PAX8 is expressed in RCC and adult normal kidney tissues. PAX8 expression was analyzed in 10 RCC sections and their normal kidney counterparts (cortex and medulla). Staining was consistently observed in the RCC cells, as well as in the cortex and medulla of adult kidney. A representative set is presented here. Abbreviations: PCT, proximal convoluted tubule; DCT, distal convoluted tubule; BC, Bowman's capsule; CD, collecting duct; RCC, renal cell carcinoma; TNL, thin loop of Henle; TKL, thick loop of Henle. Magnification: × 40. The scale bar represents 50 μm.

Depletion of PAX8 induces cell-cycle arrest through inhibition of DNA synthesis. (a) A498 and 786-O cells were transfected with the indicated siRNA. The cells were subjected to BrdU incorporation assays at 48 h post transfection (as described under Materials and methods). The percentage of cells in each cell-cycle phase is shown. (b) Experimental design for measuring S-phase progression using BrdU pulse–chase labeling assay. siRNA-transfected A498 and 786-O cells were pulse-labeled for 15 min with BrdU at 48 h post transfection. The S-phase cell population was monitored 24 h after pulse labeling. (c) FACS profiles for the indicated samples. Percentages of total cells with BrdU incorporated (R1, gray) and cells in S-phase (R2, black) were estimated. The data are the means±s.e.m. of three independent experiments. BrdU, 5-bromodeoxyuridine; siRNA, small interfering RNA.

PAX8 is required for RB protein stability. The effect of PAX8 knockdown on RB expression at (a) the transcript and (b) the protein levels. (a) Total RNA was isolated from the indicated sample at 48 h post-transfection, and subjected to qPCR analysis. Gene expression levels were normalized to the expression of the housekeeping genes, PPIB and YWHAZ, and presented relative to the siControl-transfected samples. The data are means±s.d. of three independent experiments. (b) Whole-cell lysates were extracted from siRNA-transfected A498, 786-O, IGROV-1 and K1 cells at 96 h post transfection. The lysates were subjected to immunoblotting using the indicated antibodies. (c) Effects of proteasome inhibition on RB depletion in response to PAX8 knockdown. K1 cells were transfected with the indicated siRNA. After 24 h, cells were transfected either with a vector control (−) or with pCMV-RB (+). At 48 h post DNA transfection, the cells were incubated in medium with or without MG132 for 12 h. Whole-cell lysates were then extracted and subjected to immunoblotting using the indicated antibodies. qPCR, quantitative PCR; RB, retinoblastoma; siRNA, small interfering RNA.

A model of the effect of PAX8 regulation on RB and E2F1 expression. PAX8 positively regulates both RB and E2F1 expression through different mechanisms. PAX8 binds to the E2F1 promoter, thereby transcriptionally activating E2F1 expression. In comparison, interaction of PAX8 with RB stabilizes the RB protein, which then forms a negative feedback loop, restricting PAX8-mediated E2F1 expression. We propose that this PAX8-mediated E2F1 regulation could be essential to maintain cell proliferation signal before S-phase entry. RB, retinoblastoma.

PAX8 is frequently expressed in cancer cell lines and is required for cancer cell growth. (a) Whole-cell lysates of 19 cancer cell lines were subjected to immunoblotting using the indicated antibodies. (b) Cell viability assessment. The effect of PAX8 knockdown on the viability of A498, 786-O, IGROV-1 and K1 cells was determined using trypan blue exclusion assay. (c, d) K1 cells were transfected with the indicated siRNA. At 120 h post transfection, cells were examined by (c) phase-contrast microscopy and (d) stained for SA-β-gal activity. Total cell number and stained cell number were counted in a blind manner. The data are the means±s.e.m. of two independent experiments. SA-β-gal, senescence-associated β-galactosidase; siRNA, small interfering RNA.

PAX8 directly regulates E2F1 expression through stimulation of E2F1 promoter activity. The effects of PAX8 knockdown on the expression of E2F1 targets, including E2F1 itself, were analyzed at (a) the transcript and (b) the protein level. (a) Total RNA was isolated from the indicated sample at 48 h post transfection and subjected to qPCR analysis. Gene expression levels were normalized to the expression of the housekeeping genes, PPIB and YWHAZ, and presented relative to the siControl-transfected samples. The data are means±s.d. of three independent experiments. (b) Whole-cell lysates were extracted from siRNA-transfected A498, 786-O, IGROV-1 and K1 cells at 96 h post transfection. The lysates were subjected to immunoblotting using the indicated antibodies. (c) Effect of PAX8 knockdown on E2F1 promoter activity was assessed using luciferase assay. At 24 h post siRNA transfection, K1 cells were co-transfected with the indicated luciferase-promoter plasmid and pCMV-β-gal plasmid. Relative luciferase units (RLUs) are normalized to β-gal activity. (d) The promoter sequence of pE2F1-Luc was analyzed using the ConTra web tool. Site-specific primers were designed and used for ChIP–qPCR amplification for each putative PAX8-binding site (Sites-1 to -5). A region without any putative PAX8-binding site (Site-C) was used as the negative control. A498 cells were subjected to ChIP using a PAX8 antibody. IP chromatin was analyzed by ChIP–qPCR using site-specific primers. Fold enrichment is shown relative to Site-C (=1.0). Statistical significance was assessed using one-way analysis of variance; **P<0.01. The data shown in panels c and d are the means±s.e.m. of three and two independent experiments, respectively. ChIP, chromatin immunoprecipitation; IP, immunoprecipitation; qPCR, quantitative PCR; siRNA, small interfering RNA.

PAX8 and RB co-occupy the E2F1 promoter and function antagonistically in regulating E2F1 promoter activity. (a) The effect of RB knockdown on the PAX8 transactivation of E2F1 promoter activity. At 24 h post-siRNA transfection, K1 cells were co-transfected with either vector control or pCMV-Pax8, as well as the indicated luciferase-promoter plasmid and the pCMV-β-gal plasmid. The relative luciferase units (RLUs) are normalized to β-gal activity. (b) A498 whole-cell lysate was subjected to IP using an RB antibody. IP protein complexes were analyzed using immunoblotting (IB) to detect RB and PAX8 proteins. NS indicates IP using a nonspecific antibody. (c) A498 cells were subjected to ChIP using a PAX8 antibody. IP chromatin was analyzed by ChIP–qPCR, using primers targeting the putative PAX8-binding site (Site-2). In addition, Site-1, which contains the characterized RB-E2F1-binding site, was used as a positive control. The negative control Site-C was used for comparison. Fold enrichment is shown relative to the negative control site (Site-C). Statistical significance was assessed using unpaired Student's t-test; *P<0.05. Data for a and c are the means±s.e.m. of three independent experiments for each panel. ChIP, chromatin immunoprecipitation; IP, immunoprecipitation; qPCR, quantitative PCR; RB, retinoblastoma; siRNA, small interfering RNA.