<i>H. pylori</i> infection of different HEK293 cell lines induces IκB phosphorylation at Ser-32 in a TLR2- or TLR-5-dependent fashion.

<p>(A) Western blot analysis of IκB phosphorylation in HEK293, HEK293-TLR2, and HEK293-TLR5 after 2 hours of infection. Blots for house keeping gene GAPDH were used as loading control. (B) Densitometric measurement of band intensities revealed the percentage of IκB phosphorylation per sample. The strongest band was set 100% as indicated.</p

<i>H. pylori</i> infection of THP-1 cells upregulates the secretion of IL-8 and TNF-α in a <i>cag</i>PAI-dependent manner.

<p>Concentrations of IL-8 (A) and TNF-α (B) secreted from THP-1 cells after 24 hours of infection with wild-type <i>H. pylori</i> or an isogenic <i>cag</i>PAI mutant. TNF-α and IL-8 concentrations in the culture supernatants were quantified by ELISA.</p

<i>H. pylori</i> infection of different HEK293 cell lines induces NF-κB and AP-1 activation in a TLR2- or TLR-5-dependent fashion.

<p>NF-κB and AP-1 luciferase reporter constructs were transfected into HEK293, HEK293-TLR2, and HEK293-TLR5 cells for 48 hours and followed by infection with <i>H. pylori</i> for 5 hours. The NF-κB and AP-1 luciferase reporter expression was analyzed as function of activation.</p

<i>H. pylori</i> infection induces enhanced expression of TLR-2 and TLR-5 proteins in HEK293-TLR2 and HEK293-TLR5 cells but not in HEK293 control cells.

<p>(A) Western blot analysis of TLR-2 and TLR-5 proteins in HEK293, HEK293-TLR2 and HEK293-TLR5 cell lines. (B) Fold changes of mRNA expression of TLR-2 and TLR-5 in HEK293, HEK293-TLR2 and HEK293-TLR5 cell lines infected with <i>H. pylori</i> as compared to uninfected mock control cells. The mRNA expression of TLRs was analyzed by Taqman Real Time PCR relative to the house keeping gene GAPDH using the 2−ΔΔ<i>C</i>t method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019614#pone.0019614-Livak1" target="_blank">[60]</a>.</p

<i>H. pylori</i> infection of different HEK293 cell lines induces IRAK-1 phosphorylation at Ser-376 in a TLR-2- or TLR-5-dependent fashion.

<p>(A) Western blot analysis of IRAK1 phosphorylation in HEK293, HEK293-TLR2, and HEK293-TLR5 after 2 hours of infection. Blots for house keeping gene GAPDH were used as loading control. (B) Densitometric measurement of band intensities revealed the percentage of IRAK-1 phosphorylation per sample. The strongest band was set 100% as indicated.</p

Activation of NF-κB-GFP in HEK293 cell lines is dependent on TLR-2 and TLR-5.

<p>Immunofluorescence of HEK293 cell lines transfected with NF-κB-p65 subunit (p65-GFP, green) for 48 hours followed by infection with <i>H. pylori</i> for 3 hours. Infection of HEK293-TLR2 and HEK293-TLR5 cells with <i>H. pylori</i> showed a translocation of p65-GFP into the nucleus (arrows), whereas infection of HEK293 control cells did not induce a nuclear translocation of p65-GFP. Rhodamine-phalloidine (RPH, red) was used to visualize filamentous actin in the cells and DAPI (blue) to visualize the nucleus and bacteria.</p

CagA injection by <i>H. pylori</i> cannot be achieved in infected HEK293 cell lines but in AGS gastric epithelial cells.

<p>Western blot analysis of (A) AGS, (B) HEK293-TLR2, (C) HEK293 and (D) HEK293-TLR5 cells infected with <i>H. pylori</i> wild-type strains P1, P12, P310 or 26695 for 6 hours. Phosphorylation of injected CagA was monitored using phosphotyrosine α-PY-99 and α-CagA antibodies. Red arrows indicate the position of phosphorylated CagA on the blot. Western blots for the house keeping gene GAPDH served as loading control.</p

<i>H. pylori</i> infection of THP-1 cells induces TLR-2 and TLR-5 mRNA expression.

<p>The mRNA expression of TLRs was analyzed by Taqman Real Time PCR relative to the house keeping gene GAPDH using the 2−ΔΔ<i>C</i>t method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019614#pone.0019614-Livak1" target="_blank">[60]</a>. Fold changes of mRNA expression of TLR-2 and TLR-5 in infected THP-1 cells (24 hours) were compared with that of uninfected mock control cells.</p

IL-8 and TNF-α secretion in HEK293 cell lines during <i>H. pylori</i> infection is mediated in a <i>cag</i>PAI-independent fashion.

<p>Concentrations of IL-8 (A) and TNF-α (B) secreted from HEK293, HEK293-TLR2 and HEK293-TLR5 cell lines after 24 hours of infection with wild-type <i>H. pylori</i> and an isogenic Δ<i>cag</i>PAI mutant. TNF-α and IL-8 concentrations in the culture supernatants were analyzed by ELISA.</p

Construction of lentiviral vectors.

<p>A) The HIV-1 provirus NL4.3 and the HIV-1-vector V^Hgenomic are shown. Large parts of the <i>gag, pol</i> and <i>env</i> genes are deleted in V^Hgenomic (see white bars in the deleted regions marked by shaded areas). The remaining <i>gag</i> sequence contains parts of the encapsidation signal (Psi, Ψ) and the <i>env</i> fragments contain splicing regulatory elements as well as the RRE. Due to deletions (shaded squares) and frameshift mutations (black asterisks in <i>gag</i> and <i>rev</i>) no viral genes are expressed from V^Hgenomic. Both vectors are drawn to scale. B) Schematic representation of the lentiviral vectors V^Hgenomic, V^Henv and V^Hnef. The intron between SD1 and SA5 or the introns between SD1 and SA5 and between SD4 and SA7 were deleted from V^Hgenomic in V^Henv or V^Hnef, respectively. Unspliced and spliced transcripts with splice sites (5′ splice sites in green and 3′ splice sites in blue) and <i>cis</i>-acting splicing regulatory elements (in orange) are shown. Please note that the unspliced Msd1-sa5 RNA of V^Henv is identical in sequence to the singly-spliced SD1-SA5 RNA of V^Hgenomic. Furthermore, the unspliced Msd1-sa5+Msd4-sa7 RNA of V^Hnef is identical to the fully-spliced SD1-SA5+SD4-SA7 RNA of V^Hgenomic and the singly-spliced Msd1-sa5+SD4-SA7 RNA of V^Henv. Arrowheads represent RT-PCR primers. C) and D) After cotransfection of lentiviral vectors with <i>tat</i> and <i>rev</i> expression plasmids into HEK293T cells cytoplasmic RNA was isolated and analyzed by RT-PCR with primer pairs depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048688#pone-0048688-g001" target="_blank">figure 1B</a>. Agarose gel electrophoretic analyses of PCR products are shown. The amplification products were sequenced to verify splicing between the indicated splice sites.</p