Amples was determined using the BCAFigure 1. Transient silencing of LB1 induces

Amples was determined using the BCAFigure 1. Transient silencing of LB1 induces growth arrest in U2 OS cells. A. The protein levels of LB1, LB2, and LA and C were assayed by immunoblotting at day 3 after electroporation with the vector encoding shRNA (shLB1) or a scrambled sequence (Sc). B. Relative expression levels of LMNB1, LMNB2, and LMNA mRNA in cells were determined by qRT-PCR at day 3 after silencing using GAPDH as a reference gene. The error bars represent standard deviation of the mean (n = 5). C. Growth rate of shLB1 and Sc cells were compared for 5 days following 10457188 silencing. Growth rate was evaluated as previously described [17] (n = 6, p = 5.24 61027); error bars represent standard deviations. doi:10.1371/journal.pone.0069169.gFigure 2. Activation of key signaling proteins that mediate early G1 arrest. Protein levels in silenced and control cells were detected by immunoblotting at day 3 after LB1 silencing. GAPDH served as a loading control. This experiment was repeated 4 times. doi:10.1371/journal.pone.0069169.gRole of LB1 in NERprotein assay kit (Thermo Scientific). The protein samples were separated by SDS-PAGE on 10 gels and transferred to nitrocellulose. Primary antibodies used for immunoblotting were: mouse anti-LA/C (5G4), rabbit anti-LB1 [22], mouse anti-LB1/2 (2B2); rabbit anti-CHK1, anti-pCHK1 (S345), anti-CHK2, antipCHK2 (Cell Signaling); rabbit anti-ATM, rabbit anti-pATM (Epitomics), mouse anti-p53 (DO-1), rabbit anti-ATR, rabbit antipATR, mouse anti-PCNA (PC10), rabbit anti-DDB1, goat antiCSB, rabbit anti-53BP1 (Santa Cruz Biotechnology); rabbit antipRPA32 (Bethyl Labs); mouse anti cH2AX (JBW301, Millipore); mouse anti-GAPDH (FF26A/F9, Biolegend, Inc.). Secondary antibodies conjugated with horseradish peroxidase (1 mg/mL; KPL) were used at a dilution of 1:50,000 and the peroxidase activity was detected using the SuperSignal West Pico Chemiluminescence Detection kit (Thermo Scientific). Images were quantified with Kodak Molecular Imaging software.ImmunofluorescenceU-2 OS cells grown on glass coverslips were fixed in methanol for 10 min at 220uC followed by permeabilization with 0.1 Triton X-100 in PBS for 10 min at 22uC. Primary antibodies used for immunofluorescence were mouse anti-LB1/2, rabbit anti-LB1 [22], rabbit anti-pRPA32 (Bethyl Labs), mouse anti- cH2AX (JBW301, Millipore), rabbit anti-DDB1 and rabbit anti-53BP1 (Santa Cruz Biotechnology). Secondary antibodies included goat anti mouse IgG-Alexa Fluor 488 and goat anti-mouse IgG-Alexa Fluor568 (Ion, in human genetic studies, IRAK-M has also been associated with Invitrogen). DNA was stained with 1 ng/mL Hoechst 33258 (Invitrogen). After staining, coverslips were mounted on slides in 20 mM Tris-Cl (pH 9.0) with 50 glycerol and 1 pphenylenediamine (Sigma-Aldrich). Images were obtained with a Zeiss LSM 510 microscope using oil immersion objective lenses (PlanApochromat, 63X and 100X, 1.40 NA).BrdU labelingDetection of DNA replication was carried out as described [22]. Cells were labeled with 10 mM BrdU (Sigma-Aldrich) in growth medium for 3 h at 37uC. BrdU-labeled DNA was detected with rabbit anti-BrdU (Sigma-Aldrich), followed by goat anti-rabbit IgG-Alexa Fluor 488 (Invitrogen).UV irradiationCultured cells were washed once with PBS and irradiated with 254 nm UVC using a Stratagene UV Stratalinker 1800 at a fluency of 20 J/m2 as detected by a Title Loaded From File calibrated UVC radiometer (UVC light meter 850010; Sper Scientific). Following irradiation, growth medium was replaced on the cells and they were stored in the incubator until needed.Amples was determined using the BCAFigure 1. Transient silencing of LB1 induces growth arrest in U2 OS cells. A. The protein levels of LB1, LB2, and LA and C were assayed by immunoblotting at day 3 after electroporation with the vector encoding shRNA (shLB1) or a scrambled sequence (Sc). B. Relative expression levels of LMNB1, LMNB2, and LMNA mRNA in cells were determined by qRT-PCR at day 3 after silencing using GAPDH as a reference gene. The error bars represent standard deviation of the mean (n = 5). C. Growth rate of shLB1 and Sc cells were compared for 5 days following 10457188 silencing. Growth rate was evaluated as previously described [17] (n = 6, p = 5.24 61027); error bars represent standard deviations. doi:10.1371/journal.pone.0069169.gFigure 2. Activation of key signaling proteins that mediate early G1 arrest. Protein levels in silenced and control cells were detected by immunoblotting at day 3 after LB1 silencing. GAPDH served as a loading control. This experiment was repeated 4 times. doi:10.1371/journal.pone.0069169.gRole of LB1 in NERprotein assay kit (Thermo Scientific). The protein samples were separated by SDS-PAGE on 10 gels and transferred to nitrocellulose. Primary antibodies used for immunoblotting were: mouse anti-LA/C (5G4), rabbit anti-LB1 [22], mouse anti-LB1/2 (2B2); rabbit anti-CHK1, anti-pCHK1 (S345), anti-CHK2, antipCHK2 (Cell Signaling); rabbit anti-ATM, rabbit anti-pATM (Epitomics), mouse anti-p53 (DO-1), rabbit anti-ATR, rabbit antipATR, mouse anti-PCNA (PC10), rabbit anti-DDB1, goat antiCSB, rabbit anti-53BP1 (Santa Cruz Biotechnology); rabbit antipRPA32 (Bethyl Labs); mouse anti cH2AX (JBW301, Millipore); mouse anti-GAPDH (FF26A/F9, Biolegend, Inc.). Secondary antibodies conjugated with horseradish peroxidase (1 mg/mL; KPL) were used at a dilution of 1:50,000 and the peroxidase activity was detected using the SuperSignal West Pico Chemiluminescence Detection kit (Thermo Scientific). Images were quantified with Kodak Molecular Imaging software.ImmunofluorescenceU-2 OS cells grown on glass coverslips were fixed in methanol for 10 min at 220uC followed by permeabilization with 0.1 Triton X-100 in PBS for 10 min at 22uC. Primary antibodies used for immunofluorescence were mouse anti-LB1/2, rabbit anti-LB1 [22], rabbit anti-pRPA32 (Bethyl Labs), mouse anti- cH2AX (JBW301, Millipore), rabbit anti-DDB1 and rabbit anti-53BP1 (Santa Cruz Biotechnology). Secondary antibodies included goat anti mouse IgG-Alexa Fluor 488 and goat anti-mouse IgG-Alexa Fluor568 (Invitrogen). DNA was stained with 1 ng/mL Hoechst 33258 (Invitrogen). After staining, coverslips were mounted on slides in 20 mM Tris-Cl (pH 9.0) with 50 glycerol and 1 pphenylenediamine (Sigma-Aldrich). Images were obtained with a Zeiss LSM 510 microscope using oil immersion objective lenses (PlanApochromat, 63X and 100X, 1.40 NA).BrdU labelingDetection of DNA replication was carried out as described [22]. Cells were labeled with 10 mM BrdU (Sigma-Aldrich) in growth medium for 3 h at 37uC. BrdU-labeled DNA was detected with rabbit anti-BrdU (Sigma-Aldrich), followed by goat anti-rabbit IgG-Alexa Fluor 488 (Invitrogen).UV irradiationCultured cells were washed once with PBS and irradiated with 254 nm UVC using a Stratagene UV Stratalinker 1800 at a fluency of 20 J/m2 as detected by a calibrated UVC radiometer (UVC light meter 850010; Sper Scientific). Following irradiation, growth medium was replaced on the cells and they were stored in the incubator until needed.