Hydroxyurea, aphidicolin and thymidine all cause S phase arrest.

<p>Cells were treated with different drugs for 24 h in all the panels. The distribution of cell cycle was assessed by flow cytometry. The experiments were performed in triplicate. Error bars represent standard deviation. *<i>P</i><0.05, #<i>P</i><0.01, when compared with control group.</p

Presentation_1_hsa-let-7c miRNA Regulates Synaptic and Neuronal Function in Human Neurons.PDF

Non-coding RNA, including microRNA (miRNA) serves critical regulatory functions in the developing brain. The let-7 family of miRNAs has been shown to regulate neuronal differentiation, neural subtype specification, and synapse formation in animal models. However, the regulatory role of human let-7c (hsa-let-7c) in human neuronal development has yet to be examined. Let-7c is encoded on chromosome 21 in humans and therefore may be overexpressed in human brains in Trisomy 21 (T21), a complex neurodevelopmental disorder. Here, we employ recent developments in stem cell biology to show that hsa-let-7c mediates important regulatory epigenetic functions that control the development and functional activity of human induced neuronal cells (iNs). We show that overexpression of hsa-let-7c in human iNs derived from induced pluripotent stem (iPS), as well as embryonic stem (ES), cells leads to morphological as well as functional deficits including impaired neuronal morphologic development, synapse formation and synaptic strength, as well as a marked reduction of neuronal excitability. Importantly, we have assessed these findings over three independent genetic backgrounds, showing that some of these effects are subject to influence by background genetic variability with the most robust and reproducible effect being a striking reduction in spontaneous neural firing. Collectively, these results suggest an important function for let-7 family miRNAs in regulation of human neuronal development and raise implications for understanding the complex molecular etiology of neurodevelopmental disorders, such as T21, where let-7c gene dosage is increased.</p

Newly formed MN-γ–H2AX (+) are associated with S phase cells experiencing replication stress.

<p>A. Changes in the frequency of MN-γ–H2AX (+) during the time course in MCF-7 cells treated with various agents. B. Distribution of γ–H2AX signals in cells experiencing replication stress. The cells were classified into three types, labeled as type 1, 2 and 3, by the distribution of γ–H2AX signals. MCF-7 cells were treated with thymidine (500 µM) for 24 h and were then fixed for immunofluorescence staining with antibody against γ-H2AX, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018618#s2" target="_blank">Materials and Methods</a>. C. Decline in the percentage of cells exhibiting replication stress-induced DSBs over time. MCF-7 cells were treated with various agents at indicated concentrations for 24 h and were then processed and scored for the three types of cells. Percentages, together with their standard deviations, were also shown at the bottom. D. Decline in the association of MN-γ-H2AX with type 1 and type 2 cells over time. MCF-7 cells were treated with various agents at indicated concentrations for 24 h and were then processed and scored for MN-γ-H2AX. Percentages, together with their standard deviations, were also shown at the bottom.</p

MN-γ-H2AX in formation.

<p>γ-H2AX signals are clustered in the nuclear blebs that may later form MN-γ-H2AX. Mouse skin fibroblasts at 5^th passage were processed for immunofluorescence staining with antibody against γ-H2AX. Bars, 5 µM.</p

Elevation of MN-γ–H2AX (+) in miCUL4B cells.

<p>*<i>P</i><0.05,</p><p>**<i>P</i><0.01.</p

Induction of micronuclei by agents causing replication stress.

<p>*<i>P</i><0.05,</p><p>**<i>P</i><0.01.</p

Increased MN formation in shRPA1 cells.

<p>A. Quantification of <i>RPA1</i> mRNA levels shRPA1 cells. RNA was extracted from shNeg MCF-7 cells and shRPA1 MCF-7 cells. The expression of RPA1 mRNA was quantified by real-time quantitative RT-PCR assay. The assay was performed in triplicate and relative means ± s.d. were shown. B. RPA1 protein level in shRPA1 cells. Equal amounts of protein lysates were subjected to SDS-PAGE (12%) and then detected using the antibodies against RPA1 and β-actin, respectively. C. Effect of RPA1 RNAi on cell cycle distribution of MCF-7 cells. The percentage of cells in S phase increased about 15% in shRPA1 cells when compared with shNeg cells (*<i>P</i> = 0.002). The experiments were performed in triplicate. Error bars represent standard deviation. For synchronization of cells, cells were starved in DMEM with 0.2% FBS for 48 h and then stimulated to initiate a new cell cycle in fresh media containing 10% FBS for 20 h. Afterward, cells were used for BrdU incorporation assay and cell cycle analysis. D. Reduction in cell proliferation as measured by BrdU incorporation assay. There was a marked reduction in BRDU-positive shRPA1 cells when compared with shNeg MCF-7 cells (*<i>P</i> = 0.004). The experiments were performed in triplicate. Error bars represent standard deviation. E. Increased MN-γ-H2AX (+) in shRPA1 cells. The assay was performed in triplicate and relative means ± s.d. were shown. *<i>P</i> = 0.008, when compared with shNeg MCF-7 cells.</p

MN-γ–H2AX (+) in mammalian cells.

<p>A. Examples of MN-γ–H2AX (+) and MN-γ–H2AX (−) in MCF-7 cells. B. MN-γ-H2AX (+) in various mammalian cell lines. Cells were grown on coverslips in 6-well plates 24 h before they were processed for immunofluorescence staining with anti γ–H2AX antibody.</p

Induction of micronuclei by paclitaxel.

<p>*<i>P</i><0.05,</p><p>**<i>P</i><0.01.</p

Occurrence of MN-γ–H2AX (+) in mammalian cells.

<p>NHDF, Normal Human Dermal Fibroblasts; MSF, Primary mouse skin (ear) fibroblasts.</p