Article Figures & Data
Figures
- Figure 1.
PAX2 actions in M0505 mOSE cells. A, Western blot analysis confirming that M0505 transduced with a PAX2 expression vector (PAX2) shows increased PAX2 relative to controls: WPI vector or cre-mediated PAX2 deletion (PAX2 + Cre). B, PAX2 increases M0505 proliferation. C and D, PAX2 increases viable cell number and % viability after 48 hours in growth factor- and serum-free media (starvation). E, PAX2 enhances M0505 cisplatin resistance. For all experiments, n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
- Figure 2.
PAX2 inhibits p53 induction in mOSE cells. Western blot analysis (A) and densitometric analysis (B) of p53 in M0505 and M1102 cells after cisplatin treatment (12.5 μg/mL; 48 hours), with UV-exposed mouse embryonic fibroblasts as a positive control. C, Trp53 mRNA levels after PAX2 expression. For all experiments, n = 3; *, P < 0.05; **, P < 0.01.
- Figure 3.
IPA showing biologic functions associated with gene expression changes after PAX2 expression. Relationships between genes are color-coded by predicted interaction (see legend).
- Figure 4.
PAX2 actions in RM and STOSE mouse ovarian cancer cells. A, Western blot analysis confirming PAX2 induction in both cell lines. B, proliferation of STOSE and RM cells with PAX2 induction (PAX2) versus controls (parental, WPI, PAX2-Cre). C, viability (left, alamar blue) and cleaved caspase-3 (right, Western blotting) were assessed after 48-hour cisplatin treatment (n = 3; **, P < 0.01; ****, P < 0.0001). D, PAX2 expression decreased colony formation ability of STOSE but not RM cells (n = 3; ****, P < 0.0001). E and F, Kaplan–Meier survival curves for mice injected with (E) RM + WPI or RM + PAX2 cells or (F) STOSE + WPI, STOSE + PAX2, or STOSE + PAX2 + Cre cells (****, P < 0.0001). G, PAX2 expression decreased STOSE tumor weight at endpoint (**, P < 0.01; *, P < 0.05).
- Figure 5.
PAX2 inhibited p53 accumulation in RM but not in STOSE tumors. A, PAX2 expression abolished p53 and cleaved caspase-3 (Western blotting) in all RM + PAX2 tumors examined. Positive controls for p53 and cleaved caspase-3 were cisplatin-treated normal and p53-deficient mOSE, respectively. B, Trp53 mRNA levels in RM and STOSE tumors. C, STOSE tumors did not show p53 accumulation (Western blotting). D, tumor proliferation was examined by immunohistochemistry for Ki67 (quantified in histograms). E, PAX2 expression reduced Htra1 transcripts in mOSE cells and RM tumors, but not STOSE tumors. F, PAX2 expression reduced PTEN levels in RM tumors (Western blotting). G, immunoblot detection of total and phospho-AKT following AKT immunoprecipitation in RM tumors. H, Western blot analysis shows PAX2-associated phosphorylation of ERK1/2 at Thr202 and Tyr204 in RM but not STOSE tumors; SCC25 head and neck carcinoma cells were used as a positive control for EGFR. EGF-treated SCC25 cells were used as positive and (with an EGF inhibitor) negative controls for pERK1/2.
- Figure 6.
Effect of PAX2 on COX2 and angiogenesis. A, COX2 in ovarian tumors after PAX2 induction (Western blotting). B, MVD was examined using immunohistochemistry for CD31. Bottom, CD31-positive pixel selection, quantified in the histograms on the right (*, P < 0.05). Scale bar, 100 μm. C, a proposed model for PAX2 actions in normal and cancerous ovarian cells. Dotted lines indicate predicted functional effects.
Additional Files
Supplementary Data
- Supplementary data - Supplementary Table 1: Primer pairs (forward [F] and reverse [R]) that were used to amplify murine genes. Supplementary Table 2: The primary antibodies that were used for western blot analysis in the current studies. Supplementary Table 3: The top 10 genes upregulated and downregulated by PAX2 in MO505 cells, as shown by microarray analysis, and their known relationships to ovarian tumorigenesis. Supplementary Table 4: Functional analysis of genes that were changed in M0505+PAX2 cells compared to M0505+WPI and M0505+PAX2+Cre and are involved in tumor growth and progression.
- Supplementary Figure 1 - IHC analysis shows that PAX2 is expressed in the epithelium of the murine oviduct and uterus, but not in the ovary.
- Supplementary Figure 2 - PAX2 enhances proliferation and survival in normal mOSE cells.
- Supplementary Figure 3 - Expression of PAX2 in p53-null cells enhanced proliferation but not survival.
- Supplementary Figure 4 - Western blot analysis showing there is no T-antigen expression in RM tumors.
Volume 8, Issue 12
