Ang-II induces ACE translocation to the nucleus.

<p>(A) Internalization of AT₁ and ACE in the presence of 4 nM ³H-Ang-II. Data are shown as mean from three independent experiments, each performed in duplicate. (B) CHO-ACE and CHO-AT₁ cells present the same relative protein level of each respective receptor. (Values are mean ± S.E.M, *p<0.05, n = 63 individual experiments). (C) Representative confocal images of internalized Ang-II-FITC (1 μM) in CHO-ACE and CHO-AT₁ cells after 30 seconds of Ang-II stimulation. DAPI (blue) and Wheat Germ Agglutinin (red), scale bar = 10 μm. On the right, quantification of internalized Ang-II-FITC is presented. Values are mean ± S.E.M, *p<0.05, n = 6. (D) Representative confocal images of unstimulated CHO-ACE and CHO-AT1 cells, labeled for DAPI and WGA. (E) Immunolocalization of ACE after stimulation with Ang-II (1μM), for the indicated times. ACE is shown in green, actin filaments in red, and nucleus in blue (DAPI). Right panel represents a 3D reconstruction of CHO-ACE cell after 15 minutes of incubation with Ang-II (1μM). Scale bar = 10μm. (F) Western blotting of nuclear and non-nuclear protein fractions from CHO-ACE cells, before (control) and after Ang-II (1 μM) stimulation for the indicated times. Histone-3 and GAPDH were used to shown the purification of nuclear and non-nuclear protein fractions, respectively. (G) Densitometry analysis of the western blot. Values are mean ± S.E.M., n = 3 (*** p<0.01).</p

Molecular interaction between ACE and PLC by computational analysis in silico.

<p>(A) Representation of the ten top ranked docking poses for ACE (blue) with PLCβ3 protein superimposed (the color of the PLC protein pose correspond to the colors of the labels). (B) Structure of the complex between ACE (blue) and PLC-β3 (Pose 9, red). The binding energy for the best docking pose of ACE and PLC-β3, Pose 9, is -194.06 kcal/mol. C) Amino acid residues located at the interface between the best docking pose of PLC-β3 (left panel) and ACE (right panel), Pose 9, explored through docking protocols.</p

ACE silencing inhibits the proliferative effect of Ang-II in melanoma cells.

<p>(A) Western bot (upper panel) to confirm the silencing of ACE and densitometry analysis (bottom panel). Mean ± S.E.M, n = 8. (***p<0.01 compared to respective columns). (B) BrDU uptake in Tm5 cells silenced for ACE and stimulated for 24 hours with Ang-II (1μM), showing a decrease in BrDU incorporation in the absence of ACE. Mean ± S.E.M., n = 12 (*p<0.05; ns = non-significant).</p

Ang-II promotes cellular migration and reduces focal adhesion formation in melanoma cells.

<p>(A-B) Wound healing assay using TM-5 cells stimulated with Ang-II (1μM). C) Representative confocal images of TM-5 cells double-labeled with vinculin (green) and phalloidin (red). Scale bar = 10 μm. (D-E) Quantification of the focal adhesion formation. Mean ± S.E.M., n = 6 (* p<0.05).</p

Cell proliferation induced by Ang-II/ACE involves clathrin- mediated internalization process.

<p>(A) Cell growth assay of CHO-ACE cells 12, 24 and 48 hours after stimulation with Ang-II (1μM), triplicate in 3 individual experiments. (B) Western blot to confirm the silencing of clathrin (upper panel) and densitometry analysis (bottom panel). Mean ± S.E.M., n = 5 (* p<0.05). (C) BrDU incorporation is decreased in CHO-ACE cells transfected with siRNA-Cla (Clathrin) and stimulated with Ang-II (1μM). Mean ± S.E.M., n = 6 (* p<0.05).</p

Effect of Ang-II on intracellular Ca2+ signaling in CHO-ACE cells.

<p>(A) Western immunoblotting to confirm the silencing of PLC isoforms. Densitometric analysis are shown on (B) for PLCβ3 and (C) for PLCγ1. (D) Quantitative representation of intracellular [Ca²⁺] in CHO-ACE cells transfected with siRNA-PLCβ3 and PLCγ1 using Lipofectamine and stimulated with Ang-II (1μM). (E) Line scanning of Ca²⁺ signal in CHO-ACE. Ang-II promotes Ca²⁺ increase with greater intensity in the nuclear region. (F) Time course of Ca²⁺ signaling in the nucleus (red traces) and cytosol (blue traces). (G) Quantification of fluorescence intensity signal in the nucleus and cytosol. Values are mean ± S.E.M., (* p<0.01), 45 cells, n = 3 individual experiments.</p

IFN-γ controls NOS2-mediated NO production during adapted-DENV-3 infection.

<p>(A–C) WT mice (<i>n</i> = 6 mice per group) were inoculated with 10LD₅₀ (1000 PFU) of adapted-DENV-3 (i.p) and 3, 5 or 7 days after infection, mice were culled and tissue were collected for the following analysis: (A) Determination of NOS2 RNA expression by qPCR in spleen of control and DENV-3 infected mice. Results are shown as fold increase over basal expression in naive mice. (B) Determination of NOS2 staining by immunohistochemistry in liver sections of control and DENV-3 infected mice. Results are expressed as number of positive cells per mm² of liver. (C) Esplenocytes were incubated with DAF-2DA and fluorescence determined. Results are expressed as fold increase in fluorescence over stained cells of naive mice. (D–E), WT and IFN-γ^−/− mice (n = 6 per group) were inoculated with 1LD₅₀ (100 PFU) of DENV-3 (i.p) and in the fifth day of infection mice were culled and tissues were collected for the following analysis: (D) Determination of NOS2 RNA expression by qPCR in spleen of WT and IFN-γ^−/− DENV-3 infected mice. Results are shown as fold increase over basal expression in naive mice. (E) Determination of NOS2 staining by immunohistochemistry in liver sections of WT and IFN-γ^−/− DENV-3 infected mice. Results are expressed as number of positive cells per mm² of liver. Results are expressed as mean ± SEM and are representative of at least two experiments. * for P<0.05 when compared to control naive mice. # for P<0.05 when compared to WT infected mice. 10LD₅₀ corresponds to 1000 PFU of DENV-3. 1LD₅₀ corresponds to 100 PFU of DENV-3. dpi – days post-infection. NI – Not-infected.</p

IFN-γ production is required for host resistance to adapted-DENV-3 primary infection.

<p>(A) WT mice (<i>n</i> = 4 mice per group) were inoculated with 10LD₅₀ (1000 PFU) of DENV-3 (i.p) and seven days later, mice were culled, and splenic cells isolated for assaying IFN-γ production by cellular staining with labeled antibodies and FACS analysis. Results are expressed as % of IFN-γ-positive cells in each population. (B) WT and IFN-γ^−/− mice (n = 8 per group) were inoculated with 1LD₅₀ (100 PFU) of DENV-3 (i.p) and lethality was evaluated every 12 hours during 14 days. Results are expressed as % of survival. In (C–J), WT and IFN-γ^−/− mice (n = 6 per group) were inoculated with 1LD₅₀ (100 PFU) of DENV-3 (i.p) and in the fifth day of infection mice were culled and blood and tissues were collected for the following analysis: (C–D) Viral loads were recovered from the blood (C), spleen and liver (D, left and right panels), respectively. Results are shown as the log of PFU per mL of blood or per g of tissue. (E) Serial sections from each liver were stained with anti-DV NS3 antibody E1D8 (NS3) or an isotype control mouse IgG2a (IgG2a data not shown), and multiple sections of each tissue type were thoroughly examined for staining. Positive staining for NS3 is brown while hematoxylin counterstain is blue. Results are expressed as number of NS3-positive hepatocytes. (F) Mechanical hypernociception was assessed daily. Results are shown as the difference between the force (g) necessary to induce dorsal flexion of tibio-tarsal joint, followed by paw withdraw, before and after DENV-3 inoculation. In (G), hematocrit was shown as % volume occupied by red blood cells (left panel) and the number of platelets was shown as platelets ×10³/µl of blood (right panel). (H) Changes in Systolic blood pressure from baseline until day 5 after infection expressed as Δ of blood pressure in mmHg. In (I), AST activity determination in plasma, shown as U/dL of plasma. (J) shows semi-quantitative analysis of hepatic damage (histopathological analysis performed as modified from Paes et al, 2009) and Hematoxylin & Eosin staining of liver sections of control and WT and IFN-γ^−/− DENV-3-infected mice, five days after infection. Scale bars - 400 µm. The images presented are representative of an animal on the fifth day of infection. All results are expressed as mean ± SEM (except for C–D, expressed as median) and are representative of at least two experiments. * for P<0.05 when compared to control uninfected mice. # fo P,0.05 when compared to WT infected mice. 10 LD₅₀ corresponds to 1000 PFU of adapted-DENV-3. 1LD₅₀ corresponds to 100 PFU of adapted-DENV-3. ND – not detectable. NI- Not-infected. dpi – days post-infection. HS – hepatocyte swelling. N – necrosis. D – degeneration. H – hemorrhage. OS – Overall Score.</p

NOS2^−/− mice are more susceptible to adapted-DENV-3 infection.

<p>(A) WT and NOS2^−/− mice (n = 8 per group) were inoculated with 1LD₅₀ (100 PFU) of DENV-3 (i.p) and lethality was evaluated every 12 hours during 14 days. Results are expressed as % of survival. In (B–J), WT and NOS2^−/− mice (n = 6 per group) were inoculated with 1LD₅₀ (100 PFU) of DENV-3 (i.p) and in the seventy day of infection mice were culled and blood and tissues were collected for the following analysis: (B–D) Viral loads were recovered from the blood (B), spleen (C) and liver (D), respectively. Results are shown as the log of PFU per per mL of blood or g of tissue. (E) Serial sections from liver of WT and NOS2^−/− mice were stained with anti-DV NS3 antibody E1D8 (NS3) or an isotype control mouse IgG2a (IgG2a data not shown), and multiple sections of each tissue type were thoroughly examined for staining. Positive staining for NS3 is brown while hematoxylin counterstain is blue.Results are expressed as number of NS3-positive hepatocytes. (F) Mechanical hypernociception was assessed daily. Results are shown as the difference between the force (g) necessary to induce dorsal flexion of tibio-tarsal joint, followed by paw withdraw, before and after DENV-3 inoculation. In (G), hematocrit was shown as % volume occupied by red blood cells (left panel) and the number of platelets was shown as platelets ×10³/µl of blood (right panel). (H) Changes in Systolic blood pressure from baseline until day 5 after infection expressed as Δ of blood pressure in mmHg. In (I), AST activity determination in plasma was shown as U/dL of plasma. (J) shows semi-quantitative analysis of hepatic damage (histopathological analysis performed as modified from Paes et al, 2009) and Hematoxylin & Eosin staining of liver sections of control and WT and NOS2^−/− DENV-3-infected mice, seven days after infection. Scale Bar - 400 µm. The images presented are representative of an animal on the seventh day of infection. All results are expressed as mean ± SEM and are representative of at least two experiments. * for P<0.05 when compared to control uninfected mice. # for P<0.05 when compared to WT infected mice.1LD₅₀ corresponds to 100 PFU of adapted-DENV-3. NI- Not-infected. dpi – days post-infection. HS – hepatocyte swelling. N – necrosis. D – degeneration. H – hemorrhage. OS – Overall Score.</p

Disease parameters in C57BL/6 mice infected with an adapted strain of DENV-3.

<p>(A) WT mice (<i>n</i> = 6 mice per group) were inoculated with different inoculums of adapted-DENV-3 (i.p) and lethality was evaluated every 12 hours for 14 days. Results are expressed as % of survival. In Figs (B–L) WT mice (n = 6 per group) were inoculated with 10LD₅₀ (1000 PFU) of DENV-3 (i.p) and in the third, fifth or in the seventh day of infection mice were culled and blood and tissues were collected for the following analysis: (B) Change in body weight was expressed as percentage of initial weight loss. (C) Mechanical hypernociception was assessed daily. Results are shown as the difference between the force (g) necessary to induce dorsal flexion of tibio-tarsal joint, followed by paw withdraw, before and after DENV-3 inoculation. In (D), hematocrit was expressed as % volume occupied by red blood cells (left panel) and the number of platelets was shown as platelets ×10³/µl of blood (right panel). (E) Changes in vascular permeability in the liver and lungs are shown as µg Evans blue per 100 mg of tissue (left and right panels, respectively). (F) Shows changes in Systolic blood pressure from baseline until day 7 after infection expressed as Δ of blood pressure in mmHg. (G) AST (left panel) and ALT (right panel) activity determination in plasma of control and DENV-3-infected mice was shown as U/dL of plasma. (H–L) Concentrations of IL-6, TNF-α, IFN-γ IL-12/23p40 and IL-18, quantified by ELISA. Results are shown as pg per mL (serum) or pg per 100 mg (tissue). All results are expressed as mean ± SEM and are representative of at least two experiments. * for P<0.05 when compared to control uninfected mice. 10 LD₅₀ corresponds to 1000 PFU of adapted-DENV-3. ND – not detectable. NA – not assessed. NI- Not-infected. dpi – days post-infection.</p