Arsenic combined with IFN induced capase-dependent apoptosis and latent viral proteins downregulation in BC-3 cells.

<p>(<b>A</b>) Western blot analysis of BC-3 cells treated for 48h using PARP-specific antibody. (<b>B</b>) Western blot analysis of <i>ex-vivo</i> treated (48h) ascites derived BC-3 cells using LANA-1 and LANA-2 specific antibodies.</p

Arsenic/IFN synergistically inhibited proliferation and induced apoptosis of ascites-derived BC3 (left) and BCBL-1 cells (right).

<p>(<b>A</b>) Cell Proliferation: Cells were plated in a 96 well format and treated with the single agent drugs or their combinations for 24, 48, and 72h. Results are expressed as percent of control, plotted as mean ± SD, and are representative of two independent experiments. (<b>B</b>) Annexin V staining: BC-3 and BCBL-1 ascites were treated for 48h. Histograms represent the proportion of apoptotic cells. Results are plotted as mean ± SD and are representative of at least 2 independent experiments. (<b>C</b>) TUNEL assay: BC-3 and BCBL-1 cells derived from PEL ascites were treated for 72h. Histograms represent apoptotic cells as percentage of the untreated controls and are plotted as mean ± SD.</p

Arsenic and IFN synergistically prolonged survival in PEL mice.

<p>(<b>A</b>) Mice phenotype before and after treatment with arsenic/IFN. (<b>B</b>) Solid PEL tumors in untreated mice. (<b>C</b>) PCR for V-FLIP gene expression in different organs. (<b>D</b>) Kaplan–Meier analysis of overall survival curves of PEL NOD/SCID mice. Mice (<i>n</i> = 10 for each condition) were inoculated with 2x10⁶ of BC-3 (left) and BCBL-1 (right) cells, respectively. Treatment with the single agent drugs or their combinations was initiated at 2 days post-inoculation of PEL cells for a total of 21 days. The symbol * was used to compare treatment groups to control, while the symbol ‡ was used to compare combination treatments to single treatments. (*, ‡) indicates p< 0.05; (**, ‡‡) indicates p< 0.01; and (***, ‡‡‡) indicates p< 0.001.</p

Arsenic and IFN delayed ascites formation in PEL mice.

<p>Peritoneal volume on day 45 post-treatment. PEL NOD/SCID mice (n=10 per condition) were visually monitored. Peritoneal diameter (d) was measured with a caliber to assess ascites development. Peritoneal volume was calculated according to the formula: v=4/3π(d/2)³. The symbol * was used to compare treatment groups to control (*, **, and *** indicates p< 0.05, p< 0.01, and p< 0.001, respectively).</p

Arsenic/IFN synergistically inhibited expression of latent viral transcripts.

<p>Relative expression levels of different samples were calculated by standardization of the amount of target transcript for (A) LANA-1, (B) v-Cyclin or (C) v-FLIP, in a sample to the amount of housekeeping Glucose-6-phosphate dehydrogenase (G6PDH) RNA analyzed in the same sample. In addition, the averages of the normalized control values of Glucose-6-phosphate dehydrogenase (G6PDH) for each sample were used to determine the relative changes in gene expression of the KSHV latency protein LANA-1 by the comparative CT method (2^-ΔΔC_T) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079474#B12" target="_blank">12</a>]. Untreated BC3 and BCBL-1 ascites were used as a calibrator for viral gene expression. The Y-axis represents the relative quantities expressed as percent of the control. The symbol * was used to compare treatment groups to control, while the symbol ‡ was used to compare combination treatments to single treatments. (*, ‡) indicates p< 0.05; (**, ‡‡) indicates p< 0.01; and (***, ‡‡‡) indicates p< 0.001.</p