Primers for semi-quantitative Real Time-PCR.

<p>Primers for semi-quantitative Real Time-PCR.</p

Electron microscope observations revealed details of internal organelles of undifferentiated and differentiated cells.

<p>Electron micrographs showing (A) undifferentiated ESC and (B) ESDC. Note the presence of intracellular <i>S.</i> Typhimurium in these images. Scale bars denote different sizes.</p

Gentamicin invasion assay.

<p>The ability of <i>S.</i> Typhimurium SL1344(p1C/1) to invade ESDCs was measured by the gentamicin assay described in Methods. ESDCs were exposed to either wild type <i>S</i>. Typhimurium or a <i>SipB</i> mutant for 30 minutes and then incubated with media containing 50 µg/ml of Gentamicin for 2 or 4 hours and then invading bacteria were enumerated.</p

Concentration of IL-2 in the culture supernatant of naïve MF2.2d9 T cells mixed with either BMDCs or ESDCs incubated for 24 with ovalbumin, TNFα or alone with Concavallin A (ConA).

<p>These data are representative of at least 2 independent experiments. Statistical analysis performed with one-way ANOVA.</p><p>Ovalbumin 10 µg/ml, TNFα 5000 WHOSU/ml, Concavallin A 5 µM/ml.</p

Graphic representation of the quantified expression of chosen genes versus un-infected ESDCs.

<p>Total RNA extracted from infected ESDCs was used in semi-quantitative Real Time-PCR to confirm the BioConductor analysis of up- or down-regulated genes. The data were analyzed using the ΔΔct value calculated versus the un-infected ESDCs.</p

Concentration of cytokine produced by AB2.2 mouse ESCs or ESDCs, alone and during infection with <i>S.</i> Typhimurium SL1344(p1C/1).

<p>Each value represents the average of at least two measurements ± average deviation. The detection limits of the assay were 2.5 pg/ml for IFNγ, 17.5 pg/ml for IL-10, 10.7 pg/ml for IL-12p70, 5 pg/ml for IL-6, 52.5 pg/ml for MCP-1 and 7.3 pg/ml for TNFα values below these are listed as 0 in the table).The data was analyzed using one-way ANOVA,</p>*<p>indicates a significant difference with p<0.05.</p

Innate DB pathways up-regulated by ESC derived DCs at 4 hours post-infection.

*<p>Interaction Node;</p>1<p>S-Phase Network;</p>2<p>p53-Independent DNA Damage Response Network;</p>3<p>Host interactions of HIV factors Network;</p>4<p>Signaling by Wnt Network;</p>5<p>DNA Replication.</p

Morphology of ESDC and MHC-II expression was observed by confocal microscopy.

<p>Stimulated ESDCs were imaged using a Confocal Zeiss LSM510; (A), phase contrast, (B) cell stimulated with TNFα and stained with anti-MHC Class II FITC conjugated antibody. Scale bar 10 µm.</p

The invasion assay was also evaluated by Flow Cytometry.

<p>The ability of <i>S.</i> Typhimurium SL1344(p1C/1), able to express GFP once inside the host cell, to invade ESDCs was measured by monitoring the GFP expression using FACS analysis. Cells were analyzed for GFP expressing bacteria at time 2 and 4 hours post-infection. The horizontal bar in each histogram represents the percentage of fluorescent positive cells on 10.000 events. FITC subset in non infected cells is <1%. These data are representative of at least 3 independent experiments.</p

The nature of un-differentiated or differentiated cells was tested by flow cytometry.

<p>Flow cytometry on ESCs and ESDCs was used to monitor the expression of different cell surface markers. (A) Stem cell pluripotency markers on undifferentiated ESCs and (B) Dendritic cell markers on undifferentiated ESCs confirm their pluripotency and self-renewal nature; (C) Dendritic cell markers expressed on ESDCs confirm the change in APC. Green lines represent cells stained with control isotype, red lines are cells stained with the relevant antibody.</p