Supplementary Methods from Oncolytic Herpes Simplex Virus Vector G47Δ in Combination with Androgen Ablation for the Treatment of Human Prostate Adenocarcinoma

Supplementary Methods from Oncolytic Herpes Simplex Virus Vector G47Δ in Combination with Androgen Ablation for the Treatment of Human Prostate Adenocarcinom

Supplementary Figure 1 from Oncolytic Herpes Simplex Virus Vector G47Δ in Combination with Androgen Ablation for the Treatment of Human Prostate Adenocarcinoma

Supplementary Figure 1 from Oncolytic Herpes Simplex Virus Vector G47Δ in Combination with Androgen Ablation for the Treatment of Human Prostate Adenocarcinom

Supplementary Figures 1-2 from Oncolytic Herpes Simplex Virus Vector G47Δ in Combination with Androgen Ablation for the Treatment of Human Prostate Adenocarcinoma

Supplementary Figures 1-2 from Oncolytic Herpes Simplex Virus Vector G47Δ in Combination with Androgen Ablation for the Treatment of Human Prostate Adenocarcinom

Supplementary Figure 2 from Oncolytic Herpes Simplex Virus Vector G47Δ in Combination with Androgen Ablation for the Treatment of Human Prostate Adenocarcinoma

Supplementary Figure 2 from Oncolytic Herpes Simplex Virus Vector G47Δ in Combination with Androgen Ablation for the Treatment of Human Prostate Adenocarcinom

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

Injection of senescent cells in the penis impairs erectile function.

<p>HUVECs were infected with AdRas12V (AdRas12V_HUVECs) to induce cellular senescence. HUVECs were also infected with AdGFP (AdGFP_HUVECs) as the control. These cells were injected into the cavernous body of nude mice, and ICP and MAP were measured 2 (2W), 4 (4W) and 6 (6W) weeks after injection. ICP data obtained from control nude mice that were not injected with the cells and from nude mice 2 weeks after injection with AdGFP_HUVECs were used as the positive control. A) Schematic representation of ICP traces. The dotted area represents AUC. B) Bar graphs comparing ICP/MAP among the groups (n = 5 per group). *: P<0.05 vs. the AdGFP_HUVECs injection. C) Bar graphs comparing ICP_AUC/MAP among the groups (n = 5 per group). **: P<0.001 vs. the AdGFP_HUVECs injection.</p

Senescence occurs in the penis of diabetic mice.

<p>A) The penises were isolated from STZ-induced diabetic C57BL/6J mice 7 weeks after STZ injection. Frozen penile sections were subjected to SA-β-Gal staining. The penises of age-matched control mice were also used. Scale bars = 50 μmeter. B) Immunohistochemical analysis of the penises isolated from STZ-induced diabetic C57BL/6J mice and age-matched control mice. HP-1γ was stained. Arrows indicate the positively stained area. Scale bars = 50 μmeter. C) Western blot analysis of senescence-associated markers. Proteins were extracted from the penises of STZ-induced diabetic C57BL/6J mice (STZ) 7 weeks after STZ injection. Proteins were also extracted from the penises of age-matched control mice (Control). p53 and p21^CIP1 expressions were examined. The arrow indicates the bands corresponding to the size of p21^CIP1.</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

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

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