Table 1.
Reverse transcriptase PCR primer pairs, annealing temperature (Tannealing), and predicted PCR product size for cytokeratin 18, cytokeratin 20, uroplakin II, and glyceraldehyde-3-phosphate dehydrogenase (G3PDH).
Figure 1.
The ULTI mouse urothelial cell line is conditionally immortalized under permissive conditions, but restores cell cycle control under non-permissive conditions.
A) Crystal violet proliferation assay, in which absorbances were normalized to that of 10% FBS to reduce interexperimental variability. Open circles represent cells grown under permissive conditions (33°C +IFN-γ), whereas closed circles represent cells grown under non-permissive conditions (37°C -IFN-γ). Error bars represent standard error of the mean. B) Cell cycle analysis of ULTI cells under both permissive and non-permissive conditions, and with 10% and 0.5% FBS concentration, by propidium iodide DNA labeling flow cytometry. C) Quantitation of cell cycle phase of ULTI cells under both permissive and non-permissive conditions, and with 10% and 0.5% FBS concentration. Dark grey bars indicate percent of cells in G0/G1, white bars indicate percent of cells in S phase, and light grey bars indicate percent of cells in G2/M phase. Error bars represent standard error of the mean.
Figure 2.
The ULTI mouse urothelial cell line has an intact DNA damage response under permissive conditions, but aberrant cell cycle checkpoint and apoptosis activation which normalizes under non-permissive conditions.
A) Western blot of whole cell lysates from NIH 3T3 fibroblasts or ULTI cells under both permissive and non-permissive conditions, pretreated with pifithrin-α or DMSO vehicle, then exposed to etoposide or DMSO vehicle. B) Cell cycle analysis by propidium iodide DNA labeling flow cytometry of ULTI cells treated with etoposide (ETOP) or DMSO vehicle under both permissive conditions with 10% FBS, and non-permissive conditions with 0.5% FBS. C) Quantitation of cell cycle phase of ULTI cells treated with etoposide (ETOP) or DMSO vehicle under both permissive conditions with 10% FBS and non-permissive conditions with 0.5% FBS. Dark grey bars indicate percent of cells in G0/G1, white bars indicate percent of cells in S phase, and light grey bars indicate percent of cells in G2/M phase. Error bars represent standard error of the mean.
Figure 3.
The G1/S cell cycle checkpoint activation of the ULTI mouse urothelial cell line under non-permissive conditions is sensitive to p53 inhibition.
A) Cell cycle analysis by propidium iodide DNA labeling flow cytometry of ULTI cells pretreated with pifithrin-α (PFTa) or DMSO vehicle, then treated with etoposide (ETOP) or DMSO vehicle under non-permissive conditions with 0.5% FBS. B) Quantitation of cell cycle phase of ULTI cells pretreated with pifithrin-α (PFTa) or DMSO vehicle, then treated with etoposide (ETOP) or DMSO vehicle under non-permissive conditions with 0.5% FBS. Dark grey bars indicate percent of cells in G0/G1, white bars indicate percent of cells in S phase, and light grey bars indicate percent of cells in G2/M phase. Error bars represent standard error of the mean.
Figure 4.
The ULTI mouse urothelial cell line exhibits an epithelial morphology.
A) Phase contrast micrograph obtained with 20X objective of a monolayer of ULTI cells, showing a polygonal morphology. B) Phase contrast micrograph obtained with 20X objective of ULTI cells demonstrating the organization of these cells into densely packed sheets. C) Phase contrast micrograph obtained with 20X objective of a confluent monolayer of RT4 transitional cell carcinoma cell line, also demonstrating a densely packed arrangement of smaller cells. D) Scanning electron micrograph (500X) of ULTI cells, showing a domed, polygonal morphology with smooth apical surface. Bar at the bottom right of the panel represents 100 µm.
Figure 5.
The ULTI cell line expresses epithelial markers of differentiation.
A) Cytokeratin 18 immunofluorescence of ULTI cells, obtained with 40X objective. Filamentous staining is noted characteristic of the cytokeratins. B) Cytokeratin 18 immunofluorescence of RT4 transitional cell carcinoma cells, obtained with 40X objective. Similar staining to ULTI cells is noted. Withholding primary antibody to assess nonspecific binding of the secondary antibody found no such staining. C) Confocal immunofluorescence of ULTI cells. Circumferential linear staining is noted with occludin (green, left panel) and ZO-1 (red, middle panel), which colocalizes upon merge of the images (yellow, right panel). D) Orthogonal reconstruction of axial images demonstrating colocalization of occludin and ZO-1.
Figure 6.
ULTI cells express cytokeratin 20 and uroplakin II in addition to cytokeratin 18 under hyperosmolal conditions.
A) RT-PCR of cytokeratin 18, cytokeratin 20, and G3PDH using RNA isolated from cell lines. B) RT-PCR of cytokeratin 18, cytokeratin 20, uroplakin II, and G3PDH from RNA isolated from mouse tissues and ULTI cell line. C) RT-PCR of cytokeratin 18, cytokeratin 20, uroplakin II, and G3PDH from RNA isolated from ULTI cell line under basal and hyperosmolal conditions and NIH 3T3 cells. The panel displaying the uroplakin II PCR products is composed of two images from two separate areas of the same gel image.