Figure 3. ARF attenuates response to cisplatin in a human xenograft model of MIBC.

(A) Experimental design. UMUC3 human bladder cancer cells were transduced with lentiviruses expressing exogenous ARF (or the empty vector) or an shRNA for ITGB3BP (or a control shRNA). Cells were implanted orthotopically into the bladder of host mice and tumor growth was monitored using ultrasound imaging. Cisplatin treatment was initiated when tumors reached 5 mm, and mice were treated (8 mg/kg) for 2 weeks. Note that the shRNA studies were done with two independent shRNA; data are shown from one. (B) Real-time PCR analyses showing relative levels of ARF (left) or ITGB3BP (right) in UMUC3 cells expressing the control vector (UMUC-control), a lentivirus expressing ARF (UMUC-ARF), or a lentivirus expressing an shRNA for ITGB3BP (UMUC-shITGB3BP). Expression was normalized to GADPH; p-values were calculated using a t-test. (C) Phenotypic analyses of UMUC3 human bladder tumors expressing the control vector (UMUC-control), ARF (UMUC-ARF), or an shRNA for ITGB3BP (UMUC-shITGB3BP). Shown are representative images of whole mount tumors, ultrasound imaging, H&E staining, or immunostaining with the indicated markers. Insets show high power views of nucleoli. The numbers on the ultrasound images show tumor volume; scale bars represent 50 microns. (D) Summary of tumor weights for the indicated groups. n=9–14/group; p-values were calculated using a Mann Whitney U test. (E) Quantification of cellular proliferation as assessed by the Ki67 staining of tumor cells. n=3/group; p-values were calculated using a Mann Whitney U test.

See also Figure S2 for validation of gene expression, and Tables S2 and S3 for descriptions of antibodies and primers, respectively.