Rho-A activation increases the formation of stress fibers in HGEc-1.

<p><b>(A)</b> HGEc-1 were transfected with the corresponding Rho-A constructs described above. Subsequently, 24 hours later, they were seeded on coverslips, treated with (VEGF-A + Tat + Heparin), and stained with F-actin to visualize the formation of stress fibers as described above. Scale bar = 20 μm. <b>(B)</b> The graphs show mean ± SEM values corresponding to three different experiments that assessed the formation of stress fibers in cultured HGEc-1. Results were expressed as % changes in stress fibers relative to controls. Control cells (-) were treated with serum free medium. Statistically significant differences between control cells (-) vs. VEGF-A + Tat + Heparin-treated cells (+), were highlighted with an <i>asterisk</i>, *p<0.05. Difference between cells treated with VEGF-A + Tat + Heparin vs. other groups were highlighted with a cross, ++ p < 0.01.</p

VEGF-A in combination with HIV-Tat and heparin, increases the permeability of cultured HGEc-1 through Rho-A and Src dependent pathways.

<p><b>(A)</b> Monolayers of 5 hours-starved HGEc-1 were stimulated by VEGF-A (50 ng/ml), Tat (100 ng/ml), and Heparin (50 units/ml) all combined. The Rho-A inhibitor: C3 transferase (20 ng/ml), was added 4 hours before stimulation and the inhibitor of Src family kinase SU6656 (1 μM) and ROCK inhibitor Y-27632 (10 μM) were added 1hr before stimulation. The data represent FITC-dextran permeability expresses as fold increase. <b>(B)</b> Overnight-starved HGEc-1 monolayers were treated for 5 min as described above and then harvested to assess the phosphorylation of Rho-A, Rac1, MLC and Src, as described in Methods. Panel B shows representative Western blots corresponding to the phosphorylation changes. <b>(C)</b> The graphs show mean ± SEM values corresponding to three different Western blots that assessed the phosphorylation of Rho-A, Rac1, MLC, and Src in cultured HGEc-1. Results were expressed in arbitrary optical density units expressed as a ratio of the total activity. Values significantly different from control cells treated with serum free media were marked with <i>asterisks</i> **p<0.01, and those significantly different from the cells treated with VEGF-A + Tat + Heparin were marked with crosses, + p<0.05 and ++ p<0.01.</p

Rho-A activation increases the permeability of HGEc-1.

<p><b>(A)</b> Cultured HGEc-1 cells were transfected with different plasmids, pCEFL-mock, constitutively active RhoAQL (pCEFL-AU5-RhoAQL), or dominant negative mutant RhoAN19 (pCEFL-AU5-RhoAN19). Twenty-four hours later, the cells were treated with VEGF-A (50 ng/ml) + Tat (100 ng/ml) + Heparin (50 units/ml), all together, and exposed to FITC-dextran as described in methods. <b>(B)</b> In other experiments HGEc-1 cells were treated for 5 min as described above and then harvested to assess the phosphorylation of Rho-A, MLC, Src as described in Methods. Panel B shows representative western blots corresponding to the phosphorylation changes. <b>(C)</b> The graphs show mean ± SEM values corresponding to three different Western blots that assessed the phosphorylation of Rho-A, MLC and Src in cultured HGEc-1. Results were expressed in arbitrary optical density units as a ratio of the total activity. In each group, mock, RhoAQL and RhoAN19 transfected cells were treated with either serum free media (Controls) (-), or VEGF-A + Tat + Heparin (+). Groups that were significantly different from controls (-) were labeled with <i>asterisk</i>, *p<0.05 and **p<0.01. Cells transfected with constitutively active RhoAQL that were significantly different from mock or RhoAN19 cells, were labeled with crosses: + p <0.05 and ++ p<0.01.</p

FGF-2 and VEGF-A, in combination with HIV-Tat and heparin, induce the formation of stress fibers through Rho-A dependent pathways.

<p><b>(A)</b> Panel A shows representative changes in the formation of stress fibers detected in cultured HGEc-1. Overnight-starved HGEc-1 monolayer were stimulated by thrombin (100 units/ml) as a positive control, Tat (100 ng/ml), FGF-2 (50 ng/ml), VEGF-A (50 ng/ml), and Heparin (50 units/ml) alone or in combination. The RhoA inhibitor: C3 transferase (20 ng/ml), was added 4 hrs. before stimulation and the inhibitor of Src family kinase SU6656 (1 μM) and ROCK inhibitor Y-27632 (10 μM) were added 1hr before stimulation and 20 min after treatment, F-actin fibers were visualized in cells by staining with 2 μg/ml of Alexa Fluor 488-labeled phalloidin. Cell nuclei were stained with Hoechst 33342. The scale bar is 10 μm. <b>(B)</b> The graphs show mean ± SEM values corresponding to the formation of stress fibers in three different experiments. Results were expressed as % changes in stress fibers formation relative to control cells. Values significantly different from controls were marked with an <i>asterisk</i>, *p<0.05 and **p<0.01, and those different from cells treated with VEGF-A + Tat + Heparin were marked with a cross, +p <0.05 and ++p<0.01.</p

Urine samples harvested from HIV-infected children with renal diseases increase the permeability of cultured HGEc-1 through RhoA and Src mediated mechanisms.

<p><b>(A)</b> Urine samples harvested from HIV infected children with and without renal diseases (RD) were used (1:10 dilution) to stimulate monolayers of 5 hours-starved HGEc-1. The RhoA inhibitor: C3 transferase (20 ng/ml), was added 4 hrs before stimulation. The inhibitor of Src family kinase SU6656 (1 μM) and the ROCK inhibitor Y-27632 (10 μM) were added 1hr before stimulation. The data represent FITC-dextran permeability expresses as fold increase. <b>(B)</b> Overnight-starved HGEc-1 monolayer were treated with urine (1:20 dilution) for 5 min as described above and then harvested to assess the phosphorylation of RhoA, Rac1, MLC, and Src, as described in Methods. <b>(C)</b> The graphs show mean ± SEM values corresponding to three different Western blots that assessed the phosphorylation of Rho-A, Rac1, MLC, and Src, in cultured HGEc-1. Results were expressed in optical density units expressed as a ratio of the total activity. Values significantly different from the control group (HIV-N; n = 5) were marked with <i>asterisks</i> **p<0.01, and those significantly different from the HIV-RD group (n = 5) were marked with stars ★ p<0.05 and ★★ p<0.01 (for permeability), or crosses, + p <0.05 and ++ p<0.01 (for signaling).</p

Urine samples harvested from HIV-infected children with renal diseases increase the permeability of cultured podocytes cell line (P-2) through Rho-A and Src mediated mechanisms.

<p><b>(A)</b> Urine samples harvested from HIV infected children with and without renal diseases (RD) were used (1:10 dilution) to stimulate monolayers of 5 hours-starved P-2 cells. The data represent FITC-dextran permeability changes expressed as fold increase. <b>(B)</b> Overnight-starved P-2 monolayers were treated with urine (1:20 dilution) for 5 min as described above and then harvested to assess the phosphorylation of Rho-A, MLC, and Src, as described in Methods. <b>(C)</b> The graphs show mean ± SEM values corresponding to three different Western blots that assessed the phosphorylation of Rho-A, MLC, and Src in cultured podocytes. Results were expressed in optical density units expressed as a ratio of the total activity. Values significantly different from the serum free treated control cells (Control) were marked with <i>asterisk</i>, *p<0.05 and **p<0.01, and differences between HIV-RD (n = 5) and HIV-N (n = 5) urine samples, were marked with stars ★ p< 0.05 (for permeability) or crosses + p<0.05 (for signaling).</p

FGF-2 and VEGF-A in combination with HIV-Tat and heparin increase the permeability of cultured HGEc-1 through Rho-A and Src dependent pathways.

<p><b>(A)</b> Monolayers of 5 hours-starved HGEc-1 were stimulated by thrombin (100 units/ml) as a positive control, Tat (100 ng/ml), FGF-2 (50 ng/ml), VEGF-A (50 ng/ml), and Heparin (50 units/ml) alone or in combination. The data represent FITC-dextran permeability changes expressed as fold increase. <b>(B)</b> Overnight-starved HGEc-1 monolayer were treated for 5 min as described above and then harvested to assess the phosphorylation of Rho-A, Rac1, MLC, and Src, as described in Methods. Panel B shows representative Western blots corresponding to the phosphorylation changes. <b>(C)</b> The graphs show mean ± SEM values corresponding to three different Western blots that assessed the phosphorylation of Rho-A, Rac1, MLC, and Src in cultured HGEc-1. Results were expressed in arbitrary optical density units as a ratio of the total activity. Values significantly different from control cells treated with serum free media were marked with <i>asterisk</i>, *p<0.05 and **p<0.01.</p

Free energy as a function of the fraction of native intermolecular residue-residue contacts Q

Representative conformations along the reaction coordinate are shown (blue: KIX, red: pKID-α, and green: pKID-α). Inset: The probability p(T

The Akt-mTOR pathway is upregulated during wound healing.

<p>(Upper panel). Representative histological sections of skin after incisional cutaneous wounds in mice. H&E stained section displaying the histological characteristics of the unwounded epithelium (normal adjacent) followed by an increase in the thickness of the spinous layer (acanthosis) (transitional), and the presence of migrating epithelial cells that form an epithelial tongue (epithelial tongue) at the wound edge. (second panel from top). Immunofluorescence for Cytokeratins 10 and 14 (K10-14, red) reveals the epidermal layer, which is delineated from the dermis by a white dotted line. Note the expansion of pAkt⁴⁷³ and pS6 (red) during wound healing from a single cell layer in the granular layer to multiple cell layers in the transitional epithelium, followed by expression in all cell layers including the basal layer in the migrating epithelial tongue (lower two panels). Cell nuclei were stained with DAPI (blue) and fibrin(ogen) with a FITC-conjugated antibody (green) in all panels. Scale bar, 50 µm and 100 µm, as indicated.</p

Potential of mean force as function of the number of native and non-native contacts between pKID and KIX

Free energy surfaces are shown as a function of all non-native contacts (NNC) and native contacts (NC) (A–B) at equilibrium and (C–D) in transition paths. (E–F) The probability p(T