Primers for RT-PCR and qRT-PCR.

<p>Primers for RT-PCR and qRT-PCR.</p

Homology of X. laevis TIMP2 with the ortholog of other species.

<p>GenBank accession number for nucleotide sequences: HuTIMP2: NM_003255; mTIMP2: X62622; ChTIMP2: NM_204298. The homology among the nucleotide sequences (coding sequences) was on grey background.</p

TIMP2 expression during <i>X. laevis</i> development.

<p>Total RNA was isolated from whole animals during <i>X. laevis</i> development and subjected to One-Step RT-PCR analysis with a pair of primers amplifying both TIMP2A and TIMP2B (TIMP2) or stromelysin-3 (ST3). The expression of the house-keeping gene histone H4 was similarly determined as a control.</p

Comparison of the genomic locus and organization of <i>X. tropicalis</i> and human TIMP2.

<p><i>X. tropicalis,</i> chicken, mouse and human TIMP2 genomic organizations were obtained from NCBI database. The lines represent the genomic sequences and the boxes with arrowed lines on top represent transcribed regions and the direction of the transcription. The lines and boxes were not drawn to the same scale for better visualization. The TIMP2 gene in all four species has the same intron/exon organization with 5 exons total as shown for <i>X. tropicalis</i> TIMP2 (not shown). Note that mouse and human TIMP2 loci are identical. In the chicken, CNTNAP1 gene is present on a different chromosome (shown in a darker shade).</p

There are two <i>X. laevis</i> TIMP2 genes that are highly homologous to the TIMP2 gene in other vertebrates.

<p>A) Nucleotide sequence comparison of <i>X. laevis</i> TIMP2A and TIMP2B. Dot (.): identical sequence; dash (-):a gap introduced for better alignment. Arrowed lines indicate the locations of the primers used to distinguish the two isoforms in RT-PCR analysis. B) Comparison of the protein sequences of putative <i>X. laevis</i> TIMP2A (XlTIMP2A, AAH74452), <i>X. laevis</i> TIMP2B (XlTIMP2B, NP_001087748), and <i>X. tropicalis</i> TIMP2 (XtTIMP2, NP_001015760) with their ortholog from chick (ChTIMP2, NP_989629), mouse (mTIMP2, P25785) and human (HuTIMP2, NP_003246). The conserved cysteine sequences are in bold and the signal peptide sequence is underlined. Dot (.): identical sequence; dash (-): a gap introduced for better alignment.</p

T3-inducktion of TIMP2 expression in different tissues of premetamorphic tadpoles.

<p>Tadpoles at premetamorphic stage 54 were treated with 10 nM T3 at 18°C and collected every other day for isolating total RNA from different tissues. The RNA was analyzed by qRT-PCR analysis as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036707#pone-0036707-g005" target="_blank">Fig. 5</a>. *: p<0.05 when compared to the group without T3 treatment.</p

Wild type <i>Shigella</i>, but not vaccine strains, moderately affect Caco2 monolayer viability.

<p><b>A</b>. LDH release after infection with wild-type <i>S. flexneri</i> 2a or its vaccine strain CVD 1208S at different inocula. <b>B</b>. LDH release after infection with wild-type <i>S. dysenteriae</i> 1 or its vaccine strain CVD 1256 applied at different inocula. Caco2 cells were infected with bacteria for 6 hours, washed and incubated overnight at 37°C. Apical supernatants were collected at 24 hours post-infection for LDH release measurement. Values represent LDH release from cells into the medium as a percentage of total LDH (Control). Data are expressed as means ± SEM for triplicate samples for all conditions tested. These results are representative of 3 experiments with similar results. *<i>p</i><0.05 compared to uninfected cells (ANOVA).</p

Co-regulation of TIMP2A and TIMP2B expression during intestinal metamorphosis.

<p>A) Validation of primer specificity for <i>X. laevis</i> TIMP2A or TIMP2B. The TIMP2A and TIMP2B cDNA were cloned into pCR2.1 vector, sequenced and serially diluted to serve as template DNA for PCR. Note that each primer pair specifically amplified only the intended TIMP2 gene. B) Total RNA was isolated from intestines of tadpoles during <i>X. laevis</i> metamorphosis and subjected to One-Step RT-PCR with a pair of primers recognizing both TIMP2 genes (TIMP2), specific for either TIMP2A or TIMP2B, respectively. The expression of the house-keeping gene rpL8 was similarly determined as a control.</p

Occludin is hyperphosphorylated after infection with <i>Shigella</i> strains and disassembles from tight-junctions.

<p><b>A</b>, <b>B</b>. Western blot of protein samples in the form of X-100 Triton soluble (Sol) and insoluble (Ins) fractions at 6 hours post-infection, blotted with anti-occludin (upper rows), anti-phosphothreonine (middle rows) and anti-actin (lower rows). <b>C</b>, <b>D</b>. Western blot of protein samples at 24 hours post-infection, blotted with anti-occludin (upper rows), anti-phosphothreonine (middle rows) and anti-actin (lower rows).</p

Wild type <i>Shigella</i> induce TEER decrease in Caco2 cell monolayers independent of MOIs.

<p><b>A</b>. TEER responses to wild-type <i>S. flexneri</i> 2a applied at three different inocula. <b>B</b>. TEER responses to <i>S. flexneri</i> 2a heat-killed bacteria and culture supernatants (Cond. Media). <b>C</b>. TEER responses to wild-type <i>S. dysenteriae</i> 1 applied at three different inocula. <b>D</b>. TEER responses to <i>S. dysenteriae</i> 1 heat-killed bacteria and culture supernatants. Data are expressed as means ± SEM for triplicate samples for all conditions tested. * and # indicate statistically significant differences vs. t = 0 and vs. the uninfected control at the same time point, respectively; <i>p</i><0.05 (ANOVA). These results are representative of 3 experiments with similar results.</p