Uptake of <i>S.</i> Enteritidis wild type and mutant strains into the transfected U937 cells.

<p>Wild type, <i>ssaS</i>::Km or <i>sscA</i>::Km mutant were used to infect HLA-B27-transfected cells and HLA-A2-transfected cells. The numbers of the bacteria were counted at 1 hour post-infection. The data represented the mean and standard deviation of at least three independent experiments with duplicate samples. No difference was found in the uptake of mutants and wild type bacteria in HLA-B27 cells, and no difference was found in the uptake of mutants and wild type bacteria between HLA-B27 and HLA-A2 cells.</p

Intracellular growth of wild type and mutant strains in U937 cells.

<p>The growth of wild type, the <i>ssaS</i>::Km and <i>sscA</i>::Km mutants were compared in HLA-B27-transfected (A) or HLA-A2-transfected U937 cells (B). The comparison for the mutant <i>ssaS</i>::Km growth in HLA-B27 and HLA-A2 cells (C) and for the mutant <i>sscA</i>::Km growth in HLA-B27 and HLA-A2 cells (D) was shown. Values represented the mean and standard deviation of at least three independent experiments with duplicate samples. *, ** and *** indicate <i>P</i><0.05, <0.01 and <0.001, respectively, when the mutant-infected cells compared to the wild type (WT)-infected cells (A and B) or the mutant-infected HLA-B27 cells to HLA-A2 cells (C and D). Data were compared using Student's paired 2-tailed <i>t</i>-test.</p

Primers used in the gene mutagenesis in this study.

α<p>: 80-nucleotide (nt)-long primers including 60 nt homology extensions complementary to the targeted regions of the genes and 20 nt priming sequences (underlined) for the synthesis of the kanamycin resistance cassette gene from <i>E. coli</i> MC4100 <i>ybeW</i>::Km.</p

The corresponding DNA PCR product in mutated and wild type genes.

<p>The PCR product from three mutated isolates <i>ssaS</i>::Km (lanes 1–3) and the wild type gene <i>ssaS</i> (lanes 4 and 5 (A), and one mutated isolate <i>sscA</i>::Km (lane 3) and wild type gene <i>sscA</i> (lanes 4 and 5) (B) are demonstrated. The genomic DNAs isolated from the mutant and wild type bacteria were used as templates, respectively, and the flanking primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034093#pone-0034093-t001" target="_blank">Table 1</a>) used in the PCR reaction.</p

PKR inhibition effects on HuR expression and regulation in U937 monocytic cells.

<p>Full length HuR (36 kDa) and HuR cleavage products (CP1; 24 kDa and CP2; 8 kDa) in LPS-stimulated (A) and <i>Salmonella enteritidis</i>-infected (B) PKR inhibitor (PKR+)- or negative control for PKR inhibition (PKR-)-treated U937 Mock, B27g, H9F and E45M cells at 5 h time point. Expression of HuR was determined by SDS-PAGE and Western blotting. HSPA8 was used as a loading control. Results from densitometric analysis of five independent experiments are shown in diagram. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070377#s2" target="_blank">Materials and Methods</a> for details. * = P-value ≤0.06 for HuR, ^‡ = P-value ≤0.06 for CP1 and ^† = P-value ≤0.06 for CP2 versus untreated sample at the respective cell line.</p

p38 inhibition effects on HuR expression and regulation in U937 monocytic cells.

<p>Full length HuR (36 kDa) and HuR cleavage products (CP1; 24 kDa and CP2; 8 kDa) in LPS-stimulated (A) and <i>Salmonella enteritidis</i>-infected (B) p38 inhibitor- or negative control for p38 inhibition-treated U937 Mock, B27g, H9F and E45M cells at 5 h time point (SB90; p38 inhibitor SB202190 and SB74; control for p38 inhibition SB202474) Expression of HuR was determined by SDS-PAGE and Western blotting. HSPA8 was used as a loading control. Results from densitometric analysis of five independent experiments are shown in diagram. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070377#s2" target="_blank">Materials and Methods</a> for details. * = P-value ≤0.06 for HuR and ^† = P-value ≤0.06 for CP2 versus untreated sample at the respective cell line.</p

Secretion of TNFα and IL-10 in U937 monocytic cells is controlled by PKR and p38 MAPK.

<p>Cytokine secretion in Mock, B27g and H9F transfected U937 cells was determined 6 h and 24 h after LPS stimulation and treatment with PKR (PKR+) or p38 (SB90; p38 inhibitor SB202190) inhibitor. Controls for PKR and p38 inhibition were used (PKR- and SB74; control for p38 inhibition SB202474). TNFα secretion was measured after PKR inhibition in A and after p38 inhibition in B. IL-10 secretion was measured after PKR inhibition in C and after p38 inhibition in D. Values are the mean and SD of three or four independent experiments. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070377#s2" target="_blank">Materials and Methods</a> for details.</p

Phosphorylation of STAT-1 serine 727 is only partially dependent on PKR activity.

<p>U937 transfectants were PMA-maturated, infected with <i>S. enteritidis</i>, and treated with a PKR inhibitor (PKR+) or an inhibitor control (PKR−). Cells were harvested 2 hours (A and B) or 5 hours (C and D) after excess bacteria were washed away. The Phosphorylation of STAT-1 serine 727 was studied by Western blot method with p-STAT-1 Ser727 antibody. Cell extracts (35 µg of each) were loaded on SDS-page gel. <b>A</b>, The representative figure at 2 hour time point of U937 cells transfected with genomic clone B27g, vector (mock), misfolding B27H9F or with non-misfolding B27E45 mutants. “C” denotes control <i>S. enteritidis</i>-infected cells without PKR-treatment. <b>B</b>, The relative intensity of STAT-1 serine 727 phosphorylation after PKR inhibition at 2 hour time point. The bars show the mean ± SD of 3 independent experiments. <b>C</b>, The representative figure at 5 hour time point of U937 cells transfected with genomic clone B27g, vector (mock), B27H9F or with B27E45 mutants. <b>D</b>, The relative intensity of STAT-1 serine 727 phosphorylation after PKR inhibition at 5 hour time point. The bars show the mean ± SD of 4 independent experiments. The blots were stripped and reprobed with STAT-1 antibody and Hsc70 antibody as a loading control. The relative Intensity values were normalized to the <i>S. enteritidis</i>-infected control (C, which is given value one). * = <i>P</i><0.05 versus 2 h/5 h of the respective cell line.</p

Prolonged phosphorylation of STAT-1 serine 727 in cells expressing misfolding forms of HLA-B27 molecule.

<p>U937 transfectants were PMA-maturated, infected with <i>Salmonella enteritidis</i>, and harvested at the indicated time points after excess bacteria were washed away. The phosphorylation of STAT-1 serine 727 was studied by Western blot method with p-STAT-1 Ser727 antibody. Cell extracts (35 µg of each) were loaded on SDS-page gel. <b>A</b>, The representative figure of U937 cells transfected with genomic clones of HLA-B27 (B27g) or with vector alone (mock). “C” denotes uninfected cells. <b>B</b>, The representative figure of U937 cells transfected with mutated, misfolding form of HLA-B27 (B27H9F) or with mutated, non-misfolding form of HLA-B27 (B27E45M). <b>C</b>, The relative intensity of STAT-1 serine 727 phosphorylation in B27g-expressing cells and in mock cells. The bars show the mean ± SD of 6 independent experiments. <b>D</b>, The relative intensity of STAT-1 serine727 phosphorylation in B27H9F- and B27E45M-transfected cells. The bars show the mean ± SD of 5 independent experiments. Blots were stripped and reprobed with STAT-1 antibody and Hsc70 antibody as a loading control. The relative intensity of p-STAT1 Ser 727 was calculated as the intensity of the phosphorylated band divided by the intensity of the respective loading control and normalized to the uninfected control (C, which is given value one). * = <i>P</i><0.05 and † = <i>P</i><0.07 versus 15 min time point of the respective cell line.</p

HuR expression was not affected by Src, Raf1 and CaseinI inhibition in U937 monocytic cells.

<p>Full length HuR (36 kDa) and HuR cleavage products (CP1; 24 kDa and CP2; 8 kDa) in LPS-stimulated U937 Mock, B27g, H9F and E45M cells at 5 h time point. In A, p38 inhibitor BIRB796 (Birb) was incubated with the cells 30, 60 or 90 min prior to LPS stimulation. In B, Src, Casein I (Cas) and Raf1 kinase inhibitors were incubated with the cells 15 min prior to LPS stimulation. Expression of HuR was determined by SDS-PAGE and Western blotting. HSPA8 was used as a loading control. Results from densitometric analysis of five independent experiments are shown in diagram. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070377#s2" target="_blank">Materials and Methods</a> for details. * = P-value ≤0.06 for HuR, ^‡ = P-value ≤0.06 for CP1 and ^† = P-value ≤0.06 for CP2 versus untreated sample at the respective cell line.</p