6 research outputs found
RAD18-deficiency increased apoptosis in murine thymocytes <i>in vivo</i>.
<p><i>Rad18</i><sup><i>+/+</i></sup>, <i>Rad18</i><sup><i>+/-</i></sup>, and <i>Rad18</i><sup>-/-</sup> mice were irradiated with 1Gy IR. Thymocytes were isolated at the time points indicated after irradiation. Apoptotic cell distributions in thymocytes were detected by using PE Annexin V Apoptosis Detection kit I and analyzed using flow cytometry. Each value represents the mean (+standard deviation) of the results from the individual mice.</p
Depletion of RAD18 suppressed entry into the M phase from the G2 phase after IR exposure.
<p>(A) HT1080 cells transfected with si-ctrl or si-RAD18 were exposed to 1 or 2 Gy IR, and then lysed at the indicated time points, after irradiation. Samples were analyzed by western blotting with the indicated antibodies. (B) Cells were exposed to 1 Gy of IR, fixed with ethanol at the indicated time points after irradiation, and then immunostained with phospho-histone H3 and propidium iodide (PI). The percentage of G2/M phase cells was determined by flow cytometry. Each value represents the mean (+standard deviation) of the results from three independent experiments. (C) Cells were exposed to various doses of IR and then fixed with ethanol 60 min after irradiation. The fixed cells were immunostained with phosphor-histone H3 and PI. The percentage of G2/M phase cells was determined by flow cytometry. Each value represents the mean (+standard deviation) of the results from three independent experiments.</p
Depleting RAD18 suppressed the response to DNA damage induced by IR.
<p>HEK293 cells were transfected with si-ctrl or si-RAD18, irradiated with 2 or 4 Gy, and then lysed at the indicated time points after irradiation. Samples were analyzed by western blotting with the indicated antibodies.</p
Depleting RAD18 suppressed foci formation at the G2/M phase by DNA damage signaling factors in response to IR.
<p>HEK293 cells transfected with si-ctrl or si-RAD18 were exposed to 2 Gy IR, labeled with EdU, and then fixed at 90 min after irradiation. The cells were co-immunostained with anti-Edu and anti-γH2AX, anti-phospho-ATM or anti-53BP1 antibodies. The G1, S, G2/M phase cells were distinguished using the IN Cell Analyzer. The number of foci per cell was determined using the image-analysis software of the IN Cell Developer. Each value represents the mean (+standard deviation) of the results from three independent experiments. Each value represents the mean (+standard deviation) of the results from three independent experiments. (A) γH2AX, (B) phosphor-ATM, (C) 53BP1.</p
RAD18 is required for genomic stability after ionizing radiation exposure.
<p>(A) RAD18-depletion increased the frequency of micronuclei formation in response to IR <i>in vitro</i>. HT1080 cells transfected with si-ctrl or si-RAD18 were exposed to 2 or 4 Gy IR, fixed after 18 hrs, and then stained with Hoechst 33258 and SYTO RNASelect Green Fluorescent Cell Stain. The number of micronuclei per cell was determined using the IN Cell Analyzer. Each value represents the mean (+standard deviation) derived from three independent experiments. Each value represents the mean (+standard deviation) of the results from three independent experiments. (B) RAD18-deficiency increased the frequency of micronuclei formation in response to IR <i>in vivo</i>. <i>Rad18</i><sup><i>+/+</i></sup> and <i>Rad18</i><sup>-/-</sup> mice were irradiated with 1Gy IR. Peripheral blood was withdrawn from the tail vein at the time points indicated after irradiation, and micronucleated reticulocytes were labeled using a MicroFlow<sup>PLUS</sup> kit. More than 20,000 reticulocytes from each mouse were analyzed by flow cytometry to determine the frequency of micronucleated reticulocytes. Each value represents the mean (+standard deviation) of the results for the individual mice.</p
Depleting RAD18 reduced γH2AX levels at the G2/M phase in response to IR in HT1080 cells.
<p>(A) Gating on G1, S and G2/M cells. (B) γH2AX histograms of the gated G2/M phase population of non-irradiated cells. (C) γH2AX histograms of the gated G2/M phase population of cells irradiated with 8 Gy. (D) Dose dependent response of γH2AX fluorescence intensity in HT1080 cells. HT1080 cells transfected with si-ctrl or si-RAD18 were irradiated with 0,1,2,4 or 8 Gy IR and fixed with ethanol 60min after IR exposure. Fixed cells were immunostained with γH2AX and PI and analyzed by flow cytometry. Each value represents the mean (+standard deviation) of the results from three independent experiments.</p