Increased Adenovirus Infection in Rad18-Negative Cells

<p>Rad18^−/− MEFs show a greater susceptibility to infection with adenovirus expressing EGFP as a reporter as compared to wild type. Cells were infected with serial dilutions of adenovirus EGFP and assessed by flow cytometry 40–44 h after infection. Virus titers (IU) were measured in MEFs Rad18^−/−.</p

Rad18 Influences the Susceptibility to Retroviral Infection

<div><p>(A) Rad18^−/− MEFs, demonstrate a greater sensitivity to an HIV-1–based EGFP reporter virus infection as compared to wild type. Cells were infected with serial dilutions of an HIV-1–based EGFP reporter virus and assessed after 40–44 h by flow cytometry. Virus titers (IU) were measured in NIH3T3 cyc.T cells.</p><p>(B) Rad18^−/− MEFs demonstrate a greater sensitivity to infection with MLV vector–derived virus as compared to wild type. MLV-based EGFP reporter virus was used to infect cells that were then assessed by flow cytometry 40–44 h later.</p><p>(C) Hela cells expressing human Rad18 are more resistant to HIV-1 infection as compared to cells transfected with control plasmid. Cells transiently transfected with either hRad18-IRES-HcRed vector or IRES-HcRed control vector were infected with serial dilutions of a HIV-1–based EGFP reporter virus and assessed after 40–44 h by flow cytometry.</p></div

The Increase in Infection of Rad18^−/− Cells Is Independent of Integrase Activity and Is Not Dependent on Illegitimate Integration

<div><p>(A) Rad18^−/− MEFs show a greater susceptibility to infection with an integration-inert HIV-1 virus as compared to Rad18^+/+ cells. Cells were infected with an integration-deficient HIV-1–based EGFP reporter virus using serial dilutions of p24 values equivalent to 125,000 IU of wild-type virus and then assessed after 40–44 h by flow cytometry.</p><p>(B) MEFs Rad18^−/− and Rad18^+/+ infected with an integration-deficient HIV-1–based EGFP reporter virus were assessed by flow cytometry at 2, 5, and 9 d after infection.</p><p>(C) MEFs Rad18^−/− and Rad18^+/+ infected with HIV-1–based EGFP reporter virus and assessed by flow cytometry at 2, 5, and 9 d after infection.</p></div

Rad18 Affects the Accumulation of Retroviral DNA

<div><p>(A) MEFs Rad18^−/− infected with HIV-1–based EGFP reporter virus present a higher amount of reverse transcription product as compared to wild-type cells. Cells were infected with 12,500 IU of a VSV-G pseudotyped HIV-1 integration-deficient reporter virus for 4 h, and late reverse transcripts were assessed by QPCR at 4, 8, 12, and 24 h after infection.</p><p>(B) Hela cells transfected with Rad18-IRES-HcRed and IRES-HcRed control plasmids were infected with 12,500 IU of a VSV-G pseudotyped HIV-1 integration-deficient reporter virus. HcRed-positive cells were then sorted 12 and 20 h post-infection, and late reverse transcripts were assessed by QPCR.</p></div

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

RAD18-deficiency increased apoptosis in murine thymocytes <i>in vivo</i>.

<p><i>Rad18</i>^<i>+/+</i>, <i>Rad18</i>^<i>+/-</i>, and <i>Rad18</i>^-/- 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

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>^<i>+/+</i> and <i>Rad18</i>^-/- 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^PLUS 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