Immunoblotting showing protein expression level with respect to PKCδ and PKCγ.

<p>Western blot analysis showing the expression levels of PKCδ and PKCγ in control, CCI, and ipsilateal SCDH with intramuscular injection with Ad-MOCK or Ad-GDNF (A). The expression levels of PKCδ and PKCγ with respect to each tested group were shown as bar charts of relative ratio normalized with the expression levels of β-actin (B–C). *P<0.05, **P<0.01 compared with the Ad-GDNF group.</p

Immunoblotting showing protein expression level with respect to apoptotic and autophagic marker.

<p>Western blot analysis of the effect of CCI on the expression of AIF, caspase-9, cleaved caspase-9, caspase-3, cleaved caspase-3, PARP, cleaved PARP, SPECTRIN, cleaved SPECTRIN, and Beclin-1 on ipsilateral SCDH by intramuscular injection with adenovirus plus GDNF gene (A). Ratios of AIF, cleaved caspase-9, cleaved caspase-3, cleaved PARP, cleaved SPECTRIN, Beclin-1 with β-actin on ipsilateral SCDH were measured using western blot analysis (B–G). *P<0.05, **P<0.01 compared with the CCI group.</p

Immunoblotting showing protein expression level with respect to phosphor-p38, p38 and ED-1.

<p>Western blot analysis showing the expression levels of phospho-p38, p38 and ED-1 in control, CCI, and ipsilateral SCDH with intramuscular injection of Ad-MOCK or Ad-GDNF (A). The expression levels of phospho-p38 and ED-1 with respect to each tested group were shown as bar charts of relative ratio normalized with expression levels of p38 and β-actin, respectively (B-C). *P<0.05, **P<0.01 compared with the Ad-GDNF group. Double immunofluorescence staining of OX42 (D–G), a microglia marker, and phosphor-p38 (H–K) in different tested groups. The expression levels with respect to OX42 and phospho-p38 were obviously enhanced after CCI, but phospho-p38 was no longer highly expressed after administration of Ad-GDNF as shown in merged images (L–O).</p

Immunoblotting showing protein expression level with respect to different NOS isoform.

<p>Western blot analysis showing the expression levels of iNOS, nNOS and eNOS in control, CCI, and ipsilateral SCDH with intramuscular injection with Ad-MOCK or Ad-GDNF (A). The expression levels of iNOS, nNOS and eNOS with respect to each tested group were shown as bar charts of relative ratio normalized with the expression levels of β-actin (B–D). **P<0.01 compared with Ad-GDNF group.</p

Immunoblotting showing protein expression level with respect to IL-6 and IL-1β.

<p>Western blot analysis showing the expression levels of IL-6 and IL-1β in control, CCI, and ipsilateral SCDH with intramuscular injections of Ad-MOCK or Ad-GDNF (A). The expression levels of IL-6 and IL-1β with respect to each tested group were shown as bar charts of relative ratio normalized with the expression levels of β-actin (B–C). *P<0.05, **P<0.01 compared with control group.</p

Immunoblotting showing protein expression level with respect to MMP-2 and MMP-9.

<p>Western blot analysis showing the expression levels of MMP-2 and MMP-9 in control, CCI, and ipsilateral SCDH with intramuscular injection of Ad-MOCK or Ad-GDNF (A). The expression levels of MMP-2 and MMP-9 with respect to each tested group were shown as bar charts of relative ratio normalized with the expression level of β-actin (B–C). *P<0.05 compared with control group.</p

The effect of intramuscular delivery of Ad-GDNF on allodynia (A) and thermal hyperalgesia (B) in the CCI model.

<p>*P<0.05 compared with the CCI group at each time point.</p

Double immunofluorescent staining of TUNEL and a neuronal cell marker, NeuN, in the rat ipsilateral SCDH in different treatment groups.

<p>Tissue samples were detected using antibodies against NeuN (A–D) and TUNEL staining for apoptosis (E–H). The merged images show neuron apoptosis in the ipsilateral SCDH (I–L). Yellow arrows indicate TUNEL-positive neurons. The bar chart with respect to fold increase of TUNEL staining positivity (M) and double labeling (TUNEL and NeuN, N) revealed that apoptotic events triggered by CCI were attenuated by Ad-GDNF.</p

Immunoblotting showing protein expression level with respect to GDNF and its receptor.

<p>Western blot analysis showing the expression levels of GDNF and its receptor, GDNFRa-1, under control, CCI, and ipsilateral SCDH with intramuscular injection of Ad-MOCK or Ad-GDNF (A). The expression levels of GDNF and GDNFRa-1 with respect to each tested group were shown as bar charts of relative ratio (B–C). Immunohistochemical (D–K) staining was used to confirm GDNF expression. (D–G: 200X magnification, H–K: 400X magnification) *P<0.05 **P<0.01 compared with control group.</p

Combining Paclitaxel with ABT-263 Has a Synergistic Effect on Paclitaxel Resistant Prostate Cancer Cells

<div><p>We assessed the capability of paclitaxel, one of the taxanes, to induce death in two prostate cancer lines, LNCaP and PC3. Paclitaxel drove an apoptotic pathway in LNCaP, but not in PC3 cells, in response to G2/M arrest. An examination of the levels of anti-apoptotic proteins revealed that Bcl-xl was much higher in PC3 cells than in LNCaP cells and Bcl2 could be detected only in PC3 cells, not in LNCaP cells. Knocking down Bcl-xl enhanced paclitaxel-induced apoptosis in LNCaP cells, while we were unable to knock down Bcl-xl efficiently in PC3 cells. Significantly, a comparison of ABT-263, a specific inhibitor of Bcl2 and Bcl-xl, with ABT-199, a Bcl2 selective inhibitor, disclosed that only ABT-263, not ABT-199, could induce apoptosis in LNCaP and PC3 cells. The results indicate that Bcl-xl has a protective role against paclitaxel-induced apoptosis in LNCaP and PC3 cells, and its overexpression causes the paclitaxel resistance seen in PC3 cells. Interestingly, combined paclitaxel with ABT-263 to treat LNCaP and PC3 cells demonstrated synergistic apoptosis activation, indicating that ABT-263 could enhance paclitaxel-induced apoptosis in LNCaP cells and overcome Bcl-xl overexpression to trigger paclitaxel-induced apoptosis in PC3 cells. We also observed that the activation of apoptosis in LNCaP cells was more efficient than in PC3 cells in response to paclitaxel plus ABT-263 or to ABT-263 alone, suggesting that the apoptosis pathway in PC3 cells might have further differences from that in LNCaP cells even after Bcl-xl overexpression is accounted for.</p></div