Journal: Biology

Article Title: Human Blood Serum Induces p38-MAPK- and Hsp27-Dependent Migration Dynamics of Adult Human Cardiac Stem Cells: Single-Cell Analysis via a Microfluidic-Based Cultivation Platform

doi: 10.3390/biology10080708

Figure Lengend Snippet: Increased migration behavior of hCSCs after application of human blood serum is dependent on p38-MAPK signaling. ( A ) Exemplary images of hCSCs cultivated without serum directly after medium switch, after 23:20 h and after 47:20 h. The blue line indicates the migration path. ( B ) Exemplary images of hCSCs cultivated with human blood serum directly after medium switch, after 23:20 h and after 47:20 h. The blue line indicates the migration path. ( C ) Exemplary images of hCSCs cultivated with human blood serum and p38-MAPK-inhibitor SB239063 directly after medium switch, after 23:20 h and after 47:20 h. The blue line indicates the migration path. ( D ) Migration distance of hCSCs is significantly increased by the application of human serum. This effect is reversed by the inhibition of p38. ( E ) Migration velocity of hCSCs is significantly increased by the application of human serum. This effect is reversed by inhibition of p38. Data points represent migration dynamics of single hCSCs observed in individual cultivation chambers. Measurements were performed with hCSCs from one donor and blood serum from one donor. Mann-Whitney one-tailed, **** p < 0.0001 was considered significant.

Article Snippet: In the starvation period, the medium was switched to starvation-medium consisting of DMEM/F12 (Sigma Aldrich) supplemented with 10 mg/mL penicillin/streptomycin (Sigma Aldrich), 200 mM L-glutamine (Sigma-Aldrich), 5 ng/mL basic fibroblast growth factor (bFGF) (Peprotech, Hamburg, Germany) and 10 ng/mL epidermal growth factor (EGF) (Peprotech) for 24 h. In the treatment phase, the starvation medium was supplemented with either 10% human blood serum or 10% human serum and 50 μM p38-MAPK-inhibitor SB239063 (Medchemexpress, Sollentuna, Sweden) in accordance with our previous study [ ].

Techniques: Migration, Inhibition, MANN-WHITNEY, One-tailed Test

Journal: Biology

Article Title: Human Blood Serum Induces p38-MAPK- and Hsp27-Dependent Migration Dynamics of Adult Human Cardiac Stem Cells: Single-Cell Analysis via a Microfluidic-Based Cultivation Platform

doi: 10.3390/biology10080708

Figure Lengend Snippet: Immunocytochemistry of Nestin and phospho-Hsp27 in hCSCs treated with human serum and p38-MAPK-inhibitor. ( A ) Untreated cells exhibit a basal level of Hsp27 phosphorylation. ( B ) Phosphorylation of Hsp27 in serum-treated cells is strongly increased. ( C ) Hsp27-phosphorylation is inhibited after the application of the p38-MAPK inhibitor SB239063. ( D ) Quantification of fluorescence intensity across all replicates. Measurements were performed with hCSCs from one donor and blood serum from two donors. Mann-Whitney one-tailed, * p < 0.05; **** p < 0.0001 was considered significant.

Article Snippet: In the starvation period, the medium was switched to starvation-medium consisting of DMEM/F12 (Sigma Aldrich) supplemented with 10 mg/mL penicillin/streptomycin (Sigma Aldrich), 200 mM L-glutamine (Sigma-Aldrich), 5 ng/mL basic fibroblast growth factor (bFGF) (Peprotech, Hamburg, Germany) and 10 ng/mL epidermal growth factor (EGF) (Peprotech) for 24 h. In the treatment phase, the starvation medium was supplemented with either 10% human blood serum or 10% human serum and 50 μM p38-MAPK-inhibitor SB239063 (Medchemexpress, Sollentuna, Sweden) in accordance with our previous study [ ].

Techniques: Immunocytochemistry, Phospho-proteomics, Fluorescence, MANN-WHITNEY, One-tailed Test

Journal: Biology

Article Title: Human Blood Serum Induces p38-MAPK- and Hsp27-Dependent Migration Dynamics of Adult Human Cardiac Stem Cells: Single-Cell Analysis via a Microfluidic-Based Cultivation Platform

doi: 10.3390/biology10080708

Figure Lengend Snippet: Schematic summary of hCSC-migration upon serum-treatment regulated by p38-MAPK and Hsp27.

Article Snippet: In the starvation period, the medium was switched to starvation-medium consisting of DMEM/F12 (Sigma Aldrich) supplemented with 10 mg/mL penicillin/streptomycin (Sigma Aldrich), 200 mM L-glutamine (Sigma-Aldrich), 5 ng/mL basic fibroblast growth factor (bFGF) (Peprotech, Hamburg, Germany) and 10 ng/mL epidermal growth factor (EGF) (Peprotech) for 24 h. In the treatment phase, the starvation medium was supplemented with either 10% human blood serum or 10% human serum and 50 μM p38-MAPK-inhibitor SB239063 (Medchemexpress, Sollentuna, Sweden) in accordance with our previous study [ ].

Techniques: Migration

Journal: bioRxiv

Article Title: ARHGEF18 participates in Endothelial Cell Mechano-sensitivity in Response to Flow

doi: 10.1101/2022.09.10.507283

Figure Lengend Snippet: A . Quantification of p38 phosphorylation by Western blot in HUVECs expressing a non-targeting shRNA (shNT) or a shRNA targeting Arhgef18 (shA18.1), under static condition or exposed to physiological SS (PSS). Static: N=3; PSS: N=8. **p<0.01; ***p<0.001 2-way ANOVA. B . Quantification of cell orientation and elongation with the flow in HUVECs treated with an inhibitor for p38 (SB239063, 100nM, ip38) or vehicle (CTR), exposed to physiological SS. 24h of SS. Each dot represents a cell. N=4 with in-between 200 and 450 cells analyzed by experiment. ***p<0.001; Mann-Whitney T-test. C . Quantification of Actin localized at junction in HUVECs treated with an inhibitor for p38 (SB239063, 100nM, ip38) or vehicle (CTR), exposed to physiological SS. 24h of SS. N=2, 10 to 14 areas analyzed by experiment. **p<0.01; Unpaired T-test. D . Representative images of HUVECs treated with an inhibitor for p38 (SB239063, 100nM, ip38) or vehicle (CTR) exposed to physiological SS. 24 and stained for ZO-1 (green), VE-cadherin (magenta) and actin (red). Flow direction is indicated by the arrow. E . Quantification of ZO-1 localized at junction in HUVECs treated with an inhibitor for p38 (SB239063, 100nM, ip38) or vehicle (CTR) exposed to physiological SS. 24h of SS. N=2, 10 to 15 areas analyzed by experiment. ***p<0.001; Unpaired T-test. F . Representative images of Paxillin staining in HUVECs treated with an inhibitor for p38 (SB239063, 100nM, ip38) or vehicle (CTR), exposed to physiological SS. Flow direction is indicated by the arrow. Green: paxillin; magenta: VE-cadherin; red: Actin. G . Quantification of focal adhesion type by hand classification of paxillin staining within each cell based on the representative images for each category on the left (white box: paxillin high = numerous, elongated and aligned focal adhesions; black box: paxillin low = few, short and misaligned focal adhesions). N=2. Quantified blindly.

Article Snippet: For p38 MAPK inhibition studies, confluent HUVEC were pretreated with p38 MAPK specific inhibitor (100nM, SB239063; TOCRIS) for at least 1hr prior to physiological SS, treatment was maintained during the flow stimulation.

Techniques: Phospho-proteomics, Western Blot, Expressing, shRNA, MANN-WHITNEY, Staining

Journal: bioRxiv

Article Title: ARHGEF18 participates in Endothelial Cell Mechano-sensitivity in Response to Flow

doi: 10.1101/2022.09.10.507283

Figure Lengend Snippet: A . Representative blot of ARHGEF18 binding on nucleotide-free RhoA (GST-RhoA G17A ) by pull-down assay with HUVECs exposed to physiological SS, over-expressing WT form (A18 WT ) or mutant form (A18 Y260A ) of ARHGEF18. B . Quantification of p38 phosphorylation by Western blot in HUVECs exposed to physiological SS, over-expressing WT form (A18 WT ) or mutant form (A18 Y260A ) of ARHGEF18. N=4. Paired T-test. C . Representative images of HUVECs HUVECs exposed to physiological SS, over-expressing WT form (A18 WT ) or mutant form (A18 Y260A ) of ARHGEF18 and stained VE-cadherin. Flow direction is indicated by the arrow. D . Quantification of cell orientation and elongation with the flow in HUVECs exposed to physiological SS, over-expressing WT form (A18 WT ) or mutant form (A18 Y260A ) of ARHGEF18. Each dot represents a cell. N=1 with in-between 1000 and 1350 cells analyzed by experiment. **p<0.01; Unpaired T-test. E. Representative images of ZO-1 staining in a mix population of WT cells (FLAG negative, non-red) and cells over-expressing the mutant form (A18 Y260A ) of ARHGEF18 (FLAG positive, red), exposed to physiological SS. Green: ZO-1; red: FLAG; Magenta: VE-cadherin. F. Quantification of the ZO-1 staining at junction using plot profile analysis. N=1, 25 couples of cells analyzed per experiment.

Article Snippet: For p38 MAPK inhibition studies, confluent HUVEC were pretreated with p38 MAPK specific inhibitor (100nM, SB239063; TOCRIS) for at least 1hr prior to physiological SS, treatment was maintained during the flow stimulation.

Techniques: Binding Assay, Pull Down Assay, Expressing, Mutagenesis, Phospho-proteomics, Western Blot, Staining