Fig 2: M1102-PAX2 cells form endothelial cell-independent vascular-like channels. (A) PAS staining for vascular-like channels formed by M1102-PAX2 cells showed pink positive staining compared with control cells not overexpressing PAX2. Magnification, 100X (B) Immunofluorescence staining for CD31 in vascular-like channels formed by HUVECs, M1102-PAX2 and SKOV3 ovarian cancer cell lines. M1102-PAX2 vascular-like channels were negative for CD31. HUVECs were used as a positive control for angiogenesis and SKOV3 cells as a positive control for VM. Few cells expressing CD31 were identified in vascular-like channels formed by SKOV3 cells. Magnification, 200X. PAS, periodic acid-Schiff; PAX2, paired box 2.

Fig 3: PAX2 immunohistochemical staining of human serous ovarian cancer. Human ovarian tumor sections showed tubule-like structures lined with cells that expressed PAX2 and basement membrane stained positive for PAS. Few CD31-positive vessels were observed. Scale bar, 100 µm (original) and 20 µm (magnified). PAS, periodic acid-Schiff; PAX2, paired box 2.

Fig 4: PAS and CD31 immunohistochemical staining of tumors formed in mice by RM-WPI and RM-PAX2 cell xenografts. (A) Tumors derived from RM-WPI (transfected with empty viral vector) and RM-PAX2 cells showed vascular-like channels positive for PAS staining (upper panels). CD31 staining in vascular channels in a RM-PAX2 tumor indicated that PAX2 enhanced angiogenesis [green arrows indicate the existence of regular vessels (angiogenesis); middle panels]. Double staining for CD31 and PAS (lower panels). PAS+/CD31+ vascular-channels were present in both RM-WPI and RM-PAX2 tumors (green arrows). CD31-/PAS+ lumen may have been indicative of vasculogenic mimicry in RM-PAX2 tumors (black arrow). Scale bar, 100 µm. (B) Quantification of CD31 and PAS immunohistochemical staining. Analysis was performed by measuring the percentage of positive pixels in three different areas from RM-WPI and RM-PAX2 cancers. Image analysis was performed using Aperio Positive Pixel Count Algorithm software. Statistical analysis was performed using unpaired Student's t-test. *P<0.05 vs. RM-WPI. PAS, periodic acid-Schiff; PAX2, paired box 2.

Fig 5: PAX2 influences ADAM10 expression. a Total cell lysates of NRK52E transfected with pGC–LV–PAX2, assessed by western blot. Protein levels were normalized to ß-tubulin. Data represent mean ± SD (n = 3). b ADAM10 expression (red) analyzed by immunofluorescence in NRK52E transfected with pGC–LV–PAX2 or empty vectors. The nuclei of NRK52E cells are stained with 4',6-diamidino-2-phenylindole (DAPI). Scale bars represent 50 µm. c Messenger RNA isolated from NRK52E transfected with pGC–LV–PAX2 or empty vectors. Data represent mean ± SD (n = 3). *P < 0.05 compared to vector control