<i>In vivo</i> therapeutic activity of bioconjugates.

<p>Kaplan-Meier survival curves of mice with peritoneal carcinomatosis from HT-29, MKN-45 or OE-21 tumor cells. Animals were randomly assigned to an experimental group and drug treatment was initiated according to therapeutic schedule reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112240#s2" target="_blank">Materials and Methods</a>. All experimental groups were statistically compared each other, but only significant values are reported in each panel.</p

Assessment of <i>in vivo</i> tumor growth and response to therapy.

<p>A, bioluminescence imaging of pharmacologically treated or untreated mice with peritoneal carcinomatosis induced by luciferase-transduced tumors. Panels show three representative mice per group at one month after tumor injection. B, cumulative results. Each box plot reports mean ± SD of total photon emission from 6 mice per group at one month from peritoneal carcinomatosis induction. Statistical analysis (Kruskal-Wallis test) is reported in tables at the right of each panel.</p

Confocal microscopy analysis and co-localization studies.

<p>A, accumulation of bioconjugates in HT-29, MKN-45 and KYSE-30. Cells were incubated with BODIPY-labeled ONCOFID-P (50 µg/mL in paclitaxel equivalents) or ONCOFID-S (50 µg/mL in SN-38 equivalents) for 1 hour, washed and fixed before analysis. B, co-localization analysis of bioconjugates in lysosomes. HT-29, MKN-45 and OE-33 cells were treated with LysoTracker green, incubated with BODIPY-labeled compounds and finally analyzed by confocal microscopy. Left pictures show the fluorescence of the labeled bioconjugates (red) in single cells, while central pictures illustrate signals (green) from lysosomes. The merging of the 2 components is visible in right pictures. Lysosomes were occupied by bioconjugates by ∼90% to 100%, as assessed by the Zeiss’profile software tool. Experiments were repeated at least twice with consistent results.</p

Assessment of bioconjugate mechanism of action.

<p>A, rearrangement of tumor cell microtubular architecture after drug treatment. HT-29, MKN-45 and OE-21 cells were treated with ONCOFID-P or free paclitaxel for 4 hours at 37°C. After treatment, cells were fixed, permeabilized, and stained with an anti-β-tubulin mAb and anti-mouse Ig Alexa 546-conjugated antiserum. Cells treated with free drug or bioconjugate disclosed the same interferences on the microtubular mesh. B, inhibition of Topo I activity after ONCOFID-S or SN-38 treatment in HT-29, MKN-45 and OE-21 cells. Gels show the supercoiled or relaxed forms of pBR322 plasmid after incubation with a 1∶50 dilution of nuclear protein neat extracts obtained from tumor cells treated with conjugated or free drug for 4 hours. Lane 1, marker; lane 2, relaxed pBR322 plasmid (positive control); lane 3, supercoiled plasmid (negative control); lane 4, supercoiled plasmid in the presence of nuclear protein neat extract from drug-untreated cells; lane 5, supercoiled pBR322 admixed with nuclear protein neat extract from ONCOFID-S treated cells; lane 6, supercoiled pBR322 admixed with nuclear protein neat extract from SN-38-treated cells. C, quantification of the reactions shown in B. Figure reports mean ± SD of 3 independent experiments.</p

Endocytosis pathways involved in bioconjugate cell entry.

<p>HT-29, MKN-45 and OE-21 tumor cells were left untreated (solid line) or treated (dashed line) for 1 hour with selective chemical inhibitors of different pathways involved in endocytosis (amiloride, chlorpromazine, cytochalasin D and filipin III). Subsequently, cells were exposed for 30 minutes to ONCOFID-P and then treated with hyaluronidase for 4 hours, to be finally analyzed by flow cytometry. Data at the upper-right corner of each panel report the respective geo mean values, and the percentage of reduction induced by treatment.</p

Interaction of bioconjugates with cancer cell lines.

<p>A, BODIPY-labeled ONCOFID-P (50 µg/mL in paclitaxel equivalents) or ONCOFID-S (50 µg/mL in SN-38 equivalents) were added to tumor cells and flow cytometry analysis was performed at different time points thereafter (0.5, 1, 2, 5, 10, 15, 30 or 60 minutes). Panels illustrate cytometry profiles at 3 representative time points. B, whole kinetics of interaction at all time points tested. C, kinetics of the fluorescence intensity (geo mean) detected on tumor cells at the same time points analysed as in B. Panels B and C report mean ± SD of 3 independent experiments.</p

Induction of gene expression by stimulation of LPS-activated MPs with IFN-γ and IL-4

<p><b>Copyright information:</b></p><p>Taken from "Polarized monocyte response to cytokine stimulation"</p><p>Genome Biology 2005;6(2):R15-R15.</p><p>Published online 21 Jan 2005</p><p>PMCID:PMC551535.</p><p>Copyright © 2005 Nagorsen et al.; licensee BioMed Central Ltd.</p> MPs obtained from PBMC from five normal donors were stimulated for 4 h with one cytokine representative of the alternative (IL-4) and one of the classical group (IFN-γ) and gene transcription measured by TaqMan real-time PCR. The relative quantification of 10 genes was calculated by normalizing the ratio of the mean copy number for each gene with the mean copy number of the reference AFAP gene in MPs from five donors. Statistically significant differences (-value < 0.05) between the two cytokine treatments as assessed by Student's -test are represented by an asterisk

Additional file 1: of A coordinate deregulation of microRNAs expressed in mucosa adjacent to tumor predicts relapse after resection in localized colon cancer

Supplementary information including Materials and Methods, Tables S1-S4 and Figure S1. (PDF 157 kb

Additional file 1: of Reconstruction of gene regulatory modules from RNA silencing of IFN-Îą modulators: experimental set-up and inference method

Supplementary Material including text, tables and figures. (PDF 115 kb

Functional characterization of T-bodies.

<p>(A) Lytic activity of T-body-hPSMA/eGFP and T-body-hPSMA/fluc. Cytotoxicity was analysed at 3, 11 and 60 days post-transduction; PC3, PC3-PIP and LNCaP cells were used as target cells. Untransduced PBMC served as negative control. In the upper left corner of each panel the percentages of c-myc⁺ T cells are reported. Figure shows mean +/− SD of 4 independent experiments. (B) IFN-γ secretion upon antigen stimulation. IFN-γ production was analyzed at different time points after PBMC transduction by stimulating T-body-hPSMA/fluc with PC3-PIP hPSMA⁺ or PC3 hPSMA⁻ cancer cell lines. T-bodies unstimulated or treated with PMA/Ionomycin represented the negative and positive controls, respectively. Similar results were obtained with T-body-hPSMA/eGFP. (C) c-myc expression in T-body-hPSMA/fluc populations tested for IFN-γ production.</p