Rabbit Anti-Human MCM6 (internal) Polyclonal antibody

Aim: The ATR-interacting protein (ATRIP) is responsible for the recognition of DNA damage-induced structure and regulation of cellular responses to DNA damage and replication stress. The purpose of our study was to identify the underlying mechanism with respect to chromatin loading and phosphorylation of ATRIP in mantle cell lymphoma (MCL). Methods: JeKo cells were used in our study. Differently tagged ATRIP (Myc-, hemaglutinin (HA) or Flag) and minichromosome maintenance (MCM) complex (MCM2, MCM3, MCM5, and MCM6) were transfected into 293T cells. After 48 h, ATRIP-interacting protein was identified by mass spectrometry (MS). Cell fractionation was done to localize proteins inside the cells. Immunoprecipitation (IP) and immunoblot (IB) analysis were used to identify immunoreactive species, and Glutathione S-transferase (GST) pull-down assays were performed to detect protein-protein interaction between ATRIP and MCM complex. After silencing the expression of MCM2 and MCM6 by short hairpin RNA (shRNA), chromatin fraction were analyzed. The expression of ATRIP phosphorylation (pS224-ATRIP) was determined after application of different doses of MCM2 shRNA (0.5 mu g, 1 mu g, and 2.5 mu g). Results: ATRIP directly interacts with MCM2, MCM3, MCM6, and MCM7 in JeKo cells. Downregulation of MCM2 and MCM6 significantly reduced ATRIP chromatin fraction. Downregulation of MCM2 statistically decreased the expression of ATRIP phosphorylation. The expression levels of pS224-ATRIP were regulated by MCM2 shRNA in a dose-dependent manner. Conclusion: Our results suggest that interaction between ATRIP and MCM complex is required for ATRIP chromatin loading and ATRIP phosphorylation.

Background: Cancer stem cells (CSCs), which have been isolated from various malignancies, were closely correlated with the occurrence, progression, metastasis and recurrence of the malignant cancer. Little is known about the tumor stem-like cells (TSLCs) isolated from benign tumors. Here we want to explore the global expression profile of RNA of tumor stem-like cells isolated from MMQ rat prolactinoma cells. Methods: In this study, total RNA was extracted from MMQ cells and MMQ tumor stem-like cells. RNA expression profiles were determined by Agilent Rat 8 x 60 K Microarray. Then we used the qRT-PCR to test the result of Microarray, and found VEGFA had a distinct pattern of expression in MMQ tumor stem-like cells. Then WB and ELISA were used to confirm the VEGFA protein level of tumor sphere cultured from both MMQ cell and human prolactinoma cell. Finally, CCK-8 was used to evaluate the reaction of MMQ tumor stem-like cells to small interfering RNAs intervention and bevacizumab treatment. Result: The results of Microarray showed that 566 known RNA were over-expressed and 532 known RNA were lowexpressed in the MMQ tumor stem-like cells. These genes were mainly involved in 15 different signaling pathways. In pathway in cancer and cell cycle, Bcl2, VEGFA, PTEN, Jun, Fos, APC2 were up-regulated and Ccna2, Cdc25a, Mcm3, Mcm6, Ccnb2, Mcm5, Cdk1, Gadd45a, Myc were down-regulated in the MMQ tumor stem-like cells. The expression of VEGFA were high in tumor spheres cultured from both MMQ cell and human prolactinomas. Down-regulation of VEGFA by small interfering RNAs partially decreased cell viability of MMQ tumor stem-like cells in vitro. Bevacizumab partially suppressed the proliferation of MMQ tumor stem-like cells. Conclusions: Our findings characterize the pattern of RNA expression of tumor stem-like cells isolated from MMQ cells. VEGFA may act as a potential therapeutic target for tumor stem-like cells of prolactinomas.