Journal: Cells

Article Title: RhoGEF17—An Essential Regulator of Endothelial Cell Death and Growth

doi: 10.3390/cells10040741

Figure Lengend Snippet: RhoGEF17 is essential for cell-cell contacts and adherens junctions (AJ) protein regulation in EC. ( A , B , D ) Human umbilical vein endothelial cells (HUVEC) were transduced with adenoviruses encoding EGFP alone (EGFP) or in addition to a shRNA against RhoGEF17 (sh17-1). ( A ) After 48 h, RhoGEF17 was detected in cell lysates. Shown are representative immunoblots of RhoGEF17 and α-tubulin and the quantitative analysis. Values are normalized and given as means + SEM with the single data points, n = 7, * p < 0.05 analyzed by paired t-testing. ( B ). Depicted are bright field/EGFP overlay images of transduced HUVEC. Scale bar = 100 µm. ( C ) Rat fat pad endothelial cells (RFPEC) were transduced for 48 h and then used to generate spheroids. Bright field and fluorescence images are shown. Scale bar = 200 µm. ( D ) VE-cadherin, p120-catenin, and α-tubulin were detected by immunoblot in lysates of transduced HUVEC. Shown are representative immunoblots and the quantified data normalized by α-tubulin and relative to EGFP as means + SEM with the single data points, n = 4 − 7, * p < 0.05 analyzed by paired t-testing.

Article Snippet: Antibodies and reagents —The following antibodies were used: rabbit anti-RhoGEF17 (ProSci, Poway, CA, USA), goat anti-pan-cadherin (Santa Cruz, Heidelberg, Germany), mouse anti-N-cadherin (BD Biosciences, Heidelberg, Germany), rabbit anti-p120-catenin (Epitomics by Abcam, Cambridge, UK), mouse anti-p120-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Zymo Research, Freiburg, Germany), rabbit anti-phospho-β-catenin (Ser33/Ser37/Thr41) (Cell Signaling Technology, Frankfurt, Germany), mouse anti-histone H1 (Santa Cruz, Heidelberg, Germany), mouse anti-vinculin (Sigma-Aldrich, Taufkirchen, Germany), mouse anti-α-tubulin (Sigma-Aldrich, Taufkirchen, Germany), rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-Akt (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-cyclin D1 (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-axin 1 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-GAPDH (Meridian/Biodesign, Memphis, TN, USA), rabbit anti-survivin (Abcam, Cambridge, UK), mouse anti-VE-cadherin (Enzo Life Sciences, Lörrach, Germany), rabbit anti-caspase 3 (Biorad, Feldkirchen, Germany), rabbit anti-cleaved caspase 3 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-β-actin (Sigma-Aldrich, Taufkirchen, Germany).

Techniques: Transduction, shRNA, Western Blot, Fluorescence

Journal: Cells

Article Title: RhoGEF17—An Essential Regulator of Endothelial Cell Death and Growth

doi: 10.3390/cells10040741

Figure Lengend Snippet: Loss of RhoGEF17 results in the proteasomal degradation of AJ proteins in RFPEC. RFPEC were transduced with adenoviruses encoding EGFP (EGFP), or EGFP and a shRNA against RhoGEF17 (sh17-1). ( A ) Immunoblot analysis of RhoGEF17, pan-cadherin and α-tubulin was performed at the indicated time points. The values were normalized by α-tubulin and are given as means ± SEM relative to the time point 0 h, n = 3–21. ( B ) Immunofluorescence analysis was performed 48 h after transduction. Depicted are EGFP images, immunofluorescence staining of N-cadherin (left), p120-catenin (right) and the overlays with DAPI. Scale bar = 20 µm. ( C ) The transcript levels of RhoGEF17, N-cadherin, p120-catenin and the housekeeping gene PBGD were determined by qPCR after 48 h of transduction. The values are given as means + SEM with the single data points, n = 9, * p < 0.05 assessed by paired t-testing. ( D ) The proteasome was inhibited with 100 nM Bortezomib for 2 h. Non-transduced (nt) cells were used as additional control. RhoGEF17, N-cadherin and p120-catenin were detected by immunoblot in whole cell lysates. Shown are representative immunoblots of RhoGEF17, N-cadherin, p120-catenin and α-tubulin (left) and the quantitative analyses. Values were normalized by α-tubulin and are given relative to non-transduced cells treated with DMSO only. Shown are means + SEM and the single data points; n = 3–5, * p < 0.05 assessed by 2-way ANOVA with Tukey’s multiple comparison testing.

Article Snippet: Antibodies and reagents —The following antibodies were used: rabbit anti-RhoGEF17 (ProSci, Poway, CA, USA), goat anti-pan-cadherin (Santa Cruz, Heidelberg, Germany), mouse anti-N-cadherin (BD Biosciences, Heidelberg, Germany), rabbit anti-p120-catenin (Epitomics by Abcam, Cambridge, UK), mouse anti-p120-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Zymo Research, Freiburg, Germany), rabbit anti-phospho-β-catenin (Ser33/Ser37/Thr41) (Cell Signaling Technology, Frankfurt, Germany), mouse anti-histone H1 (Santa Cruz, Heidelberg, Germany), mouse anti-vinculin (Sigma-Aldrich, Taufkirchen, Germany), mouse anti-α-tubulin (Sigma-Aldrich, Taufkirchen, Germany), rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-Akt (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-cyclin D1 (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-axin 1 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-GAPDH (Meridian/Biodesign, Memphis, TN, USA), rabbit anti-survivin (Abcam, Cambridge, UK), mouse anti-VE-cadherin (Enzo Life Sciences, Lörrach, Germany), rabbit anti-caspase 3 (Biorad, Feldkirchen, Germany), rabbit anti-cleaved caspase 3 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-β-actin (Sigma-Aldrich, Taufkirchen, Germany).

Techniques: Transduction, shRNA, Western Blot, Immunofluorescence, Staining

Journal: Cells

Article Title: RhoGEF17—An Essential Regulator of Endothelial Cell Death and Growth

doi: 10.3390/cells10040741

Figure Lengend Snippet: Loss of RhoGEF17 results in the accumulation of phosphorylated β-catenin and increases β-catenin target gene expression in EC. ( A ) HUVEC or ( B ) RFPEC were transduced for 48 h. Representative immunoblots of β-catenin and α-tubulin are shown. The intensity of both detected β-catenin bands (β-cat) as well as the intensity of the upper β-catenin band (mod-β-cat) were quantified and normalized by α-tubulin. The values are given relative to the EGFP control as means + SEM with the single data points, n = 3 (HUVEC), n = 3–14 (RFPEC), * p < 0.05 vs. EGFP control (not shown) assessed by paired t-testing. ( C ) The proteasome was inhibited with 100 nM Bortezomib for 2 h in RFPEC. β-catenin and phosphorylated β-catenin were detected by immunoblot. Shown are representative immunoblots of β-catenin (Ab1 = Santa Cruz, Ab2 = Zymo Research), p -β-catenin (S33/37/T41) and α-tubulin (left) and the quantitative analyses. The values are normalized and given as means + SEM with the single data points, n = 8–11, * p < 0.05 assessed by 2-way ANOVA with Tukey’s multiple comparison test. ( D ) Axin1, survivin, cyclin D1 and α-tubulin were detected by immunoblot in RFPEC lysates. Shown are representative immunoblots (left) and the corresponding analyses. The values are given relative to the EGFP control as means + SEM with the single data points, n = 3, * p < 0.05 vs. EGFP control (not shown) assessed by paired t-testing. ( E ) Cell fractionation experiments were performed with transduced RFPEC. β-catenin, histone H1 and GAPDH were detected by immunoblot in different cell fractions.

Article Snippet: Antibodies and reagents —The following antibodies were used: rabbit anti-RhoGEF17 (ProSci, Poway, CA, USA), goat anti-pan-cadherin (Santa Cruz, Heidelberg, Germany), mouse anti-N-cadherin (BD Biosciences, Heidelberg, Germany), rabbit anti-p120-catenin (Epitomics by Abcam, Cambridge, UK), mouse anti-p120-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Zymo Research, Freiburg, Germany), rabbit anti-phospho-β-catenin (Ser33/Ser37/Thr41) (Cell Signaling Technology, Frankfurt, Germany), mouse anti-histone H1 (Santa Cruz, Heidelberg, Germany), mouse anti-vinculin (Sigma-Aldrich, Taufkirchen, Germany), mouse anti-α-tubulin (Sigma-Aldrich, Taufkirchen, Germany), rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-Akt (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-cyclin D1 (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-axin 1 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-GAPDH (Meridian/Biodesign, Memphis, TN, USA), rabbit anti-survivin (Abcam, Cambridge, UK), mouse anti-VE-cadherin (Enzo Life Sciences, Lörrach, Germany), rabbit anti-caspase 3 (Biorad, Feldkirchen, Germany), rabbit anti-cleaved caspase 3 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-β-actin (Sigma-Aldrich, Taufkirchen, Germany).

Techniques: Expressing, Western Blot, Cell Fractionation

Journal: Cells

Article Title: RhoGEF17—An Essential Regulator of Endothelial Cell Death and Growth

doi: 10.3390/cells10040741

Figure Lengend Snippet: The RhoGEF17 knockdown alters the adhesion and migration behavior of RFPEC. RFPEC were transduced for 48 h. ( A ) The cells were detached and reseeded. Adhesion was monitored by fluorescence microscopy over a time course of 24 h. Depicted are the percentages of transduced (EGFP + ), adherent cells given as means ± SEM, n = 6, * p < 0.05. ( B ) The surface area of the cells was determined at the end (24 h) of the adhesion assay. Given are the means + SEM with the single data points, n = 4, * p < 0.05 assessed by t-testing. ( C ) Shown are EGFP images, immunofluorescence staining of vinculin and the merges of EGFP (green), vinculin (red), and DAPI (blue). Scale bar = 20 µm. ( D ) The quantification of the number of central focal adhesions in transduced (EGFP + ) and non-transduced (EGFP - ) adjacent cells are given as means + SEM with the single data points, n = 4, p < 0.05, * vs. EGFP-transduced, # EGFP + vs. EGFP - in sh17-1 transduced cells. ( E ) Vinculin was detected by immunoblot. Shown are representative immunoblots of vinculin and α-tubulin. ( F ) Fluorescence imaging of the transduced cells was performed. Shown are EGFP and TRITC-phalloidin images together with the merges of EGFP (green), phalloidin (red), and DAPI (blue). Scale bar = 20 µm. ( G ) Confluent transduced cells were scratched and imaged at the indicated time points. Left: Representative bright field/fluorescent images are show. Middle: The migration distance of the sheet was measured as indicated in the left images by the arrow. Given are the quantified data as mean ± SEM, n = 3 with 10 replicates per experiment, * p < 0.05 vs. EGFP assessed by 2-way ANOVA with Sidak’s multiple comparison test. Single, EGFP + cells in the wound were counted at the end of the assay. The number of cells per mm 2 are given as means + SEM of all measured 30 wells, * p < 0.05 assessed an unpaired t-test.

Article Snippet: Antibodies and reagents —The following antibodies were used: rabbit anti-RhoGEF17 (ProSci, Poway, CA, USA), goat anti-pan-cadherin (Santa Cruz, Heidelberg, Germany), mouse anti-N-cadherin (BD Biosciences, Heidelberg, Germany), rabbit anti-p120-catenin (Epitomics by Abcam, Cambridge, UK), mouse anti-p120-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Zymo Research, Freiburg, Germany), rabbit anti-phospho-β-catenin (Ser33/Ser37/Thr41) (Cell Signaling Technology, Frankfurt, Germany), mouse anti-histone H1 (Santa Cruz, Heidelberg, Germany), mouse anti-vinculin (Sigma-Aldrich, Taufkirchen, Germany), mouse anti-α-tubulin (Sigma-Aldrich, Taufkirchen, Germany), rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-Akt (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-cyclin D1 (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-axin 1 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-GAPDH (Meridian/Biodesign, Memphis, TN, USA), rabbit anti-survivin (Abcam, Cambridge, UK), mouse anti-VE-cadherin (Enzo Life Sciences, Lörrach, Germany), rabbit anti-caspase 3 (Biorad, Feldkirchen, Germany), rabbit anti-cleaved caspase 3 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-β-actin (Sigma-Aldrich, Taufkirchen, Germany).

Techniques: Migration, Fluorescence, Microscopy, Cell Adhesion Assay, Immunofluorescence, Staining, Western Blot, Imaging

Journal: Cells

Article Title: RhoGEF17—An Essential Regulator of Endothelial Cell Death and Growth

doi: 10.3390/cells10040741

Figure Lengend Snippet: The reduction of RhoGEF17 prevents apoptosis and induces a cell cycle block in EC. HUVEC ( A , E , H ) or RFPEC ( B – D , F , G ) were transduced. ( A , B ) Apoptotic cells were detected by annexin-V in semi-efficiently transduced cells after 48 h. The number of apoptotic transduced (EGFP + ) and non-transduced (EGFP - ) adjacent cells are given as means + SEM with the single data points, n = 3–4, p < 0.05 * vs. EGFP-transduced, # EGFP + vs. EGFP - in sh17 transduced cells assessed by 1-way ANOVA with Tukey’s multiple comparison test. C ) Pro- and cleaved caspase 3 expression was analyzed by immunoblot in 100% transduced cells after 48 h. Representative immunoblots of both variants and β-actin ( B ) and the analyses ( C ) are shown. Quantified values are normalized by β-actin and are given relative to EGFP. Shown are the means + SEM with the single data points, n = 5, * p < 0.05 assessed by paired t-test. ( D ) Phosphorylation of Akt was detected in whole cell lysates by immunoblot 48h after transuction. Shown are representative immunoblots of pAkt (S473), total Akt and α-tubulin and the analysis. Quantified p-Akt was normalized by Akt and is given relative to EGFP. Shown are the means + SEM with the single data points, n = 7, * p < 0.05 assessed by paired t-test. ( E , F ) Cell cycle progression was analyzed by flow cytometry in cells transduced for 48 h. Calculation of G1, S, and G2/M phases are given as means + SEM with the single data points, n = 4–8, * p < 0.05 assessed by 2-way ANOVA with Sidak’s or Tukey’s multiple comparison test. ( G ) RFPEC were 100% transduced and replated. After the indicated time points cell proliferation was assessed by automated nuclei counting. The absolute cell number is given as means + SEM with single data points, n = 3, * p < 0.05 assessed by 2-way ANOVA with Sidak’s multiple comparison test. ( H ) HUVEC were seeded and transduced with different amounts of viruses in 24-well plates. One, two, and three days later images were taken and the non-transduced (EGFP - ) and transduced (EGFP + ) cells were counted manually in 160x magnification images. Left: The results were clustered according to the transduction efficiencies in low, medium, and high transduction experiments. Right: Given are the number of cells per field of view in the different conditions, n = 3–4, * p < 0.05 vs. the corresponding Day1 data as detected by 1-way ANOVA with a Dunnett’s multiple comparison test.

Article Snippet: Antibodies and reagents —The following antibodies were used: rabbit anti-RhoGEF17 (ProSci, Poway, CA, USA), goat anti-pan-cadherin (Santa Cruz, Heidelberg, Germany), mouse anti-N-cadherin (BD Biosciences, Heidelberg, Germany), rabbit anti-p120-catenin (Epitomics by Abcam, Cambridge, UK), mouse anti-p120-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Zymo Research, Freiburg, Germany), rabbit anti-phospho-β-catenin (Ser33/Ser37/Thr41) (Cell Signaling Technology, Frankfurt, Germany), mouse anti-histone H1 (Santa Cruz, Heidelberg, Germany), mouse anti-vinculin (Sigma-Aldrich, Taufkirchen, Germany), mouse anti-α-tubulin (Sigma-Aldrich, Taufkirchen, Germany), rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-Akt (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-cyclin D1 (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-axin 1 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-GAPDH (Meridian/Biodesign, Memphis, TN, USA), rabbit anti-survivin (Abcam, Cambridge, UK), mouse anti-VE-cadherin (Enzo Life Sciences, Lörrach, Germany), rabbit anti-caspase 3 (Biorad, Feldkirchen, Germany), rabbit anti-cleaved caspase 3 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-β-actin (Sigma-Aldrich, Taufkirchen, Germany).

Techniques: Blocking Assay, Expressing, Western Blot, Flow Cytometry, Transduction

Journal: Cells

Article Title: RhoGEF17—An Essential Regulator of Endothelial Cell Death and Growth

doi: 10.3390/cells10040741

Figure Lengend Snippet: Scheme of RhoGEF17 function in EC. RhoGEF17 stabilizes AJ in EC. Its loss leads to AJ protein degradation via the proteasome and an accumulation of β-catenin in its destruction complex (phosphorylated and ubiquitinated) form. A part of the β-catenin pool, which might be phosphorylated by Akt, is translocated to the nucleus and induces β-catenin/TCF dependent gene transcription of, for example, survivin and cyclin D1. Besides impaired cell adhesion and migration due to the disruption of the AJ, RhoGEF17-depleted EC can escape anoikis and end in cell cycle arrest. In contrast neighboring cells, without RhoGEF17 knockdown, enter apoptosis due to the loss of cell–cell contacts. EC = Endothelial cell, RhoGEF17 = Rho-specific guanine nucleotide exchange factor 17, p120 = p120-catenin, α = α-catenin, TCF = Transcription factor, Ub = ubiquitinated, P = phosphorylated.

Article Snippet: Antibodies and reagents —The following antibodies were used: rabbit anti-RhoGEF17 (ProSci, Poway, CA, USA), goat anti-pan-cadherin (Santa Cruz, Heidelberg, Germany), mouse anti-N-cadherin (BD Biosciences, Heidelberg, Germany), rabbit anti-p120-catenin (Epitomics by Abcam, Cambridge, UK), mouse anti-p120-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Santa Cruz, Heidelberg, Germany), rabbit anti-β-catenin (Zymo Research, Freiburg, Germany), rabbit anti-phospho-β-catenin (Ser33/Ser37/Thr41) (Cell Signaling Technology, Frankfurt, Germany), mouse anti-histone H1 (Santa Cruz, Heidelberg, Germany), mouse anti-vinculin (Sigma-Aldrich, Taufkirchen, Germany), mouse anti-α-tubulin (Sigma-Aldrich, Taufkirchen, Germany), rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-Akt (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-cyclin D1 (Cell Signaling Technology, Frankfurt, Germany), rabbit anti-axin 1 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-GAPDH (Meridian/Biodesign, Memphis, TN, USA), rabbit anti-survivin (Abcam, Cambridge, UK), mouse anti-VE-cadherin (Enzo Life Sciences, Lörrach, Germany), rabbit anti-caspase 3 (Biorad, Feldkirchen, Germany), rabbit anti-cleaved caspase 3 (Cell Signaling Technology, Frankfurt, Germany), mouse anti-β-actin (Sigma-Aldrich, Taufkirchen, Germany).

Techniques: Migration