Expression of NMB by AdNMB.

<p>(A) Expression of NMB protein by AdNMB. HEK293 cells plated on type I collagen-coated dishes, were infected with AdNMB. After 24 h, the medium was replaced with serum-free DMEM, and the medium was collected 2 hours later for ELISA (n = 6 per group). *: P<0.001 vs. AdGFP infection. (B) Expression of NMB mRNA in the penis after AdNMB injection. AdGFP and AdNMB were injected into the penis and total RNA was extracted 7 days after injection. Real time PCR analysis was performed in order to examine the expression of NMB mRNA in the penis (n = 5 per group). *: P<0.01 vs. AdGFP infection.</p

Elastica van Gieson staining of the cavernous body isolated from age-matched positive control rats and STZ-induced diabetic rats injected with AdGFP or AdNMB.

<p>AdGFP (STZ/AdGFP) and AdNMB (STZ/AdNMB) were injected into the penises of STZ-induced diabetic rats and the penises were harvested for Elastica van Gieson staining 4 weeks later. Age-matched non-diabetic rats were used as positive controls (PC). (A) The histology of the root portion of penis (longitudinal section) is shown. Bars are 300 μm. (B) The histology of the cross section of penis is shown. Arrowheads indicate the dorsal penile nerves. Bars are 300 μm.</p

Overexpression of NMB restores erectile function.

<p>AdGFP (STZ/AdGFP) and AdNMB (STZ/AdNMB) were injected into the penises of STZ-induced diabetic rats, and ICP was measured 4 weeks later. Age-matched non-diabetic control rats were also used as positive controls (PC). (A) Representative traces of ICP and systemic arterial pressure (SAP). The stimulus interval (10 s) is indicated by a solid bar. (B) Histograms comparing the ICP/MAP among the groups (n = 5 per group). * and **: P<0.01 and P<0.001, respectively vs. PC. †: P<0.001 vs. AdGFP-injection. (C) Histograms comparing the AUC/MAP (n = 5 per group). * and **: P<0.01 and P<0.001, respectively vs. PC. †: P<0.001 vs. AdGFP-injection.</p

Effects of NMB on the differentiation and survival of SH-SY5Y cells.

<p>(A) Effect of NMB on the differentiation of SH-SY5Y cells. SH-SY5Y cells were cultured in DMEM-F12 (1:1) containing 1% FBS in the presence of NMB (10⁻⁷ M) or NGF (25 ng/mL) for 3 days. SH-SY5Y cells cultured in DMEM-F12 (1:1) containing 1% FBS were used as negative controls (NC). Representative photographs of the neurites are shown. Scale bars are 50 μmeter. (B) Bar graphs comparing the length of neurites. One hundred of neurites were randomly selected in each group and the length of the neurites was measured. *: P<0.01 vs. NC. (C) Effect of NMB on the survival of SH-SY5Y cells. SH-SY5Y cells were cultured in serum-free DMEM-F12 (1:1) in the presence of NMB (10⁻⁸ M or 10⁻⁷ M) or NGF (25 ng/mL) for 2 days. SH-SY5Y cells cultured in serum-free DMEM-F12 (1:1) were used as negative controls (NC) and those cultured in DMEM-F12 (1:1) containing 5% FBS (5% FBS) were used as positive controls. The cell viability observed in SH-SY5Y cells cultured in DMEM-F12 (1:1) containing 5% FBS was calculated as 100%, and the relative viability is shown (n = 8 per group). * and **: P<0.05 and P<0.01, respectively vs. negative control.</p

Expression of NMB in ASCs.

<p>(A) Expression of NMB mRNA. Rat ASCs were cultured in EBM and EGM for 7 days. Total RNA was extracted from ASCs and real time PCR analysis was performed in order to examine the expression of NMB (n = 6 per group). *: P<0.01 vs. EBM culture. (B) Expression of NMB protein by ASCs. After culturing ASCs in EBM and EGM for 7 days, the cells were washed with PBS, and the medium was changed to serum-free DMEM. After 2 h, the medium was collected and used for ELISA (n = 6 per group). *: P<0.01 vs. EBM culture.</p

Expression of VE-Cad, eNOS, SMA, and nNOS proteins in the penis.

<p>AdGFP (STZ/AdGFP) and AdNMB (STZ/AdNMB) were injected into the penises of STZ-induced diabetic rats and the penises were harvested for protein extraction 4 weeks later. Age-matched non-diabetic rats were used as positive controls (PC). Western blot analysis was performed to detect the expression of VE-Cad, total eNOS (T-eNOS), phospho-eNOS (P-eNOS), SMA, and nNOS. (A) The representative images of the expression of VE-Cad, total eNOS, phospho-eNOS, SMA, and nNOS are shown. (B) Bar graphs comparing the expression levels of VE-Cad, total eNOS, phospho-eNOS, SMA, and nNOS among the groups (n = 4 per group). * and **: P<0.05 and P<0.01, respectively vs. AdGFP-injected diabetic rats.</p

Western blot analysis of VE-Cad, SMA, phospho-eNOS, total eNOS, and nNOS expressions.

<p>A) The penises were isolated for protein extraction 2 weeks after the AdGFP_HUVECs injection, and 2 (2W), 4 (4W), and 6 (6W) weeks after the AdRas12V_HUVECs injection. Proteins were also extracted from age-matched control mice (Control). Representative photographs are shown. B) Histograms showing the relative intensity of the bands (n = 4 per group). * and **: P<0.05 and P<0.01, respectively vs. AdGFP_HUVECs injection. T-eNOS: total eNOS.</p

Immunohistochemical staining of nNOS in the dorsal penile nerve.

<p>Experiments were performed in the same way as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124129#pone.0124129.g003" target="_blank">Fig 3</a>. nNOS was stained. The photo of the negative control (Negative Cont) is also shown in which the incubation with primary antibody against nNOS was omitted. Scale bar = 40 μmeter.</p

The expression of proinflammatory cytokines in the penis.

<p>AdGFP_HUVECs (AdGFP) and AdRas12V_HUVECs (AdRas12V) were injected in the penises of nude mice and RNA was extracted from the penises 1 (1D), 7 (7D), 14 (14D), and 28 (28D) days after injection for real time PCR analysis. Nontreated mice (Control) were also analyzed. A) The expression of human IL-1β that was produced from injected HUVECs. The ratio of human IL-1β to mouse GAPDH was calculated to demonstrate human IL-1β expression in the penises of nude mice. The expression of human IL-1β in AdRas12V_HUVECs–injected penises on day 1 was calculated as 1.0 and the fold induction was shown in other groups (n = 5 per group). * and **: P<0.05 and P<0.01, respectively vs. AdRas12V_HUVECs–injected penises on day 1, †: P<0.01 vs. AdRas12V_HUVECs–injected penises at each time point. B) The expression of mouse IL-1β, mouse IL-6, and mouse TNF-α in the AdGFP_HUVECs- or AdRas12V_HUVECs-injected penises. The expression of these cytokines in nontreated control mice (Control) was calculated as 1.0 and fold induction was shown in other groups (n = 5 per group). *: P<0.05 vs. Control, †: P<0.05 vs. AdGFP_HUVECs–injected penises at each time point.</p

Elastica van Gieson staining of the cavernous body isolated from age-matched nude mice and nude mice injected with AdGFP_HUVECs or AdRas12V_HUVECs.

<p>The penises were isolated 2 weeks after AdGFP_HUVECs injection, and 2 (2W), 4 (4W), and 6 (6W) weeks after the AdRas12V_HUVECs injection. The penises were also isolated from nude mice that were not injected with HUVECs as the positive control (Positive Cont). The boxed areas corresponding to the dorsal penile nerve are enlarged and shown at the right lower corners. Scale bar = 200 μmeter.</p