ABSTRACT
Detection and imaging RNAs in live cells is in high demand. Methodology for such a purpose is still a challenge, particularly for single RNA detection and imaging in live cells. In this study, a type of quantum dot (QD) nanobeacon with controllable valencies was constructed by precisely conjugating the black hole quencher (BHQ1) and phosphorothioate comodified DNA onto CdTe:Zn2+ QDs via a one-pot hydrothermal method. The nanobeacon with only one conjugated DNA was used to label and detect low-abundance nucleic acids in live cells, and single HIV-1 RNAs were detected and imaged in live HIV-1 integrated cells. Additionally, QD nanobeacon-labeled HIV-1 genomic RNAs were encapsulated in progeny viral particles, which can be used to track the uncoating process of single viruses. The current study provides a platform for nucleic acid labeling and imaging with high sensitivity, being especially meaningful for tracking of individual RNAs in live cells.
Subject(s)
Cadmium Compounds/chemistry , DNA/chemistry , Optical Imaging/methods , Quantum Dots/chemistry , RNA/analysis , Tellurium/chemistry , Cell Line , HIV-1/isolation & purification , Humans , Microscopy, Confocal/methods , RNA, Viral/analysisABSTRACT
Bisphenol A (BPA) acts as xenoestrogen and has a great impact on disorders of human reproductive system. However, the mechanism through which BPA can affect human testicular function remains to be identified. GPR30 is a novel membrane estrogen receptor with high-affinity and low-capacity binding to estrogens. We demonstrated that estrogen receptor α (ERα), estrogen receptor ß (ERß) as well as GPR30 are expressed in mouse spermatocyte-derived GC-2 cells using Real-time PCR. We treated the cells with different doses of BPA and found that even low doses of BPA can inhibit GC-2 cell growth using MTT assay. To make sure which receptor is responsible for the biological function of BPA, we used ER down-regulator ICI and indicated that BPA could bind to GPR30. We also observed that BPA was able to induce Erk1/2 phosphorylation in GC-2 cells and proved that this process was mediated by GPR30-related EGFR-MAPK pathway using western blot. By Real-time PCR, we found that the expression of c-Fos was up-regulated and Cyclin D1 gene was down-regulated, in the presence of BPA and ICI. The results of MTT assay, comet assay and flow cytometry indicated that the activation of GPR30 induced by BPA inhibited the cell growth and induced cell apoptosis and ICI, GPR30 siRNA, EGFR inhibitor (AG), and MAPK (PD) inhibitor could partially reverse this effect. Immunohistochemistry on the testis of BPA -damaged mice showed that BPA induced spermatocyte apoptosis without affecting the seminiferous tubules and spermatocyte. In conclusion, BPA triggered spermatocyte apoptosis via GPR30.