Uropathogenic <i>E. coli</i> strain NU14 adheres to HUCs.

<p>HUCs were exposed to NU14 (A), the isogenic <i>fimH</i> mutant NU14-1 (B) or media alone (C). After 2 h, the HUCs were washed and stained for the presence of adherent bacteria (green). Host cell actin (red) and nuclei (blue) were counterstained. Merged images show NU14 (A, arrows) adhering to urothelial cells. The percentage of urothelial cells with adherent bacteria was quantified (D) by scoring at least 200 urothelial cells from random fields of view for the presence of adherent bacteria. The percentage represents the number of urothelial cells with adherent bacteria divided by the total number of urothelial cells examined. Data are representative of at least three (A–C) or two (D) independent experiments performed with cells derived from independent biopsies. Between experiments, the average percentage of urothelial cells with adherent NU14 ranged from 8 to 23%. The errors bars represent the standard deviation of two coverslips per strain per experiment. Scale bars, 10 µm. *Indicates p<0.05, Student’s t-test.</p

Primer Sequences.

<p>Primer Sequences.</p

Antibodies Used in the Study.

#<p>A volume of 5 µL of the indicated dilution was used.</p><p>Antibodies Used in the Study.</p

Flow cytometric analysis of primary HUCs.

<p>Cells were stained with eight fluorochrome-conjugated monoclonal antibodies direct against cell surface makers. Cells were analyzed for the expression of: (A) HLA-ABC and CD54 (ICAM-1); (B) CD104 and EpCAM; (C) HLA-DR, and CD44– expression of CD44 shows two distinct populations of HUCs: CD44^lo and CD44^hi; (D, E) TLR and CD14. Representative data from 3 experiments performed with HUCs generated from 3 independent bladder biopsies.</p

Expression of cytokines, cytokine receptors, chemokines in primary HUCs (^#Number of copies/µg total RNA).

<p>Data were from three independent cultures of HUCs established from three different human bladder biopsies. Transcript levels were determined by qRT-PCR.</p>#<p>Values represent the average of triplicates; numbers in parentheses are standard errors of the mean.</p><p>Expression of cytokines, cytokine receptors, chemokines in primary HUCs (^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111375#nt102" target="_blank">#</a>}Number of copies/µg total RNA).</p

Ultrastructures of <i>ex vivo</i> human bladder urothelium.

<p>Bladder tissue biopsy was immediately processed for TEM study. (A) Surface umbrella cells with numerous DFVs. Arrowheads point to tight junctions (TJ) between two adjacent umbrella cells; dotted line demarcates junction between the upper umbrella cells and the underlying urothelial cells. (B) A higher magnification photograph of umbrella cells showing DVFs and uroplakins plaques (Uro P), which appear as darker lines associated with the cell membrane. (C, E) Higher magnification of TJ structures shown in A. (E, F) enlargement of areas shown in C and E (square boxes) showing DFVs, Uro P, TJ and desmosomes (Des). Bar scale = 1 µm.</p

Effect of IL22 on the expression of MR1, S100A9, lipocalin-2, and CarAT by primary HUCs.

<p>Transcript levels were measured by qRT-PCR and expressed as fold change of treated versus untreated HUC cultures. Values represent the average of two independent experiments performed with two different HUC cultures. Errors bars are standard deviations.</p

Ultrastructures of primary HUCs.

<p>Cultured HUCs were analyzed by TEM. (A, B) a HUC with two nuclei (N) and many discoid fusiform vesicles (DVF) - *denotes lumens of DFV; B is a higher magnification of the square area identified in A - MVB denotes multivesicular body; (C) another example of MVB and DFV in a HUC; (D) presence of uroplakins plaques (Uro P, dark line) on the cell membrane of an umbrella cell; (E) formation of lateral interdigitation between two adjacent HUCs, a higher magnification of the identified was shown in F; (F) presence of numerous desmosomes (Des, arrowheads) within a well-formed lateral interdigitation of two adjacent HUCs. Bar scale is equal to 1 µm (in A, B, E and F) and 0.1 µm (in C and D).</p

Immunofluorescent staining of cultured HUCs.

<p>HUCs were fixed and double stained with anti-uroplakins (PE, Red) and keratin-20 (FITC, green). DAPI (blue) was used to stain the nucleus. Lower right panel is a negative control stained with mouse and rabbit IgG. Representative images from 3 experiments from 3 independent biopsies.</p

Cellular morphology of human urothelial cells (HUCs) generated from bladder tissue explant.

<p>(A, B) Cells growing out from the edge of two tissue explants obtained from two different biopsies; cells with typical coble-stone morphology were clearly identifiable. Arrow points to a larger cell (60–150 µm) with four nuclei and prominent nucleolus. (C, D) cells with uniform size and morphology (25–40 µm) surround a cluster of larger cells (70–100 µm); several are bi-nucleated (arrows). (E, F) clusters of larger cells (80–150 µm), many of these cells are bi-nucleated (arrows). The dotted lines demarcate the edge of the tissue explants. Scale bar = 50 µm.</p