Systemic administration of NBD peptide inhibits IKK and IκBα phosphorylation in malignant lymphoid tissue.

<p>Biopsies of malignant lymph nodes taken before (pre-) and 24 hours after (post-) NBD peptide administration were evaluated for the presence of phospho-IKKα/β and phospho-IκBα by immunoblot. <b>A</b>. Immunoblots for phospho-IKKα/β and IKKβ and phospho-IκBα and IκBα. Immunoblots were evaluated by densitometry and the ratios of <b>B</b>. phospho-IKKα/β:IKKβ and <b>C</b>. phospho-IκBα:IκBα were calculated. ND (Not Determined).</p

NBD peptide promotes apoptosis and reduces tumor burden in a subset of dogs with ABC-like DLBCL.

<p><b>A</b>. Malignant lymph node tissue sections taken before (pre-) and 24 hours after (post-) NBD peptide treatment were stained using TUNEL and counterstained with DAPI. <b>B</b>. Apototic cells were counted in 10 high power fields and the average number of positive cells was determined for both pre- and post-treatment sections. <b>C</b>. Calculated percent decrease in tumor mass 24 hours after NBD peptide treatment. Measureable tumor burden was determined pre and 24 hours post NBD peptide treatment and the percentage change in tumor burden was calculated. ND (Not Determined).</p

NBD peptide inhibits malignant cell proliferation and Cyclin D expression in a subset of dogs with ABC-like DLBCL

<p><b>A.</b> Malignant lymph node tissue sections taken before (pre-) and 24 hours after (post-) NBD peptide treatment were evaluated for expression of Ki67 by IHC. <b>B</b>. 10 high power fields were evaluated for Ki67 positive cells in each pre- and post-NBD peptide tissue section. The average number of Ki67 positive cells per high power field (hpf) was determined. <b>C</b>. Quantitative RT-PCR analysis of CyclinD expression in malignant tumor samples post-NBD peptide. The relative quantification of gene expression in post treatment samples was normalized to pre-treatment values for each dog. All assays were performed in triplicate and β-actin was used as an endogenous control. ^★p<0.05, ^★★p<0.005, ND (Not Determined).</p

NBD inhibits the expression of canonical NF-κB target genes in a subset of dogs with ABC-like DLBCL.

<p>NF-κB target gene expression within malignant lymph node tissues before and 24 hours after NBD peptide administration was determined by qRT-PCR. The relative quantification of gene expression in post treatment samples was normalized to pre-treatment values for each dog. β-actin was used as an endogenous control and all assays were performed in triplicate. p values are calculated on dCt data, ^★p<0.05, ^★★p<0.005.</p

Constitutive canonical NF-κB activity is present in dogs with large B cell lymphoma.

<p><b>A</b>. Whole cell extracts from malignant lymph node biopsies were analyzed by immunoblot for the presence of phospho-IKKα/β, IKKβ, phospho-IκBα, IκBα, phospho-p65 and β-actin. WCE from TNF-α activated canine PBMCs and from the human ABC-DLBCL cell line Ocl-Ly3 were used as positive controls and WCE from unstimulated canine PBMCs and the GCB-DLBCL cell line SUDHL-6 were used as negative controls. Integrated densitometry was used to quantify amounts of phospho-IKKα/β and IKKβ and phospho-p65 and β-actin. Relative canonical NF-κB activity in each sample was expressed as <b>B</b>. phospho-IKKα/β:IKKβ and <b>C</b>. phospho-p65:β-actin. The horizontal line in <b>B</b> and <b>C</b> marks the phosphorylation status of IKKα/β and phospho-p65 respectively in TNF-α activated canine PBMCs.</p