Supplementary Material for: Alterations of UHRF family expression and UHRF1/ICBP90 inhibits PTEN expression in endometrial cancer

Introduction: The altered protein expression of inverted CCAAT box-binding protein of 90 kDa/ubiquitin-like with PHD and RING finger domains 1 (ICBP90/UHRF1) and Np95-like ring finger protein (NIRF)/UHRF2, which belong to the ubiquitin-like with PHD and RING finger domains (UHRF) family, is linked to tumor malignancy and the progression of various cancers. To determine the role of NIRF and ICBP90 in endometrial tumorigenesis, we evaluated ICBP90 and NIRF expression levels in endometrial cancers. Also molecular alterations of PTEN expression is the important event for endometrial carcinogenesis, therefore we investigated the involvement between ICBP90 and PTEN expression. Methods: We used Western blot for NIRF, ICBP90, and PTEN expression, mutation analysis of NIRF gene, and immunohistochemical staining for the expression of NIRF and ICBP90. For immunohistochemical staining, we examined atypical endometrial hyperplasia, endometrial cancers, and non-cancerous samples. Results: Our data showed that the reduced expression of NIRF and overexpression of ICBP90 occurred in atypical endometrial hyperplasia and endometrial cancer compared to the normal endometrium. The decrease in NIRF expression was significantly correlated with histological grade. Expression of ICBP90 was high, especially in the peripheral margin of a cancer nest. Western blot analysis of endometrial cancer cell lines referred an opposite correlation between ICBP90 and phosphatase and tensin homolog (PTEN) expression. Conclusion: Our findings suggested that continually overexpressed ICBP90 may contribute to the inhibition of PTEN expression, which is a frequent and important event in endometrial carcinogenesis. We propose that the reduced NIRF expression and ICBP90 overexpression is an early event in endometrial carcinogenesis; thus ICBP90 may be useful as a therapeutic target in this disease.