The effects of NAT1 knock-down on cell morphology.

<p>(<b>A</b>) Light microscopy of control (Control 4) and knock-down (shRNA 3.2) cells at low density (upper panels) and high density (lower panels). (<b>B</b>) Confocal microscopy of actin polymerization in cells at low (upper panels) and high (lower panels) density. Actin was stained with Alexa Fluor 647 conjugated phalloidin (red) and the cell nuclei were stained with DAPI (blue). White arrows show sub-cortical actin associated with sites of cell-cell contact (lower right panel). (<b>C</b>) Transmission electron microscopy of control and NAT1 knock-down cells at low and high density. Black arrows show vacuoles. (<b>D</b>) Longitudinal sections of control and NAT1 knock-down cells by transmission electron microscopy at high density. Arrowheads indicate the bottom of the culture flask.</p

Up-regulation of E-cadherin following NAT1 knock-down is not cell-type specific.

<p>(<b>A</b>) 22Rv1 cells were stably transfected with the NAT1-directed shRNA construct (Clones 1 and 2) and knock-down determined by NAT1 activity assay. Results are presented as mean ± SEM (<i>n</i> = 3). Asterisks denote significant difference compared to the control (p<0.05). (<b>B</b>) Western blot analysis of E-cadherin protein in 22Rv1 control and NAT1 knock-down cells. Immunoblotting was performed with E-cadherin antibody and tubulin was probed as the loading control. The blot is representative of 3 independent experiments. Immunoblots were quantified by densitometry after normalization to tubulin. Results are presented as means ± SEM (<i>n</i> = 3). A representative blot is shown above the graph. Asterisks denote significant difference compared to control (<i>P</i><0.05). (<b>C</b>) Immunocytochemistry of E-cadherin expression in 22Rv1 control and NAT1 knock-down cells. E-cadherin was detected with anti-E-cadherin antibody followed by Alexa Fluor 488-conjugated secondary antibody (green) and visualized under a fluorescence microscope. The nuclei were stained with DAPI (blue).</p

Growth of control and NAT1 knock-down cells <i>in vivo</i>.

<p>(<b>A</b>) Female Balb/c nude mice were injected subcutaneously with parental HT-29 cells (◊), Control 4 cells (•), or shRNA3.2 cells (○). Tumor volume was determined as outlined in the Methods. Results are mean ± SEM (<i>n</i> = 7–8). (<b>B</b>) Representative H&E staining of tumors (upper panels) from Control 4 (left panel) or shRNA 3.2 (right panel) showing large areas of necrosis (*) along with loosely organized columnar cells (small arrows). In the tumors derived from the shRNA 3.2 cells, more organized glandular structures were evident (large arrow). Representative immunohistochemistry for Ki67 in Control 4 and shRNA 3.2 tumors is shown in the middle and lower panels. (<b>C</b>) NAT1 activity in individual tumors from Control 4 and shRNA3.2 tumor tissue. The results are mean ± SEM (<i>n</i> = 3). (<b>D</b>) Real-time PCR of human NAT1 mRNA in individual tumors from Control 4 and shRNA3.2 tumor tissues. Results were normalized to β-actin and then expressed relative to Control 4. The results are mean ± SEM (<i>n</i> = 3).</p

The effects of NAT1 knock-down by shRNA are not due to off-target effects.

<p>(<b>A</b>) NAT1 was reintroduced into the shRNA 3.2 cells (shRNA 3.2+NAT1) and NAT1 activity measured. Each value is mean ± SEM (<i>n</i> = 3). The asterisk denotes a significant difference compared to Control 4 (p<0.05). (<b>B</b>) Control 4, shRNA3.2, and shRNA3.2+NAT1 cell lysates were immunoblotted for E-cadherin. Tubulin was probed as the loading control. The blot is representative of 3 independent experiments. (<b>C</b>) NAT1 mRNA levels were quantified by real-time PCR using primers that detect endogenous NAT1 mRNA but not NAT1 mRNA derived from the exogenous plasmid. The data are presented as mean ± SEM (<i>n</i> = 3). Results were normalized to β-actin levels and then expressed relative to Control 4 mRNA. Asterisks denote significant difference compared to Control 4 (p<0.05). (<b>D</b>) Different independent non-overlapping shRNA targeting the NAT1 open reading frame were used to knock-down NAT1 and then NAT1 activity was assessed for each of the different clones. Each value is mean ± SEM (<i>n</i> = 3). Asterisks denote significant difference compared to Control 4 (p<0.05). (<b>E</b>) The effect of NAT1 knock-down by the different shRNA sequences on E-cadherin protein expression was determined by immunoblotting. Tubulin was probed as the loading control.</p

NAT1 knock-down up-regulates E-cadherin mRNA and alters DNA methylation status in the cell.

<p>(<b>A</b>) E-cadherin mRNA levels in NAT1 knock-down cells. Each value is mean ± SEM (<i>n</i> = 3). The asterisk denotes significant difference from control (p<0.05). (<b>B</b>) Western blot analysis of E-cadherin suppressor proteins in control and NAT1 knock-down cells. Twist and Slug were not detected in either cell-line and there was no difference in the expression of Snail. Tubulin was probed as the loading control.</p

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Additional file 3: of Immunomodulatory activities of pixatimod: emerging nonclinical and clinical data, and its potential utility in combination with PD-1 inhibitors

The incidence and severity of noteworthy microscopic findings in kidneys, liver, spleen and thymus. Perivascular mixed cell infiltrate was present in most mid- and high-dose individuals. Minimal to mild dilatation of renal tubules was apparent in most treated individuals though minimal or mild glomerular vacuolation or sclerosis was only reported in high-dose individuals. Minimal to mild hepatocellular hypertrophy was evident across dose levels whereas incidence and severity of hypertrophy of Kupffer cells in the liver was dose-dependent. A minimal to mild increase in cell infiltrate was apparent some high-dose individuals. There was also evidence of diffuse mixed cellular infiltrate in the spleen and minimal to mild lymphoid atrophy of the thymus observed in mid and high dose animals. (DOCX 13 kb

Strand breakage by decay of DNA-bound ¹²⁴I provides a basis for combined PET imaging and Auger endoradiotherapy

<p><b>Purpose</b> DNA ligands labelled with ¹²⁵I induce cytotoxic DNA double-strand breaks (DSB), suggesting a potential for Auger endoradiotherapy. Since the 60-day half-life of ¹²⁵I is suboptimal for therapy, we have investigated another Auger-emitter ¹²⁴I, with shorter half-life (4.18 days), and the additional feature of positron-emission, enabling positron emission tomography (PET) imaging. The purpose of this study was to compare the two radionuclides on the basis of DNA DSB per decay.</p> <p><b>Materials and methods</b> Using a ¹²⁴I- (or ¹²⁵I)-labelled minor groove binding DNA ligand, we investigated DNA breakage using the plasmid DNA assay. Biodistribution of the conjugate of the labelled ligand with transferrin was investigated in nude mice bearing a K562 human lymphoma xenograft.</p> <p><b>Results</b> The probability of DSB per decay was 0.58 and 0.85 for ¹²⁴I and ¹²⁵I, respectively, confirming the therapeutic potential of the former. The crystal structure of the ligand DNA complex shows the iodine atom deep within the minor groove, consistent with the high efficiency of induced damage. Biodistribution studies, including PET imaging, showed distinctive results for the conjugate, compared to the free ligand and transferrin, consistent with receptor-mediated delivery of the ligand.</p> <p><b>Conclusions</b> Conjugation of ¹²⁴I-labelled DNA ligands to tumor targeting peptides provides a feasible strategy for Auger endoradiotherapy, with the advantage of monitoring tumor targeting by PET imaging.</p

Diagnostic Imaging Agents for Alzheimer’s Disease: Copper Radiopharmaceuticals that Target Aβ Plaques

One of the pathological hallmarks of Alzheimer’s disease is the presence of amyloid-β plaques in the brain and the major constituent of these plaques is aggregated amyloid-β peptide. New thiosemicarbazone-pyridylhydrazine based ligands that incorporate functional groups designed to bind amyloid-β plaques have been synthesized. The new ligands form stable four coordinate complexes with a positron-emitting radioactive isotope of copper, ⁶⁴Cu. Two of the new Cu^II complexes include a functionalized styrylpyridine group and these complexes bind to amyloid-β plaques in samples of post-mortem human brain tissue. Strategies to increase brain uptake by functional group manipulation have led to a ⁶⁴Cu complex that effectively crosses the blood-brain barrier in wild-type mice. The new complexes described in this manuscript provide insight into strategies to deliver metal complexes to amyloid-β plaques

Diagnostic Imaging Agents for Alzheimer’s Disease: Copper Radiopharmaceuticals that Target Aβ Plaques

One of the pathological hallmarks of Alzheimer’s disease is the presence of amyloid-β plaques in the brain and the major constituent of these plaques is aggregated amyloid-β peptide. New thiosemicarbazone-pyridylhydrazine based ligands that incorporate functional groups designed to bind amyloid-β plaques have been synthesized. The new ligands form stable four coordinate complexes with a positron-emitting radioactive isotope of copper, ⁶⁴Cu. Two of the new Cu^II complexes include a functionalized styrylpyridine group and these complexes bind to amyloid-β plaques in samples of post-mortem human brain tissue. Strategies to increase brain uptake by functional group manipulation have led to a ⁶⁴Cu complex that effectively crosses the blood-brain barrier in wild-type mice. The new complexes described in this manuscript provide insight into strategies to deliver metal complexes to amyloid-β plaques