higher and less than 0.71 with p -value predicted by EdgeR of less than 0.05.

Article Snippet: The fixed DOR-HEK293 and HEK293 cells were then incubated with a rabbit anti-DOR antibody (GeneTex, Irvine, CA, USA) after dilution with 1% casein in PBS (1/1000) and stained with a secondary antibody (Cy3-conjugated anti-rabbit IgG, Rockland Inc., PA, USA).

Techniques: Expressing, Staining, Gene Expression

Journal: Life

Article Title: Casomorphine-10 (CM-10) Peptide Orchestrates Circadian and Neurodevelopmental Gene Clusters via δ-Opioid Receptor Signaling: Insights from Transcriptome Analysis with δ-Opioid Receptor-Expressing HEK293 Cells

doi: 10.3390/life15101636

Figure Lengend Snippet: Predicted DOR agonistic signaling networks in DOR-HEK293 cells after 1 h treatment with CM-10. Fourteen genes with suggested involvement in cAMP-dependent protein kinases, transcriptional regulators in response to cAMP, circadian rhythm, stress and depression are shown in and were applied for network analysis by STRING. Red: circadian rhythm, Green: regulation of transcription of Notch receptor target, Yellow: PKA activation in glucagon signalling.

Article Snippet: The fixed DOR-HEK293 and HEK293 cells were then incubated with a rabbit anti-DOR antibody (GeneTex, Irvine, CA, USA) after dilution with 1% casein in PBS (1/1000) and stained with a secondary antibody (Cy3-conjugated anti-rabbit IgG, Rockland Inc., PA, USA).

Techniques: Activation Assay

Journal: Cells

Article Title: Jamming Transitions in Astrocytes and Glioblastoma Are Induced by Cell Density and Tension

doi: 10.3390/cells12010029

Figure Lengend Snippet: Collective migration properties of astrocytes and glioblastoma cells. ( a ) Graph of the mean speed per field of view over time, evaluated for 81, 33 or 18 fields of views for LN229, U138 or astrocytes, respectively. ( b ) 4-point susceptibility as obtained from the velocity fields over the whole measurement time. Peak positions of the 4-point susceptibility represent the average life time of collectively moving packs of cells. Sample sizes were identical as reported in ( a ). ( c ) Analysis of layer reorganization in terms of cells without making new neighbors. Sample sizes were identical as reported in ( a ). ( d ) Differences in shape factor between the cell types measured during live-cell imaging. The dotted line shows the critical value of 3.81 predicted by the vertex model. Below cells are considered to be jammed. “+” and “−” depict if shape factors are significantly larger or smaller than the critical value, with p < 0.0001 for all cell types, as tested with sign test. For shape factor calculation 2645, 772 or 269 LN229, U138 cells or astrocytes were measured from the fields of view. ( b , c ) Error bars and shaded areas depict the standard error of the mean. ( c , d ) Box plots show the median (red line), 25 and 75 percentile (box), non-outlier range (whiskers) and outliers (red dots).

Article Snippet: LN229 cells were purchased from the American Type Culture Collection (ATCC, CRL-261, Manassas, VA, USA) and U138 cells were obtained from Cell Lines Service (Cell Lines Service, 300363, Eppelheim, Germany).

Techniques: Migration, Live Cell Imaging

Journal: Cells

Article Title: Jamming Transitions in Astrocytes and Glioblastoma Are Induced by Cell Density and Tension

doi: 10.3390/cells12010029

Figure Lengend Snippet: Cell density dependence of collective migration. ( a ) Measured mean speeds of astrocytes per field of view when seeded in different densities of 300k, 400k, 600k, 800k cells or subjected to a scratch, measured for 18 or 20 (scratch) fields of view. ( b ) Illustration of the 4-point susceptibility over the whole measurement time. Peak positions of the 4-point susceptibility represent the average life time of collectively moving packs of cells. Sample sizes are identical to ( a ). ( c ) Illustrates the difference in shape factor for different seeding densities of astrocytes and during the wound healing assay. The dotted line shows the critical value of 3.81 predicted by the vertex model. Below cells are considered to be jammed. “+” and “−“ depicts if shape factors are significantly larger or smaller than the critical value, with p < 0.0001 for all instances, as tested by sign test for 256, 269, 234, 183 or 386 astrocytes for seeding densities of 300k, 400k, 600k, 800k cells or the scratch. ( d – f ) Plot of the cell density over the speed for astrocytes, LN229 and U138 cells. Formulas show the scaling behavior of the speed v with cell density δ. Values in brackets denote 95% confidence intervals. ( a , b , d – f ) Error bars and shaded areas depict the standard error of the mean. ( c ) Box plots show the median (red line), 25 and 75 percentile (box), non-outlier range (whiskers) and outliers (red dots).

Article Snippet: LN229 cells were purchased from the American Type Culture Collection (ATCC, CRL-261, Manassas, VA, USA) and U138 cells were obtained from Cell Lines Service (Cell Lines Service, 300363, Eppelheim, Germany).

Techniques: Migration, Wound Healing Assay

Journal: Cells

Article Title: Jamming Transitions in Astrocytes and Glioblastoma Are Induced by Cell Density and Tension

doi: 10.3390/cells12010029

Figure Lengend Snippet: Effect of myosin II and ROCK inhibition on collective migration. ( a , e , i ) Mean speed per field of view for astrocytes, LN229 and U138 cells treated with ROCK inhibitor Y-27632 or myosin II inhibitor blebbistatin. For astrocytes, 15 fields of view were measured for all conditions. For LN229 and U138 cells 81 or 33 fields of view were assessed in control conditions and 15 otherwise. ( b , f , j ) 4-point susceptibility over the whole measurement time for astrocytes, LN229 and U138. Peak positions of the 4-point susceptibility represent the average life time of collectively moving packs of cells. Sample sizes are the same as in ( a , e , i ), respectively. ( c , g , k ) cell layer reorganization in terms of changed neighborhood. Sample sizes are the same as in ( a , e , i ), respectively. Here p < 0.001 for all instances of U138 and LN229 cells except for LN229 treated with 10 µM Y-27632 ( p < 0.05). ( d , h , l ) illustrate the difference in shape factor for astrocytes, LN229 and U138 cells. The dotted line shows the critical value of 3.81 predicted by the vertex model. Below cells are considered to be jammed. “+” and “−” depict if shape factors are significantly larger or smaller than the critical value, as assessed by sign test. Here p < 0.0001 holds for all instances of astrocytes, U138 cells and LN229 cells in control conditions or when treated with 10 µM blebbistatin. p < 0.01 for LN229 cells treated with 5 µM Y-27632 and p < 0.05 for LN229 treated with 10 µM Y-27632. For astrocytes 365, 284, 261, 279, 296, 390, 413 or 396 cells were measured for control conditions, Y-27632 (5, 10, 20, 40 µM) or blebbistatin (5, 10, 20 µM) treatment. For LN229 cells 2645, 298, 301, 454, 302 or 460 cells were measured for control conditions, Y-27632 (5, 10, 20 µM) or blebbistatin (5, 10 µM) treatment, respectively. For U138 cells 772, 448, 448, 450, 150, 300, 302 or 150 cells were measured for control conditions, Y-27632 (5, 10, 20, 40 µM) or blebbistatin (5, 10, 20 µM) treatment, respectively. ( a , b , e , f , i , j ) error bars and shaded areas depict the standard error of the mean. ( c , d , g , h , k , l ) box plots show the median (red line), 25 and 75 percentile (box), non-outlier range (whiskers) and outliers (red dots). Stars (*) depict statistically significant results.

Article Snippet: LN229 cells were purchased from the American Type Culture Collection (ATCC, CRL-261, Manassas, VA, USA) and U138 cells were obtained from Cell Lines Service (Cell Lines Service, 300363, Eppelheim, Germany).

Techniques: Inhibition, Migration, Control

Journal: Cells

Article Title: Jamming Transitions in Astrocytes and Glioblastoma Are Induced by Cell Density and Tension

doi: 10.3390/cells12010029

Figure Lengend Snippet: Biomechanical properties of astrocytes and glioblastoma cells. ( a ) Illustration of a typical 140 × 140 µm 2 dense layer of LN229 cells used for measuring elastic moduli (left) and the respective map of the elastic moduli (right). Scale bar represents 20 µm. ( b ) Measurements of the Young moduli for astrocytes and both GBM cell lines, when treated with the ROCK inhibitor Y-27632 or myosin II inhibitor blebbistatin. p < 0.0001 for all significant results, as assessed by one way ANOVA with Tukey post hoc test. For astrocytes 1187 sample curves were obtained. For LN229 1568, 1566 or 1585 and for U138 cells, 1161, 1164 or 1148 measurement curves were evaluated for control conditions, Y-27632 or blebbistatin treatment, respectively. ( c ) Cortical tension of astrocytes, LN229 and U138 cells, when subjected to ROCK or myosin II inhibition. p < 0.05 for all LN229 measurements and U138 control conditions vs. 10 µM blebbistatin. p < 0.01 for U138 vs. 40 µM Y-27632. One way ANOVA with Tukey post hoc test was used. For astrocytes, 45 cells were measured. For LN229, 55, 55 or 53 and for U138, 55, 53 or 54 cells were measured for control conditions or when treated with Y-27632 or blebbistatin, respectively. ( d ) A typical actin staining of LN229 cells in control conditions (left) and when treated with 20 µM of Y-27632 is shown. Scale bars depict 10 µm. ( e ) Estimation of the tension generated by stress fibers by astrocytes and GBM cell lines. 75 cells in 15 fields of view were analyzed in each group. p < 0.0001 for all significantly different treatments, as assessed by one way ANOVA with Tukey post hoc test. ( f ) Normalized bead displacements obtained from the TFM measurements for LN229 cells. p < 0.0001 for all significantly different treatments. ( g ) Sample images and the respective displacement maps for (un-)treated LN229 cells. Scale bars depict 50 µm. ( b , c , e , f ) Box plots show the median (red line), 25 and 75 percentile (box), non-outlier range (whiskers) and outliers (red dots). Stars (*) depict statistically significant results.

Article Snippet: LN229 cells were purchased from the American Type Culture Collection (ATCC, CRL-261, Manassas, VA, USA) and U138 cells were obtained from Cell Lines Service (Cell Lines Service, 300363, Eppelheim, Germany).

Techniques: Control, Inhibition, Staining, Generated

Journal: Cells

Article Title: Jamming Transitions in Astrocytes and Glioblastoma Are Induced by Cell Density and Tension

doi: 10.3390/cells12010029

Figure Lengend Snippet: ( a ) Representative examples of actin and β-catenin staining for LN229 cells. Scale bar depicts 10 µm. ( b ) Cell–cell adhesion strength as estimated by the fluorescence images. For all statistically significant differences: p < 0.0001. For astrocytes 73 cell–cell connections were measured. For LN229 70, 49 or 72 and for U138 75, 74 or 67 cell–cell junctions were analyzed for control conditions or when treated with Y-27632 or blebbistatin, respectively. ( c ) Cell–cell adhesion forces as measured using AFM. For LN229 treated with Y-27632 and U138 treated with blebbistatin p < 0.01, p < 0.05 otherwise. The sample size for astrocytes was 30, for LN229 39, 35, 34 and for U138 32, 27, 26 for control conditions or after treatment with Y-27632 or blebbistatin, respectively. ( d ) Values calculated for the cell–cell-adhesion energies obtained from the spheroid aggregation. p < 0.0001 for all significantly different treatments, as assessed by one way ANOVA with Tukey post hoc test. For astrocytes 46 spheroids were assessed. For LN229 31, 23 or 17 and for U138 cells 33, 39 or 27 spheroids were measured for control conditions or Y-27632 or blebbistatin treatment, respectively. ( e ) Evolution of the spheroid area over time for astrocytes and both GBM cell lines for all used conditions. Inlets show typical spheroids at the beginning and end of the measurement. Sample size is identical to d. Scale bars depict 300 µm. ( b – d ) Box plots show the median (red line), 25 and 75 percentile (box), non-outlier range (whiskers) and outliers (red dots). ( e ) Error bars depict the standard error of the mean. Stars (*) depict statistically significant results.

Article Snippet: LN229 cells were purchased from the American Type Culture Collection (ATCC, CRL-261, Manassas, VA, USA) and U138 cells were obtained from Cell Lines Service (Cell Lines Service, 300363, Eppelheim, Germany).

Techniques: Staining, Fluorescence, Control

Journal: Cells

Article Title: Jamming Transitions in Astrocytes and Glioblastoma Are Induced by Cell Density and Tension

doi: 10.3390/cells12010029

Figure Lengend Snippet: Ratio of median adhesion and tension to control conditions.

Article Snippet: LN229 cells were purchased from the American Type Culture Collection (ATCC, CRL-261, Manassas, VA, USA) and U138 cells were obtained from Cell Lines Service (Cell Lines Service, 300363, Eppelheim, Germany).

Techniques: Control