B Serum of placensin glucagon at different of pregnancy measured using specific ELISA in normal GDM . Numbers in parentheses represent number of .

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B Serum levels of placensin and glucagon at different weeks of pregnancy measured using specific ELISA in normal and GDM patients. Numbers in parentheses represent number of patients.


IFNα dose - dependency of STAT1 STAT2 phosphorylation in Huh7 . 5 . were seeded 24 hours prior to start of experiment . Three hours before stimulation , were growth factor - depleted were subsequently stimulated with indicated concentrations of IFNα . lysates were collected one hour after stimulation phosphorylation of STAT1 STAT2 was detected by immunoblot utilizing antibodies recognizing STAT1 phosphorylated on tyrosine residue 701 , or STAT2 phosphorylated on tyrosine residue 690 . Data points are displayed as dots with 1σ confidence interval estimated from biological replicates ( N = 1 to N = 38 ) using a combined scaling error model . Data is approximated with a sigmoidal function signals corresponding to a low dose ( 2 . 8 pM IFNα ) , a medium dose ( 28 pM IFNα ) a high dose ( 1400 pM IFNα ) are displayed with dashed lines .

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IFNα dose-dependency of STAT1 and STAT2 phosphorylation in Huh7.5 cells. Cells were seeded 24 hours prior to the start of the experiment. Three hours before stimulation, cells were growth factor-depleted and were subsequently stimulated with the indicated concentrations of IFNα. Cytoplasmic protein lysates were collected one hour after the stimulation and phosphorylation of STAT1 and STAT2 was detected by immunoblot utilizing antibodies recognizing STAT1 phosphorylated on tyrosine residue 701, or STAT2 phosphorylated on tyrosine residue 690. Data points are displayed as dots with 1σ confidence interval estimated from biological replicates (N=1 to N=38) using a combined scaling and error model. Data is approximated with a sigmoidal function and signals corresponding to a low dose (2.8 pM IFNα), a medium dose (28 pM IFNα) and a high dose (1400 pM IFNα) are displayed with dashed lines.


Representative immunoblot of IFNα - induced phosphorylation of STAT1 STAT2 upon stimulation of Huh7 . 5 prestimulated for 24 hours with 2 . 8 pM IFNα , 1400 pM IFNα or without prestimulation . Time points after presimulation are indicated . 20 µg of lysates were analyzed using antibodies for indicated targets . Phosphorylation of STAT1 STAT2 was detected by immunoblot utilizing antibodies recognizing STAT1 phosphorylated on tyrosine residue 701 , or STAT2 phosphorylated on tyrosine residue 690 . An asterisk indicates pSTAT1β . Calnexin served as loading control . Molecular weights are indicated on left . Immunoblot detection was performed with chemiluminescence employing a CCD camera device ( ImageQuant ) . of immunoblots of IFNα - induced phosphorylation of STAT1 STAT2 in Huh7 . 5 prestimulated with 2 . 8 pM IFNα , 1400 pM IFNα or without pretreatment . Time points after presimulation are displayed . Data is displayed as fold change relative to untreated . Errors were estimated with a combined scaling error model , comprising 1σ confidence interval estimated from biological replicates ( N = 3 ) . Dashed lines indicate smoothing splines .

O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative immunoblot of IFNα-induced phosphorylation of STAT1 and STAT2 upon stimulation of Huh7.5 cells prestimulated for 24 hours with 2.8 pM IFNα, 1400 pM IFNα or without prestimulation. Time points after presimulation are indicated. 20 µg of cytoplasmic lysates were analyzed using antibodies for the indicated targets. Phosphorylation of STAT1 and STAT2 was detected by immunoblot utilizing antibodies recognizing STAT1 phosphorylated on tyrosine residue 701, or STAT2 phosphorylated on tyrosine residue 690. An asterisk indicates pSTAT1β. Calnexin served as loading control. Molecular weights are indicated on the left. Immunoblot detection was performed with chemiluminescence employing a CCD camera device (ImageQuant).   Quantification of immunoblots of IFNα-induced phosphorylation of cytoplasmic and nuclear STAT1 and STAT2 in Huh7.5 cells prestimulated with 2.8 pM IFNα, 1400 pM IFNα or without pretreatment. Time points after presimulation are displayed. Data is displayed as fold change relative to untreated cells. Errors were estimated with a combined scaling and error model, comprising 1σ confidence interval estimated from biological replicates (N=3). Dashed lines indicate smoothing splines.


Induction of interferon - stimulated genes upon prestimulation with 1400 pM IFNα ( yellow background ) stimulation with 1400 pM IFNα ( background ) in Huh7 . 5 , assessed by qRT - PCR . were normalized to geometric mean of reference genes GAPDH , HPRT TBP were displayed as fold change . Peak of gene expression is indicated . Data points displayed as dots with 1σ confidence - interval estimated from biological replicates ( N = 4 to N = 14 ) using a combined scaling error model . Dashed lines indicate smoothing splines . Induction of interferon - stimulated genes upon prestimulation with 2 . 8 pM IFNα ( yellow background ) stimulation with 1400 pM IFNα ( background ) in Huh7 . 5 , assessed by qRT - PCR . were normalized to geometric mean of reference genes GAPDH , HPRT TBP were displayed as fold change . Peak of gene expression is indicated . Data points are displayed as dots with 1σ confidence interval estimated from biological replicates ( N = 4 to N = 6 ) using a combined scaling error model . Dashed lines indicate smoothing splines .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Induction of interferon-stimulated genes upon prestimulation with 1400 pM IFNα (yellow background) and stimulation with 1400 pM IFNα (white background) in Huh7.5 cells, assessed by qRT-PCR. RNA levels were normalized to the geometric mean of reference genes GAPDH, HPRT and TBP and were displayed as fold change. Peak of gene expression is indicated. Data points displayed as dots with 1σ confidence-interval estimated from biological replicates (N=4 to N=14) using a combined scaling and error model. Dashed lines indicate smoothing splines.   Induction of interferon-stimulated genes upon prestimulation with 2.8 pM IFNα (yellow background) and stimulation with 1400 pM IFNα (white background) in Huh7.5 cells, assessed by qRT-PCR. RNA levels were normalized to the geometric mean of reference genes GAPDH, HPRT and TBP and were displayed as fold change. Peak of gene expression is indicated. Data points are displayed as dots with 1σ confidence interval estimated from biological replicates (N=4 to N=6) using a combined scaling and error model. Dashed lines indicate smoothing splines.


Growth factor - depleted Huh7 . 5 were prestimulated with 2 . 8 pM , 28 pM , 1400 pM IFNα or left untreated were stimulated with 1400 pM IFNα 24 hours later . IFNα - induced signaling was measured by time - resolved quantitative immunoblotting detected with chemiluminescence using a CCD - camera . Data was normalized to reference Calnexin or HDAC1 , scaled subjected to model calibration . Model calibration with time - resolved IFNα - induced phosphorylation of STAT1 STAT2 induced feedback upon prestimulation with 0 , 2 . 8 pM , 28 pM or 1400 pM IFNα . lysates were subjected to quantitative immunoblotting . Experimental data is represented by filled circles with errors representing 1σ confidence intervals estimated from biological replicates ( N = 3 to N = 23 ) using a combined scaling error model . Model trajectories are represented by lines . pSTAT1 , pSTAT2 represent phosphorylated STAT1 STAT2 on residue Tyr701 Tyr690 , respectively . tSTAT1 tSTAT2 represent total form of STAT1 STAT2 comprising both phosphorylated unphosphorylated STAT1 , STAT2 , respectively .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O

Growth factor-depleted Huh7.5 cells were prestimulated with 2.8 pM, 28 pM, 1400 pM IFNα or left untreated and were stimulated with 1400 pM IFNα 24 hours later. IFNα-induced signaling was measured by time-resolved quantitative immunoblotting and detected with chemiluminescence using a CCD-based camera. Data was normalized to reference proteins Calnexin or HDAC1, scaled and subjected to model calibration.   Model calibration with time-resolved IFNα-induced phosphorylation of STAT1 and STAT2 and induced feedback proteins upon prestimulation with 0, 2.8 pM, 28 pM or 1400 pM IFNα. Cytoplasmic lysates were subjected to quantitative immunoblotting. Experimental data is represented by filled circles with errors representing 1σ confidence intervals estimated from biological replicates (N=3 to N=23) using a combined scaling and error model. Model trajectories are represented by lines. pSTAT1, pSTAT2 represent phosphorylated STAT1 and STAT2 on residue Tyr701 and Tyr690, respectively. tSTAT1 and tSTAT2 represent total form of STAT1 and STAT2 comprising both phosphorylated and unphosphorylated STAT1, STAT2, respectively.


Model calibration with time - resolved IFNα - induced feedback transcripts upon prestimulation with 0 , 2 . 8 pM , 28 pM or 1400 pM IFNα , assessed by qRT - PCR . mRNA were normalized to geometric mean of reference genes GAPDH , HPRT TBP . Experimental data is represented by filled circles with errors representing 1σ confidence - interval estimated from biological replicates ( N = 3 to N = 14 ) using a combined scaling error model . Model trajectories are represented by lines .

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Model calibration with time-resolved IFNα-induced feedback transcripts upon prestimulation with 0, 2.8 pM, 28 pM or 1400 pM IFNα, assessed by qRT-PCR. mRNA levels were normalized to the geometric mean of reference genes GAPDH, HPRT and TBP. Experimental data is represented by filled circles with errors representing 1σ confidence-interval estimated from biological replicates (N=3 to N=14) using a combined scaling and error model. Model trajectories are represented by lines.


Model calibration with amount of molecules per of STAT1 , STAT2 , IRF9 USP18 determined 24 hours after prestimulation with 0 , 2 . 8 pM , 28 pM or 1400 pM IFNα . Calibrator were spiked into 10 µg of total lysate subjected to quantitative immunoblotting . Immunoblot detection was performed by chemiluminescence using a CCD - camera - device . Averaged values ( N = 4 ) are displayed with standard deviations . Green squares indicate amounts estimated by mathematical model .

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Model calibration with the amount of molecules per cell of STAT1, STAT2, IRF9 and USP18 determined 24 hours after prestimulation with 0, 2.8 pM, 28 pM or 1400 pM IFNα. Calibrator proteins were spiked into 10 µg of total protein lysate and subjected to quantitative immunoblotting. Immunoblot detection was performed by chemiluminescence using a CCD-camera-based device. Averaged values (N=4) are displayed with standard deviations. Green squares indicate amounts estimated by the mathematical model.


Model calibration of IFNα - induced phosphorylation of STAT1 STAT2 upon stimulation of Huh7 . 5 prestimulated with 0 , 2 . 8 pM , 28 pM or 1400 pM IFNα . lysates were subjected to quantitative immunoblotting . Experimental data is represented by filled circles with errors representing 1σ confidence - interval estimated from biological replicates ( N = 4 to N = 22 ) using a combined scaling error model . Model trajectories are represented by lines .

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Model calibration of IFNα-induced phosphorylation of STAT1 and STAT2 upon stimulation of Huh7.5 prestimulated with 0, 2.8 pM, 28 pM or 1400 pM IFNα. Nuclear lysates were subjected to quantitative immunoblotting. Experimental data is represented by filled circles with errors representing 1σ confidence-interval estimated from biological replicates (N=4 to N=22) using a combined scaling and error model. Model trajectories are represented by lines.


Model predictions of IFNα - induced dynamics of occupied bindings sites ( OccGASbs ) ( left panel ) , of sum of pSTAT1 : pSTAT2 heterodimers pSTAT1 : pSTAT2 : IRF9 trimers ( middle panel ) of pSTAT1 : pSTAT2 : IRF9 trimers ( right panel ) in untreated Huh7 . 5 in Huh7 . 5 prestimulated for 24 hours with 280 pM IFNα that were subsequently stimulated with 1400 pM IFNα . Lines with shading represent model predictions with 68 % confidence intervals using prediction profile likelihood method . For experimental validation of dynamics of OccGASbs , electrophoretic mobility shift assays ( EMSA ) were performed using lysates obtained from untreated Huh7 . 5 or Huh7 . 5 that were prestimulated for 24 hours with 280 pM IFNα then stimulated with 1400 pM IFNα . Lysates were incubated with radioactively - labeled oligonucleotides probes harboring - binding region of human IRF1 promoter . Samples were resolved on a native polyacrylamide gel radioactivity was visualized quantified from three independent experiments ( left panel ) . For experimental validation of dynamics of sum of pSTAT1 : pSTAT2 heterodimers pSTAT1 : pSTAT2 : IRF9 trimers , immunoprecipitations ( IP ) were performed using total lysates obtained from untreated Huh7 . 5 or Huh7 . 5 that were prestimulated for 24 hours with 280 pM IFNα then stimulated with 1400 pM IFNα . Lysates were subjected to immunoprecipitation with antibodies recognizing STAT2 phosphorylated STAT1 was detected with quantitative immunoblotting ( IB ) ( middle panel ) . For experimental validation of dynamics of pSTAT1 : pSTAT2 : IRF9 trimers , immunoprecipitations were performed using total lysates obtained from untreated Huh7 . 5 or Huh7 . 5 that were prestimulated for 24 hours with 280 pM IFNα then stimulated with 1400 pM IFNα . Lysates were subjected to immunoprecipitation ( IP ) with antibodies recognizing IRF9 phosphorylated STAT1 was detected with quantitative immunoblotting ( IB ) ( right panel ) . Antibodies corresponding in complexes are underlined . Experimental data is represented by filled circles with errors representing 1σ confidence - interval estimated from biological replicates ( N = 3 ) using a combined scaling error model .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Model predictions of IFNα-induced dynamics of occupied GAS bindings sites (OccGASbs) (left panel), of the sum of the pSTAT1:pSTAT2 heterodimers and the pSTAT1:pSTAT2:IRF9 trimers (middle panel) and of the pSTAT1:pSTAT2:IRF9 trimers (right panel) in untreated Huh7.5 cells and in Huh7.5 cells prestimulated for 24 hours with 280 pM IFNα that were subsequently stimulated with 1400 pM IFNα. Lines with shading represent model predictions with 68% confidence intervals using the prediction profile likelihood method. For experimental validation of the dynamics of OccGASbs, electrophoretic mobility shift assays (EMSA) were performed using nuclear protein lysates obtained from untreated Huh7.5 cells or Huh7.5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα. Lysates were incubated with radioactively-labeled oligonucleotides probes harboring the GAS-binding region of the human IRF1 promoter. Samples were resolved on a native polyacrylamide gel and radioactivity was visualized and quantified from three independent experiments (left panel). For experimental validation of the dynamics of the sum of the pSTAT1:pSTAT2 heterodimers and the pSTAT1:pSTAT2:IRF9 trimers, immunoprecipitations (IP) were performed using total cell lysates obtained from untreated Huh7.5 cells or Huh7.5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα. Lysates were subjected to immunoprecipitation with antibodies recognizing STAT2 and phosphorylated STAT1 was detected with quantitative immunoblotting (IB) (middle panel). For experimental validation of the dynamics of the pSTAT1:pSTAT2:IRF9 trimers, immunoprecipitations were performed using total cell lysates obtained from untreated Huh7.5 cells or Huh7.5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα. Lysates were subjected to immunoprecipitation (IP) with antibodies recognizing IRF9 and phosphorylated STAT1 was detected with quantitative immunoblotting (IB) (right panel). Antibodies and the corresponding proteins in the complexes are underlined. Experimental data is represented by filled circles with errors representing 1σ confidence-interval estimated from biological replicates (N=3) using a combined scaling and error model.


Model predictions of IFNα - induced dynamics of occupied bindings sites ( OccGASbs ) of occupied ISRE binding sites ( OccISREbs ) in Huh7 . 5 without prestimulation in prestimulated for 24 hours with 28 pM 280 pM IFNα that were subsequently stimulated with 1400 pM IFNα . Model predictions were performed using prediction profile likelihood method . Lines with shading represent model predictions with 68 % confidence intervals . For experimental validation , growth factor - depleted Huh7 . 5 were prestimulated with 0 pM , 28 pM 280 pM IFNα . After 24 hours were stimulated with 1400 pM IFNα IFNα - induced expression of target genes was measured by qRT - PCR . were normalized to geometric mean of reference genes GAPDH , HPRT TBP , averaged displayed as fold change , represented by filled circles with errors representing standard error of mean calculated from biological replicates ( N = 3 ) . Except for gene - specific parameters ( mRNA synthesis degradation rates , time - delay parameter Hill coefficient ) , qRT - PCR data were used for model validation but not for model calibration .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Model predictions of IFNα-induced dynamics of occupied GAS bindings sites (OccGASbs) and of occupied ISRE binding sites (OccISREbs) in Huh7.5 cells without prestimulation and in cells prestimulated for 24 hours with 28 pM and 280 pM IFNα that were subsequently stimulated with 1400 pM IFNα. Model predictions were performed using the prediction profile likelihood method. Lines with shading represent model predictions with 68% confidence intervals. For experimental validation, growth factor-depleted Huh7.5 cells were prestimulated with 0 pM, 28 pM and 280 pM IFNα. After 24 hours cells were stimulated with 1400 pM IFNα and IFNα-induced expression of target genes was measured by qRT-PCR. RNA levels were normalized to the geometric mean of reference genes GAPDH, HPRT and TBP, averaged and displayed as fold change, represented by filled circles with errors representing standard error of the mean calculated from biological replicates (N=3). Except for gene-specific parameters (mRNA synthesis and degradation rates, time-delay parameter and Hill coefficient), qRT-PCR data were used for model validation but not for model calibration.


Dose - dependent sensitization of signal transduction induced by therapeutic interferon Roferon . Growth factor - depleted Huh7 . 5 were prestimulated with 0 , with 1 . 2 or 608 pM Roferon stimulated with 608 pM Roferon 24 hours later . Concentrations of Roferon correspond to equipotent concentrations of IFNα . lysates were subjected to quantitative immunoblotting Roferon - induced phosphorylation of STAT1 STAT2 was detected by chemiluminescense utilizing a CCD - camera device ( ImageQuant ) . Filled circles represent scaled data with errors representing 1σ confidence intervals estimated from biological replicates ( N = 3 to N = 8 ) using a combined scaling error model . Model trajectories are represented by lines . Dose - dependent sensitization of signal transduction induced by Roferon in HepG2 - hNTCP . Growth factor - depleted HepG2 - hNTCP were prestimulated with 0 , 1 . 8 or 887 pM Roferon stimulated with 887 pM Roferon 24 hours later . lysates were subjected to quantitative immunoblotting Roferon - induced phosphorylation of STAT1 STAT2 was detected by chemiluminescence utilizing a CCD - camera device ( ImageQuant ) . Filled circles represent scaled data with errors representing 1σ confidence intervals estimated from biological replicates ( N = 3 ) using a combined scaling error model . Model trajectories are represented by lines .

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Dose-dependent sensitization of signal transduction induced by the therapeutic interferon Roferon. Growth factor-depleted Huh7.5 were prestimulated with 0, with 1.2 or 608 pM Roferon and stimulated with 608 pM Roferon 24 hours later. Concentrations of Roferon correspond to equipotent concentrations of IFNα. Nuclear lysates were subjected to quantitative immunoblotting and Roferon-induced phosphorylation of STAT1 and STAT2 was detected by chemiluminescense utilizing a CCD-based camera device (ImageQuant). Filled circles represent scaled data with errors representing 1σ confidence intervals estimated from biological replicates (N=3 to N=8) using a combined scaling and error model. Model trajectories are represented by lines.   Dose-dependent sensitization of signal transduction induced by Roferon in HepG2-hNTCP. Growth factor-depleted HepG2-hNTCP were prestimulated with 0, 1.8 or 887 pM Roferon and stimulated with 887 pM Roferon 24 hours later. Nuclear lysates were subjected to quantitative immunoblotting and Roferon-induced phosphorylation of STAT1 and STAT2 was detected by chemiluminescence utilizing a CCD-based camera device (ImageQuant). Filled circles represent scaled data with errors representing 1σ confidence intervals estimated from biological replicates (N=3) using a combined scaling and error model. Model trajectories are represented by lines.


Knockdown of USP18 results in sustained signaling . USP18 knockdown efficiency determined by quantitative immunoblotting of lysates of Huh7 . 5 transfected with USP18 siRNA relative to parental Huh7 . 5 Huh7 . 5 transfected with non - targeting siRNA . were stimulated with 1400 pM IFNα for 24 hours . Error bars represent 1σ confidence intervals estimated from biological replicates ( N = 6 to N = 10 ) ( left ) . Model fit experimental data of Huh7 . 5 transfected with control siRNA or USP18 siRNA are shown . were transfected with USP18 siRNA or control siRNA 1 day prior to growth factor - depletion . Next , were prestimulated with 1400 pM IFNα ( yellow background ) stimulated with 1400 pM IFNα at 24 hours or untreated ( background ) . IFNα - induced phosphorylation of STAT1 STAT2 induction of USP18 tSTAT1 , comprising both phosphorylated unphosphorylated STAT1 , are displayed . Experimental data were obtained by quantitative immunoblotting using chemiluminescence CCD camera device ( ImageQuant ) . For model purposes data in control siRNA untransfected Huh7 . 5 are combined to one condition . Data from multiple time courses scaled together is displayed as filled circles with errors representing 1σ confidence interval estimated from biological replicates ( N = 2 to N = 10 ) using a combined scaling error model . Lines represent model trajectories .

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Knockdown of USP18 results in sustained signaling. USP18 knockdown efficiency determined by quantitative immunoblotting of cytoplasmic lysates of Huh7.5 transfected with USP18 siRNA relative to parental Huh7.5 cells and Huh7.5 cells transfected with non-targeting siRNA. Cells were stimulated with 1400 pM IFNα for 24 hours. Error bars represent 1σ confidence intervals estimated from biological replicates (N=6 to N=10) (left). Model fit and experimental data of Huh7.5 cells transfected with control siRNA or USP18 siRNA are shown. Cells were transfected with USP18 siRNA or control siRNA 1 day prior to growth factor-depletion. Next, cells were prestimulated with 1400 pM IFNα (yellow background) and stimulated with 1400 pM IFNα at 24 hours or untreated (white background). IFNα-induced phosphorylation of STAT1 and STAT2 and induction of USP18 and tSTAT1, comprising both phosphorylated and unphosphorylated STAT1 protein, are displayed. Experimental data were obtained by quantitative immunoblotting using chemiluminescence and CCD camera device (ImageQuant). For model purposes data in control siRNA and untransfected Huh7.5 are combined to one condition. Data from multiple time courses scaled together is displayed as filled circles with errors representing 1σ confidence interval estimated from biological replicates (N=2 to N=10) using a combined scaling and error model. Lines represent model trajectories.


Overexpression of Huh7 . 5 is not sufficient to explain negative memory . Induced expression of USP18 after treatment of Huh7 . 5 - TetON - USP18 Huh7 . 5 - TetON - control with doxycycline for 24 hours in comparison with parental Huh7 . 5 treated with 1400 pM IFNα for 24 hours . was performed by quantitative immunoblotting . Error bars represent 1σ confidence intervals estimated from biological replicates ( N = 3 to N = 5 ) ( left ) . Model fits experimental data of Huh7 . 5 - TetON - USP18 treated with doxycycline for 24 hours stimulated with 1400 pM IFNα or parental Huh7 . 5 prestimulated with 0 or 1400 pM IFNα stimulated with 1400 pM IFNα after 24 hours are shown . dynamics of IFNα - induced phosphorylation of STAT1 STAT2 induction of USP18 are depicted . Experimental data was obtained by quantitative immunoblotting using chemiluminescence CCD camera device ( ImageQuant ) . For modeling purposes data from Huh7 . 5 - TetON empty vector control untransduced Huh7 . 5 are combined to one condition . Data is displayed as filled circles with errors representing 1σ confidence - interval estimated from biological replicates ( N = 3 to N = 4 ) using a combined scaling error model . represents model trajectories .

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Overexpression of Huh7.5 is not sufficient to explain negative memory. Induced expression of USP18 after treatment of Huh7.5-TetON-USP18 and Huh7.5-TetON-control cells with doxycycline for 24 hours in comparison with parental Huh7.5 cells treated with 1400 pM IFNα for 24 hours. Analysis was performed by quantitative immunoblotting. Error bars represent 1σ confidence intervals estimated from biological replicates (N=3 to N=5) (left). Model fits and experimental data of Huh7.5-TetON-USP18 treated with doxycycline for 24 hours and stimulated with 1400 pM IFNα or parental Huh7.5 cells prestimulated with 0 or 1400 pM IFNα and stimulated with 1400 pM IFNα after 24 hours are shown. The dynamics of IFNα-induced phosphorylation of STAT1 and STAT2 and induction of USP18 are depicted. Experimental data was obtained by quantitative immunoblotting using chemiluminescence and CCD camera device (ImageQuant). For modeling purposes data from Huh7.5-TetON empty vector control and untransduced Huh7.5 are combined to one condition. Data is displayed as filled circles with errors representing 1σ confidence-interval estimated from biological replicates (N=3 to N=4) using a combined scaling and error model. Line represents model trajectories.


number of molecules per for STAT1 , STAT2 , IRF9 USP18 were determined experimentally in human hepatocytes from six different . were harvested without pretreatment different amounts of calibrators were spiked in total lysates lysates were subjected to immunoblotting . Detection was performed with antibodies specific to STAT1 , STAT2 , IRF9 or USP18 using chemiluminescense on a CCD camera - device ( ImageQuant ) . Average of at least N = 3 are displayed with standard deviations . Data was used for calibration of a human hepatocyte - specific mathematical model estimated values are shown with green squares .

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The number of molecules per cell for STAT1, STAT2, IRF9 and USP18 were determined experimentally in primary human hepatocytes from six different patients. Cells were harvested without pretreatment and different amounts of protein calibrators were spiked in total cell lysates and lysates were subjected to immunoblotting. Detection was performed with antibodies specific to STAT1, STAT2, IRF9 or USP18 using chemiluminescense on a CCD camera-based device (ImageQuant). Average of at least N=3 are displayed with standard deviations. Data was used for calibration of a primary human hepatocyte-specific mathematical model and estimated values are shown with green squares.


Experimental data model fits of IFNα - Roferon - induced phosphorylation of or STAT1 STAT2 in growth factor - depleted human hepatocytes prestimulated with 0 , 2 . 8 or 1400 pM IFNα ( 1 - 3 ) or 0 , 1 . 2 or 608 pM Roferon ( 4 - 6 ) . human hepatocytes from same 1 - 6 as in ( B ) were used . Experimental data is represented by filled circles ( N = 1 per ) . Experimental errors were estimated from signal variance of hepatocytes prestimulated with 1400 pM IFNα . Lines indicate model fits .

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Experimental data and model fits of IFNα- and Roferon-induced phosphorylation of cytoplasmic or cellular STAT1 and STAT2 in growth factor-depleted primary human hepatocytes prestimulated with 0, 2.8 or 1400 pM IFNα (patients 1-3) or 0, 1.2 or 608 pM Roferon (patients 4-6). Primary human hepatocytes from the same patients 1-6 as in (B) were used. Experimental data is represented by filled circles (N=1 per patient). Experimental errors were estimated from the signal variance of the hepatocytes prestimulated with 1400 pM IFNα. Lines indicate model fits.


Plasma of IFNα were measured in a cohort of 36 with chronic HBV .

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Plasma levels of IFNα were measured in a cohort of 36 patients with chronic HBV.


( C ) Western blot showing that Hel2 ( 1 - 315 ) is defective in RQC but not in NGD . arrest products derived from R ( CGN ) 12 reporter in ltn1 ∆ expressing truncated Hel2 mutant were detected with an anti - GFP antibody .

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(C) Western blot showing that Hel2(1-315) is defective in RQC but not in NGD. The arrest products derived from the R(CGN)12 reporter in ltn1∆ cells expressing truncated Hel2 mutant protein were detected with an anti-GFP antibody.


( D ) Northern blot showing 5 ' NGD - IM derived from R ( CGN ) 12 reporter in ski2 ∆ expressing indicated Hel2 mutant . 5 ' NGD - IMs were detected with a DIG - labelled GFP probe .

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(D) Northern blot showing the 5' NGD-IM derived from the R(CGN)12 reporter in ski2∆ cells expressing the indicated Hel2 mutant proteins. 5' NGD-IMs were detected with a DIG-labelled GFP probe.


( E ) Primer extension mapping of 5 ' ends of 3 ' NGD - intermediates in Hel2 - WT or Hel2 ( 1 - 315 ) mutant at nucleotide resolution . . primer extension samples were analysed using 5 % TBE - Urea - PAGE detected by . Non - specific reverse transcription ( ReTr ) products are indicated by asterisks .

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(E) Primer extension mapping of 5' ends of 3' NGD-intermediates in Hel2-WT or Hel2(1-315) mutant cells at nucleotide resolution. . The primer extension samples were analysed using 5% TBE-Urea-PAGE and detected by fluorescence. Non-specific reverse transcription (ReTr) products are indicated by asterisks.


( A ) Northern blot demonstrating that K63 - linked ubiquitination was required for an endonucleolytic cleavage by R ( CGN ) 12 . ski2 ∆ UB - WT ski2 ∆ ub - K63R were transformed with R ( CGN ) 12 reporter , total samples were separated by 2 % agarose / MOPS gel . 5 ' NGD - IMs in were detected as in Fig 1D .

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(A) Northern blot analysis demonstrating that K63-linked ubiquitination was required for an endonucleolytic cleavage by R(CGN)12. The ski2∆UB-WT and ski2∆ub-K63R cells were transformed with the R(CGN)12 reporter, and total RNA samples were separated by 2% agarose/MOPS gel. 5'NGD-IMs in the cells were detected as in Fig 1D.


( B ) Western blot showing that K63 - linked ubiquitination was required for RQC . samples from UB - WT , UB - WT ltn1 ∆ , ub - K63R , ub - K63R ltn1 ∆ expressing GFP - R ( CGN ) 12 - HIS3 reporter were subjected to Western blot using an anti - GFP antibody to detect arrest products . Note accumulation of RQC - specific CAT - tails in lane 2 .

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(B) Western blot showing that K63-linked ubiquitination was required for RQC. Protein samples from UB-WT, UB-WT ltn1∆, ub-K63R, ub-K63R ltn1∆ cells expressing the GFP-R(CGN)12-HIS3 reporter were subjected to Western blot analysis using an anti-GFP antibody to detect the arrest products. Note the accumulation of RQC-specific CAT-tails in lane 2.


( C ) Western blot demonstrating polyubiquitination of uS10 : Affinity - tagged Hel2 - Flag - TEV - ProteinA ( FTP ) was co - purified with ribosomes harbouring HA - tagged wild type uS10 ( uS10 - 3HA ribosomes ) or uS10 mutated in its ubiquitination sites ( K6 / 8R ) . Western blots of whole extracts were performed using an anti - HA antibody to detect polyubiquitinated uS10 - 3HA .

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(C) Western blot analysis demonstrating polyubiquitination of uS10: Affinity-tagged Hel2-Flag-TEV-ProteinA (FTP) was co-purified with ribosomes harbouring HA-tagged wild type uS10 (uS10-3HA ribosomes) or uS10 mutated in its ubiquitination sites (K6/8R). Western blots of whole protein extracts were performed using an anti-HA antibody to detect polyubiquitinated uS10-3HA.


( D ) Western blot showing that uS10 - polyubiquitination in Hel2 - bound ribosomes was mainly K63 - linked : Affinity tagged Hel2 ( Hel2 - ProteinA - TEV - His6 ; PTH - Hel2 ) was co - purified with ribosomes from a expressing wild type ubiquitin ( UB - WT ) or K63R mutant ubiquitin ( ub - K63R ) HA - tagged uS10 ( uS10 - 3HA ) . Western blot of whole extracts was performed using anti - HA antibody .

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(D) Western blot analysis showing that uS10-polyubiquitination in Hel2-bound ribosomes was mainly K63-linked: Affinity tagged Hel2(Hel2-ProteinA-TEV-His6; PTH-Hel2) was co-purified with ribosomes from a strain expressing wild type ubiquitin (UB-WT) or K63R mutant ubiquitin (ub-K63R) and HA-tagged uS10 (uS10-3HA). Western blot analysis of whole protein extracts was performed using anti-HA antibody.


( E ) Western blot of in vitro polyubiquitination assays of uS10 . Reactions were performed with indicated components including His - tagged ubiquitin several ubiquitin mutants . Polyubiquitinated HA - tagged uS10 ( uS10 - 3HA ) was detected by Western blot using an anti - HA antibody .

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(E) Western blot of in vitro polyubiquitination assays of uS10. Reactions were performed with the indicated components including His-tagged ubiquitin and several ubiquitin mutants. Polyubiquitinated HA-tagged uS10 (uS10-3HA) was detected by Western blot analysis using an anti-HA antibody.


( F - H ) Northern blot of NGD - cleavage sites in absence of uS10 ubiquitination or RQT components : full - GFP - R ( CGN ) 12 - FLAG - HIS3 mRNA 5 ' NGD - intermediates ( 5 ' NGD - IM ) or 3 ' NGD - intermediates ( 3 ' NGD - IM ) were detected in indicated mutant by Northern blotting with DIG - labelled probes . 5 ' NGD - intermediates were detected by DIG - labelled GFP probe 3 ' NGD - intermediates were detected by DIG - labelled HIS3 probe . SCR1 was used as loading control . FL = full - . Note upstream shift of NGD cleavage sites in lanes F4 , G4 , H6 , H8 .

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(F-H) Northern blot analysis of NGD-cleavage sites in the absence of uS10 ubiquitination or RQT complex components: The full-length GFP-R(CGN)12-FLAG-HIS3 mRNA and 5' NGD-intermediates (5' NGD-IM) or 3' NGD-intermediates (3' NGD-IM) were detected in the indicated mutant cells by Northern blotting with DIG-labelled probes. 5' NGD-intermediates were detected by DIG-labelled GFP probe and 3' NGD-intermediates were detected by the DIG-labelled HIS3 probe. SCR1 was used as loading control. FL = full-length. Note the upstream shift of NGD cleavage sites in lanes F4, G4, H6, and H8.


( I ) Mapping of 5 ' ends of 3 ' NGD - intermediates as demonstrated in Fig 1E . Non - specific reverse transcription ( ReTr ) products are indicated by asterisks . Note that both uS10 ubiquitination Slh1 / Rqt2 were required for an endonucleolytic cleavage within road - blocked ribosome ( X1 - X4 ) .

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(I) Mapping of 5' ends of 3' NGD-intermediates as demonstrated in Fig 1E. Non-specific reverse transcription (ReTr) products are indicated by asterisks. Note that both uS10 ubiquitination and Slh1/Rqt2 were required for an endonucleolytic cleavage within the road-blocked ribosome (X1-X4).


Both RQC NGDRQC + are triggered by a ( CGA - CCG ) dicodon containing arrest in vivo . ( B ) Western blot showing arrest products derived from GFP - X - FLAG - HIS3 reporter . Translation products were detected by western blot using an anti - GFP antibody .

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Both RQC and NGDRQC+ are triggered by a (CGA-CCG) dicodon containing arrest sequence in vivo. (B) Western blot showing the arrest products derived from the GFP-X-FLAG-HIS3 reporter. Translation products were detected by western blot using an anti-GFP antibody.


Both RQC NGDRQC + are triggered by a ( CGA - CCG ) dicodon containing arrest in vivo . ( C ) Northern blot for 5 ' NGD - IM derived from ( CGA - CCG ) reporter in ski2 ∆ . 5 ' - NGD - IMs were detected with a DIG - labelled GFP probe . SCR1 was used as a load control .

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Both RQC and NGDRQC+ are triggered by a (CGA-CCG) dicodon containing arrest sequence in vivo. (C) Northern blot for the 5' NGD-IM derived from the (CGA-CCG) reporter in ski2∆ cells. 5'-NGD-IMs were detected with a DIG-labelled GFP probe. SCR1 was used as a load control.


( D ) Western blot of translations using ( CGA - CCG ) dicodon stalling mRNA reporter shown in ( A ) . mRNA reporter was added to a yeast in vitro translation extract obtained from a ski2 ∆ uS10 - 3HA . Expression of translation products ( free His - V5 tagged truncated uL4 same attached to tRNA ) was visualized with an anti - V5 antibody .

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(D) Western blot of test translations using the (CGA-CCG) dicodon stalling mRNA reporter shown in (A). The mRNA reporter was added to a yeast in vitro translation extract obtained from a ski2∆uS10-3HA strain. Expression of the translation products (free His- and V5 tagged truncated uL4 protein and the same protein attached to tRNA) was visualized with an anti-V5 antibody.


( E ) Sucrose gradient fractions ( top ) western blot ( bottom ) of ( CGA - CCG ) dicodon - stalled RNC samples . After translation reaction , RNCs were affinity - purified as indicated in ( C ) . eluate was loaded on a 10 - 50 % sucrose gradient fractionated . Each collected fraction was analysed using anti - HA antibody to detect uS10 - HA . Note that disomes are preferentially polyubiquitinated over monosomes .

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(E) Sucrose gradient fractions (top) and western blot analysis (bottom) of the (CGA-CCG) dicodon-stalled RNC samples. After the translation reaction, the RNCs were affinity-purified as indicated in (C). The eluate was loaded on a 10-50 % sucrose gradient and fractionated. Each collected fraction was analysed using anti-HA antibody to detect uS10-HA. Note that disomes are preferentially polyubiquitinated over monosomes.


( A ) Western blot after overexpression of Hel2 in strains expressing HA - tagged ribosomal using an anti - HA antibody . Note increase of polyubiquitinated eS7 as well as uS10 uS3 .

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(A) Western blot analysis after overexpression of Hel2 in strains expressing HA-tagged ribosomal proteins using an anti-HA antibody. Note the increase of the polyubiquitinated eS7 as well as uS10 and uS3.


( C ) Western blot showing role of Hel2 Not4 in eS7A ubiquitination : Both copies of eS7 ( eS7A eS7B ) were deleted transformed with a plasmid containing HA - tagged eS7A ( eS7 - WT ) or eS7A mutated in four potential ubiquitination sites ( 4KR ) . Whole extracts we obtained from eS7AΔeS7BΔ or with an additional deletion in E3 ligases Hel2 Not4 ( hel2Δ not4Δ ) . Note that four mutated lysine residues were responsible for Not4 - dependent monoubiquitination .

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(C) Western blot analysis showing the role of Hel2 and Not4 in eS7A ubiquitination: Both copies of eS7 (eS7A and eS7B) were deleted and transformed with a plasmid containing HA-tagged eS7A (eS7-WT) or eS7A mutated in the four potential ubiquitination sites (4KR). Whole protein extracts we obtained from eS7AΔeS7BΔ cells or cells with an additional deletion in E3 ligases Hel2 and Not4 (hel2Δ and not4Δ). Note that the four mutated lysine residues were responsible for Not4-dependent monoubiquitination.


( D ) Western blot showing that Not4 was required for Hel2 - mediated polyubiquitination of eS7 in Hel2 - bound ribosomal complexes : expressing HA - tagged eS7A or eS7 - 4KR mutant , as well as PTH - tagged Hel2 in eS7AΔeS7BΔ eS7AΔeS7BΔnot4Δ background were probed for eS7 - ubiquitination . Either lysates or affinity - purified Hel2 - ribosome complexes were used . of ubiquitinated eS7A in lysates in affinity - purified samples with PTH - Hel2 were determined by Western blot using an anti - HA antibody to detect eS7 .

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(D) Western blot showing that Not4 was required for Hel2-mediated polyubiquitination of eS7 in Hel2-bound ribosomal complexes: Cells expressing HA-tagged eS7A or the eS7-4KR mutant, as well as PTH-tagged Hel2 in eS7AΔeS7BΔ and eS7AΔeS7BΔnot4Δ background were probed for eS7-ubiquitination. Either cell lysates or affinity-purified Hel2-ribosome complexes were used. The levels of the ubiquitinated eS7A in the lysates and in the affinity-purified samples with PTH-Hel2 were determined by Western blot analysis using an anti-HA antibody to detect eS7.


( E ) Western blotting of in vitro ubiquitination assays of eS7A . reactions were performed with indicated components including His - tagged ubiquitin . Ribosomes were purified from Hel2Δ expressing HA - tagged eS7A . Polyubiquitinated HA - tagged eS7A ( eS7A - 3HA ) was detected by Western blot using an anti - HA antibody . We observed Hel2 - mediated polyubiquitination of monoubiquitinated eS7A .

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(E) Western blotting of in vitro ubiquitination assays of eS7A. The reactions were performed with the indicated components including His-tagged ubiquitin. Ribosomes were purified from Hel2Δ cells expressing HA-tagged eS7A. Polyubiquitinated HA-tagged eS7A (eS7A-3HA) was detected by Western blot analysis using an anti-HA antibody. We observed Hel2-mediated polyubiquitination of the monoubiquitinated eS7A.


( F ) Western blot of eS7A in vitro ubiquitination assays showing that Hel2 - mediated polyubiquitination of eS7 was mainly K63 - linked required Not4 - dependent monoubiquitination : These assays were performed similarly as described in ( E ) except yeast strains were lacking Not4 ( not4Δ ) . Reactions were performed with indicated components including His - tagged ubiquitin several ubiquitin mutants eS7A - ubiquitination was monitored using an anti - HA antibody .

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(F) Western blot analysis of eS7A in vitro ubiquitination assays showing that Hel2-mediated polyubiquitination of eS7 was mainly K63-linked and required Not4-dependent monoubiquitination: These assays were performed similarly as described in (E) except yeast strains were lacking Not4 (not4Δ). Reactions were performed with the indicated components including His-tagged ubiquitin and several ubiquitin mutants and eS7A-ubiquitination was monitored using an anti-HA antibody.


( A - C ) Northern blots probing for 5 ' NGD - IM in mutant expressing R ( CGN ) 12 reporter . ( A ) 5 ' NGD - IM detection in not4 ∆ ski2 ∆ , hel2 ∆ ski2 ∆ , slh1 ∆ ski2 ∆ , not4 ∆ slh1 ∆ ski2 ∆ . of full 5 ' NGD - IM relative to loading control ( SCR1 ) is given below . Note difference of 5 ' - NGD IMs resulting from NGDRQC + NGDRQC - that shorter 5 ' - NGD intermediate ( representing intermediates of NGDRQC - pathway ) was reduced in not4 ∆ slh1 ∆ ski2 ∆ mutant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O

(A-C) Northern blots probing for the 5' NGD-IM in mutant cells expressing the R(CGN)12 reporter. (A) 5' NGD-IM detection in not4∆ski2∆, hel2∆ski2∆, slh1∆ski2∆, not4∆ slh1∆ski2∆. Quantification of full length and the 5'NGD-IM relative to the loading control (SCR1) is given below. Note the size difference of 5'-NGD IMs resulting from NGDRQC+ and NGDRQC- and that the shorter 5'-NGD intermediate (representing intermediates of the NGDRQC- pathway) was reduced in not4∆slh1∆ski2∆ mutant cells.


Northern blots probing for 5 ' NGD - IM in mutant expressing R ( CGN ) 12 reporter . ( B ) Northern blot as in ( A ) except uS10 - WTski2 ∆ or uS10 - K6 / 8Rski2 ∆ mutant with or without a deletion of Not4 ( not4 ∆ ) were used . Note that shorter 5 ' - NGD intermediates are also reduced in ski2 ∆ not4 ∆ uS10 - K6 / 8R .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O

Northern blots probing for the 5' NGD-IM in mutant cells expressing the R(CGN)12 reporter. (B) Northern blot as in (A) except uS10-WTski2∆ or uS10-K6/8Rski2∆ mutant cells with or without a deletion of Not4 (not4∆) were used. Note that the shorter 5'-NGD intermediates are also reduced in ski2∆not4∆uS10-K6/8R cells.


Northern blots probing for 5 ' NGD - IM in mutant expressing R ( CGN ) 12 reporter . ( C ) expressing HA - tagged eS7A or eS7A - 4KR mutant in a ski2ΔeS7AΔeS7BΔ or ski2ΔeS7AΔeS7BΔslh1Δ backgrounds were probed for 5 ' - NGD intermediates by Northern blotting . Note that four mutated lysine residues in eS7 were responsible for NGDRQC - in R ( CGN ) 12 reporter .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

Northern blots probing for the 5' NGD-IM in mutant cells expressing the R(CGN)12 reporter. (C) Cells expressing HA-tagged eS7A or the eS7A-4KR mutant in a ski2ΔeS7AΔeS7BΔ or ski2ΔeS7AΔeS7BΔslh1Δ backgrounds were probed for 5'-NGD intermediates by Northern blotting. Note that the four mutated lysine residues in eS7 were responsible for NGDRQC- in the R(CGN)12 reporter.


( D ) Western blot showing that K83 K84 of eS7A are main target sites for Hel2 Not4 - mediated ubiquitination : Similarly , as in Fig 6C , eS7AΔeS7BΔ mutant were used wild type single mutants of HA - tagged eS7A were expressed . Ubiquitinated eS7 - HA was monitored using an anti - HA antibody .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O

(D) Western blot analysis showing that K83 and K84 of eS7A are main target sites for Hel2 and Not4-mediated ubiquitination: Similarly, as in Fig 6C, eS7AΔeS7BΔ mutant cells were used and wild type and single mutants of HA-tagged eS7A were expressed. Ubiquitinated eS7-HA was monitored using an anti-HA antibody.


( E ) Northern Blot probing for of 5 ' NGD - IM derived from R ( CGN ) 12 reporter expressed in eS7AΔeS7BΔski2Δ HA - tagged eS7 or eS7 mutants . We observed , that ubiquitination at K83 or K84 of eS7A is mainly responsible for NGDRQC - .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

(E) Northern Blot analysis probing for the levels of 5' NGD-IM derived from the R(CGN)12 reporter expressed in eS7AΔeS7BΔski2Δ cells and HA-tagged eS7 or eS7 mutants. We observed, that ubiquitination at K83 or K84 of eS7A is mainly responsible for NGDRQC-.


( F ) of polyubiquitinated eS7A were increased by overproduction of wild - type Hel2 ( FL ) but neither Hel2ΔRING nor Hel2 ( 1 - 315 ) mutants . eS7A - 3HAhel2Δ mutant harbouring indicated plasmids expressing wild - type Hel2 ( FL ) , Hel2ΔRING or Hel2 ( 1 - 315 ) mutant were harvested . samples were analysed by Western blotting with an anti - HA ( Top panel ) or anti - FLAG antibody ( Bottom panel ) .

O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) The levels of polyubiquitinated eS7A were increased by the overproduction of wild-type Hel2 (FL) but neither Hel2ΔRING nor Hel2(1-315) mutants. eS7A-3HAhel2Δ mutant cells harbouring the indicated plasmids expressing wild-type Hel2 (FL), Hel2ΔRING or Hel2(1-315) mutant proteins were harvested. Protein samples were analysed by Western blotting with an anti-HA (Top panel) or anti-FLAG antibody (Bottom panel).


( G ) Hel2 ( 1 - 315 ) polyubiquitinates eS7A but not uS10 . Western blotting after an in vitro ubiquitination using Hel2 - ( 1 - 315 ) mutant purified ribosomes containing HA - tagged uS10 .

O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O

(G) Hel2(1-315) polyubiquitinates eS7A but not uS10. Western blotting after an in vitro ubiquitination assay using the Hel2-(1-315) mutant and purified ribosomes containing HA-tagged uS10.


M - O : Schematic describing measurement of a ' s approach angle to nearest point on AER ( M ) . Merged , immunofluorescent of WT ( top ) , slit3sq49 ( centre ) s1pr2te273 ( below ) mesenchymal in sqet37Et background stained for EGFP ( green ) , Gamma tubulin ( red ) DAPI ( blue ) . lines run from centre of nucleus to nearest point on AER , through MTOC ( N ) . Graph depicting approach angles to AER of leading ( Tier 1 ) , following ( Tier 2 ) trailing ( Tier 3 ) of WT , slit3sq49 s1pr2te273 ( O ) . A minimum of 30 were measured for each tier of each genotype . * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 001 ; ANOVA with Fisher ' s post - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

M-O: Schematic describing the measurement of a cell's approach angle to the nearest point on the AER (M). Merged, immunofluorescent images of WT (top), slit3sq49 (centre) and s1pr2te273 (below) mesenchymal cells in sqet37Et background stained for EGFP (green), Gamma tubulin (red) and DAPI (blue). White lines run from the centre of nucleus to the nearest point on the AER, through the MTOC (N). Graph depicting the approach angles to AER of leading (Tier 1), following (Tier 2) and trailing (Tier 3) cells of WT, slit3sq49 and s1pr2te273 embryos (O). A minimum of 30 cells were measured for each tier of each genotype. *p<0.05; **p<0.005; ***p<0.001; ANOVA with Fisher's post-test.


C - E : Lateral Nomarski of 48hpf larval fins which are WT ( C ) or slit3 + / sq49 heterozygotes ( D ) are injected with 300µM fn1a morpholino . Blisters are observed only when there is reduced Fn1a in slit3 heterozygotes ( quantified in E ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O

C-E: Lateral Nomarski images of 48hpf larval fins which are WT (C) or slit3+/sq49 heterozygotes (D) and are injected with 300µM fn1a morpholino. Blisters are observed only when there is reduced Fn1a in slit3 heterozygotes (quantified in E).


F - G : Lateral Nomarski of 48hpf WT larval fins injected with 200µM gna13b alone ( F ) or with 300µM fn1a morpholino ( G ) . H : of proportion of larvae with fin blisters when low amounts of gna13 morpholinos ( 125µM each gna13a gna13b MO combined or 200µm gna13b MO alone ) are injected into WT , fn1a morphants ( 300µM MO ) , fn1a + / - heterozygotes fn1a - / - mutants . Loss of a single or both copies of fn1a exacerbates reduced gna13 , as does knockdown of fn1a .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O

F-G: Lateral Nomarski images of 48hpf WT larval fins injected with 200µM gna13b alone (F) or with 300µM fn1a morpholino (G). H: Quantification of the proportion of larvae with fin blisters when low amounts of gna13 morpholinos (125µM each gna13a and gna13b MO combined or 200µm gna13b MO alone) are injected into WT, fn1a morphants (300µM MO), fn1a+/- heterozygotes and fn1a-/- mutants. Loss of a single or both copies of fn1a exacerbates reduced gna13 levels, as does knockdown of fn1a.


Immunoblot analyses of TDG TDG - SUMO in whole extracts of mESCs MEFs . TDG TDG - SUMO conjugates before ( 2i , 0 h ) during differentiation of TDG wildtype ( TDGwt ) TDG deficient ( TDGnull ) mESC MEFs ( 5 µM - trans retinoic acid ( RA , 24 h , 48 h ) . Percentages of TDG - SUMO relative to total TDG signal are indicated .

O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O

Immunoblot analyses of TDG and TDG-SUMO in whole cell extracts of mESCs and MEFs. TDG and TDG-SUMO conjugates before (2i, 0 h) and during differentiation of TDG wildtype (TDGwt) and TDG deficient (TDGnull) mESC and MEFs (5 µM all-trans retinoic acid (RA, 24 h, 48 h). Percentages of TDG-SUMO relative to total TDG signal are indicated.


Immunoblot analyses of TDG , TDG - SUMO in whole extracts of mESCs TDG TDG - SUMO before after H2O2 treatment ( 10 mM , 15 min ) .

O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O

Immunoblot analyses of TDG, TDG-SUMO in whole cell extracts of mESCs TDG and TDG-SUMO before and after H2O2 treatment (10 mM, 15 min).


Immunoblot analyses of TDG TDG - SUMO in whole extracts of mESCs fractionation from mESCs expressing wildtype ( ESC - TDGwt ) or SUMOylation - deficient TDG ( ESC - TDGsnm ) . Sol , unbound ; Chr1 , associated ; Chr2 , bound fraction ; Histone H2B , stably bound marker .

O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O

Immunoblot analyses of TDG and TDG-SUMO in whole cell extracts of mESCs Chromatin fractionation from mESCs expressing wildtype (ESC-TDGwt) or SUMOylation-deficient TDG (ESC-TDGsnm). Sol, chromatin unbound proteins; Chr1, chromatin associated proteins; Chr2, chromatin bound fraction; Histone H2B, stably chromatin bound marker.


Immunoblot analyses of XRCC1 , XRCC1 - SUMO , in whole extracts of mESCs MEFs . XRCC1 XRCC1 - SUMO conjugates before ( 2i , 0 h ) during RA - induced differentiation ( 24 h , 48 h ) in mESCs MEFs . Percentages of XRCC1 - SUMO relative to total XRCC1 signal are indicated .

O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O

Immunoblot analyses of XRCC1 and , XRCC1-SUMO, in whole cell extracts of mESCs and MEFs. XRCC1 and XRCC1-SUMO conjugates before (2i, 0 h) and during RA-induced differentiation (24 h, 48 h) in mESCs and MEFs. Percentages of XRCC1-SUMO relative to total XRCC1 signal are indicated.


Immunoblot analyses of XRCC1 XRCC1 - SUMO , in whole extracts of mESCs XRCC1 SUMOylation in native whole extracts before after incubation with purified SUMO protease ( SENP2364 - 489 ) .

O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O

Immunoblot analyses of XRCC1 and XRCC1-SUMO, in whole cell extracts of mESCs XRCC1 SUMOylation in native whole cells extracts before and after incubation with purified SUMO protease (SENP2364-489).


Immunoblot analyses of , XRCC1 XRCC1 - SUMO in whole extracts of mESCs XRCC1 SUMOylation before after exposure to H2O2 ( 5 mM , 15 min ) .

O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O

Immunoblot analyses of , XRCC1 and XRCC1-SUMO in whole cell extracts of mESCs XRCC1 SUMOylation before and after exposure to H2O2 (5 mM, 15 min).


Immunoblot analyses of TET1 isoforms in whole extracts of mESCs MEFs . TET1 TET1s isoform in mESCs during RA induced differentiation in MEFs .

O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O

Immunoblot analyses of TET1 isoforms in whole cell extracts of mESCs and MEFs. TET1 and TET1s isoform protein levels in mESCs during RA induced differentiation and in MEFs.


microscopy of GFP - TDGwt GFP - TDGpip in mESCs . Scale bar : 100µM . Quantitation of signals of GFP - TDGwt GFP - TDGpip in mESCs . median with interquartile range . > 470 were analysed of each GFPTDGwt GFPTDGpip ESCs .

O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

Fluorescence microscopy of GFP-TDGwt GFP-TDGpip in mESCs. Scale bar: 100µM. Quantitation of fluorescence signals of GFP-TDGwt and GFP-TDGpip in mESCs. median with interquartile range. >470 cells were analysed of each GFPTDGwt and GFPTDGpip ESCs.


Immunoblot of XRCC1 XRCC1 - SUMO in GFP - TDGwt GFP - TDGpip mESCs .

O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O

Immunoblot analysis of XRCC1 and XRCC1-SUMO in GFP-TDGwt and GFP-TDGpip mESCs.


" Far Western " blot analyses of BER interactions . Recombinant purified human TDG ( 10 µg ) , POLß ( 1 µg ) , APE1 ( 1 µg ) , LIGIII ( 1 µg ) probed with XRCC1 XRCC1 - SUMO ( 20 µg ) immune - stained for XRCC1 .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O

"Far Western" blot analyses of BER protein interactions. Recombinant purified human TDG (10 µg), POLß (1 µg), APE1 (1 µg), and LIGIII (1 µg) probed with XRCC1 and XRCC1-SUMO (20 µg) and immune-stained for XRCC1.


Immunoblot of interactions of XRCC1 XRCC1 - SUMO with endogenous TDG . Purified 6hisXRCC1 or in vitro modified purified 6hisXRCC1 - SUMO was coupled to Ni - NTA agarose incubated with mESC extracts . Input ( I ) , last wash ( LW ) elution ( E ) fractions .

O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot analysis of interactions of XRCC1 and XRCC1-SUMO with endogenous TDG. Purified 6hisXRCC1 or in vitro modified and purified 6hisXRCC1-SUMO was coupled to Ni-NTA agarose and incubated with mESC extracts. Input (I), last wash (LW) and elution (E) fractions.


0 . 5 µM TDGwt , were incubated with biotinylated 5caC substrate ( G • 5caC , 1 µM ) homoduplex competitor ( G • C , 10 µM ) equimolar amounts of XRCC1 , POLβ LIGIII .

O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O

0.5 µM TDGwt, were incubated with biotinylated 5caC substrate (G•5caC DNA, 1 µM) and homoduplex competitor DNA (G•C DNA, 10 µM) and equimolar amounts of XRCC1, POLβ and LIGIII proteins.


Schematic of biotin - 5caC substrate pulldown experiment . 0 . 5 µM TDGwt or TDGcat ( TDG ( N140A ) ) incubated with biotinylated 5caC substrate ( G • 5caC , 1 µM ) homoduplex competitor ( G • C , 10 µM ) as indicated equimolar amounts of XRCC1 - SUMO ( > 50 % SUMOylated ) , POLβ LIGIII .

O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O

Schematic of biotin-5caC DNA substrate pulldown experiment. 0.5 µM TDGwt or TDGcat (TDG(N140A)) incubated with biotinylated 5caC substrate (G•5caC DNA, 1 µM) and homoduplex competitor DNA (G•C DNA, 10 µM) as indicated and equimolar amounts of XRCC1-SUMO (>50% SUMOylated), POLβ and LIGIII.


1 µM biotinylated 5caC substrate ( G • 5caC ) 10 µM homoduplex competitor ( G • C ) were incubated with XRCC1 - SUMO ( 50 % SUMOylated ) BER as indicated .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O

1 µM biotinylated 5caC substrate (G•5caC DNA) and 10 µM homoduplex competitor DNA (G•C DNA) were incubated with XRCC1-SUMO (50% SUMOylated) and BER proteins as indicated.


0 . 5 µM human TDGwt were incubated with 1 µM biotinylated 5caC substrate ( G • 5caC ) , 0 . 5 µM SUMO 10 µM homoduplex competitor ( G • C ) as indicated . Shown is an immunoblot of TDG free SUMO .

O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O

0.5 µM human TDGwt protein were incubated with 1 µM biotinylated 5caC substrate (G•5caC DNA), 0.5 µM SUMO and 10 µM homoduplex competitor DNA (G•C DNA) as indicated. Shown is an immunoblot analysis of TDG and free SUMO.


" Far Western " blot analyses of TDGwt TDGE310Q with 6hisXRCC1 6hisXRCC1 - SUMO . TDGwt or TDGE310Q was dot blotted on nitrocellulose membranes probed with XRCC1 or XRCC1 - SUMO ( probe : XRCC1 or XRCC1SUMO ) where indicated . TDG XRCC1 probes were detected by immunoblotting with anti TDG anti XRCC1 antibodies as indicated .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O

"Far Western" blot analyses of TDGwt and TDGE310Q with 6hisXRCC1 and 6hisXRCC1-SUMO. TDGwt or TDGE310Q was dot blotted on nitrocellulose membranes and probed with XRCC1 or XRCC1-SUMO (probe:XRCC1 or XRCC1SUMO) where indicated. TDG and XRCC1 probes were detected by immunoblotting with anti TDG and anti XRCC1 antibodies as indicated.


Schematic of biotin - 5caC substrate pulldown experiment . Immunoblot of purified TDGwt or TDG SUMO - binding mutant ( TDGE310Q ) , 6hisXRCC1 / XRCC1 - SUMO 6his BER bound to Streptavidin ( St ) Biotin - 5caC substrate . 0 . 5 µM TDGwt or TDG SUMO - binding mutant ( TDGE310Q ) were incubated with biotinylated 5caC substrate ( G • 5caC , 1 µM ) homoduplex competitor ( G • C , 10 µM ) as indicated equimolar amounts of XRCC1 , POLβ , APE1 LIGIII . Grey ovals , BERosome .

O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O

Schematic of biotin-5caC DNA substrate pulldown experiment. Immunoblot analysis of purified TDGwt or TDG SUMO-binding mutant (TDGE310Q), 6hisXRCC1/XRCC1-SUMO and 6his BER proteins bound to Streptavidin (St) Biotin-5caC DNA substrate. 0.5 µM TDGwt or TDG SUMO-binding mutant (TDGE310Q) were incubated with biotinylated 5caC substrate (G•5caC DNA, 1 µM) and homoduplex competitor DNA (G•C DNA, 10 µM) as indicated and equimolar amounts of XRCC1, POLβ, APE1 and LIGIII proteins. Grey ovals, BERosome.


Reconstitution SUMO conjugation / de - conjugation reaction with purified SAE1 / 2 , UBC9 , SUMO protease SENP2364 - 489 ) , free SUMO in equimolar amounts 20 - fold molar excess over TDG . TDG SUMOylation was performed in presence or absence of AP - site containing synthetic double - strands as ( as indicated ( G • AP ) . TDG TDG - SUMO were detected by immunoblotting . SUMOylation of ( G • AP ) bound TDG is strongly reduced but a 20 - fold molar excess of free SUMO compensates inhibitory effect of .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O

Reconstitution SUMO conjugation/de-conjugation reaction with purified SAE1/2, UBC9, SUMO protease SENP2364-489), free SUMO in equimolar amounts and 20-fold molar excess over TDG. TDG SUMOylation was performed in the presence or absence of AP-site containing synthetic DNA double-strands as (as indicated (G•AP). TDG and TDG-SUMO were detected by immunoblotting. SUMOylation of DNA (G•AP) bound TDG is strongly reduced but a 20-fold molar excess of free SUMO compensates the inhibitory effect of DNA.


Reconstitution SUMO conjugation / de - conjugation reaction with purified SAE1 / 2 , UBC9 , SUMO protease SENP2364 - 489 ) , free SUMO in equimolar amounts 20 - fold molar excess over TDG . TDG SUMOylation was performed in presence or absence of AP - site containing synthetic double - strands as ( as indicated ( G • AP ) . TDG TDG - SUMO were detected by immunoblotting . SUMOylation of ( G • AP ) bound TDG is strongly reduced but a 20 - fold molar excess of free SUMO compensates inhibitory effect of . SUMOylation as in ( A ) with SUMO provided as XRCC1 - SUMO instead of free SUMO .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O

Reconstitution SUMO conjugation/de-conjugation reaction with purified SAE1/2, UBC9, SUMO protease SENP2364-489), free SUMO in equimolar amounts and 20-fold molar excess over TDG. TDG SUMOylation was performed in the presence or absence of AP-site containing synthetic DNA double-strands as (as indicated (G•AP). TDG and TDG-SUMO were detected by immunoblotting. SUMOylation of DNA (G•AP) bound TDG is strongly reduced but a 20-fold molar excess of free SUMO compensates the inhibitory effect of DNA. SUMOylation assay as in (A) with SUMO provided as XRCC1-SUMO instead of free SUMO.


Reconstitution SUMO conjugation / de - conjugation reaction with purified SAE1 / 2 , UBC9 , SUMO protease SENP2364 - 489 ) , free SUMO in equimolar amounts 20 - fold molar excess over TDG . TDG SUMOylation was performed in presence or absence of AP - site containing synthetic double - strands as ( as indicated ( G • AP ) . TDG TDG - SUMO were detected by immunoblotting . SUMOylation of ( G • AP ) bound TDG is strongly reduced but a 20 - fold molar excess of free SUMO compensates inhibitory effect of . SUMO transfer as depicted in ( B ) with additional APE1 , POLß , LIGIII , TDG .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-MEASURED_VAR O

Reconstitution SUMO conjugation/de-conjugation reaction with purified SAE1/2, UBC9, SUMO protease SENP2364-489), free SUMO in equimolar amounts and 20-fold molar excess over TDG. TDG SUMOylation was performed in the presence or absence of AP-site containing synthetic DNA double-strands as (as indicated (G•AP). TDG and TDG-SUMO were detected by immunoblotting. SUMOylation of DNA (G•AP) bound TDG is strongly reduced but a 20-fold molar excess of free SUMO compensates the inhibitory effect of DNA. SUMO transfer as depicted in (B) with additional APE1, POLß, LIGIII, and TDG.


Immunoblot of XRCC1 , TDG , TDG - SUMO in TDGwt ( wt ) , TDGsnm ( snm ) XRCC1null mESCs ( null ) .

O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O

Immunoblot analysis of XRCC1, TDG, TDG-SUMO in TDGwt (wt), TDGsnm (snm) and XRCC1null mESCs (null).


Immunoblot of TDG TDG - SUMO in TDGwt ( wt ) XRCC1null mESCs ( null ) exposed to H2O2 as indicated .

O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O

Immunoblot analysis of TDG and TDG-SUMO in TDGwt (wt) and XRCC1null mESCs (null) exposed to H2O2 as indicated.


Top : Schematic of SUMO - mediated TDG - BERosome ( grey oval ) formation processing . Bottom : Reconstitution of active demethylation on a 5caC containing synthetic double - strands in presence of dynamic SUMO - conjugation / de - conjugation . Left panels lanes 3 - 14 : BER reconstitution with XRCC1 or XRCC1 - SUMO as indicated . Right panels lanes 15 - 21 : BER reconstitution with free SUMO or XRCC1 - SUMO as SUMO donor as indicated . ds59merDNA substrates : lane 1 - 2 CG / CG HpaII methylation sensitive restriction site , cleavable by HpaII ; lane 3 - 6 , 8 - 14 , 15 - 17 , 21 HpaII methylation sensitive restriction site restriction site with a caC ( 5caCG / CG ) , not cleavable by HpaII ; lane 7 20 repaired Product ( CC / CG ) that is cleavable by HpaII after excision / repair of caC / CG to CG / CG by BER . of total signal of fully repaired product is indicated . ss59merDNA , single stranded .

O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Top: Schematic of the SUMO-mediated TDG-BERosome (grey oval) formation and DNA processing. Bottom: Reconstitution of active DNA demethylation on a 5caC containing synthetic DNA double-strands in presence of dynamic SUMO-conjugation/de-conjugation. Left panels lanes 3-14: BER reconstitution with XRCC1 or XRCC1-SUMO as indicated. Right panels lanes 15-21: BER reconstitution with free SUMO or XRCC1-SUMO as SUMO donor as indicated. ds59merDNA substrates: lane 1-2 CG/CG HpaII methylation sensitive restriction site, cleavable by HpaII; lane 3-6, 8-14, 15-17, 21 HpaII methylation sensitive restriction site restriction site with a caC (5caCG/CG), not cleavable by HpaII; lane 7 and 20 repaired Product (CC/CG) that is cleavable by HpaII after excision/repair of caC/CG to CG/CG by BER. Percentage of total signal of fully repaired product is indicated. ss59merDNA, single stranded DNA.


Bright field captures of TDGwt , TDGcat TDGsnm mESCs at indicated stages of differentiation . DAPI , Alexa Fluor ® 680 phalloidin actin at bottom . Scale bars 25µM , 50µM , 500µM as indicated . of neural with neurites of total differentiating TDGwt , TDGcat TDGsnm mESCs . n ≥ 5 . Means with standard deviation ( SD ) . * * : P < 0 . 01 . Non parametric , Mann - Whitney .

O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bright field captures of TDGwt, TDGcat and TDGsnm mESCs at indicated stages of differentiation. DAPI, Alexa Fluor® 680 phalloidin actin staining at the bottom. Scale bars 25µM, 50µM, 500µM as indicated. Quantification of neural cells with neurites of total differentiating TDGwt, TDGcat and TDGsnm mESCs. n ≥ 5. Means with standard deviation (SD). **: P<0.01. Non parametric, Mann-Whitney test.


Relative changes of lineage marker expression between NPCs undifferentiated mESCs ( ESCM LIF , 0 h ) , quantified with Gapdh as normalizer . n ≥ 3 . Means with standard deviation ( SD ) ) . * : P < 0 . 05 ; * * : P < 0 . 01 by non parametric Mann - Whitney .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Relative changes of lineage marker expression between NPCs and undifferentiated mESCs (ESCM LIF, 0 h), quantified with Gapdh as normalizer. n ≥ 3. Means with standard deviation (SD)). *: P<0.05; **: P<0.01 by non parametric Mann-Whitney test.


LC - MS / MS of global cytosine - modification in from ground - state mESCs ( 2i ) EBs . Means with standard deviation ( SD ) of 5mC , 5hmC , 5fC 5caC per 106 bases ) . n ≥ 3 . * : P ≤ 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 0001 by one - way ANOVA , Dunnett ' s multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LC-MS/MS analysis of global cytosine-modification levels in DNA from ground-state mESCs (2i) and EBs. Means with standard deviation (SD) of 5mC, 5hmC, 5fC and 5caC per 106 bases). n ≥ 3. *: P≤0.05, **: P<0.01, ***: P<0.0001 by one-way ANOVA, Dunnett's multiple comparisons test.


Alkaline comet with pluripotent ( 0 h ) differentiating ( 24 h 5 µM RA ) mESC as indicated . Olive Tail Moments % in tail . n ≥ 3 . Medians with standard deviation ( SD ) . * : P ≤ 0 . 05 ; * * : P ≤ 0 . 01 , * * * : P ≤ 0 . 0001 by one - way ANOVA , Sidak ' s multiple comparisons .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Alkaline comet assay with pluripotent (0 h) and differentiating (24 h 5 µM RA) mESC as indicated. Olive Tail Moments and % DNA in tail. n ≥ 3. Medians with standard deviation (SD). *: P≤0.05; **: P≤0.01, ***: P≤0.0001 by one-way ANOVA, Sidak's multiple comparisons test.


proliferation . Relative viability of Talazoparib treated to untreated . n = 3 . Means with standard deviation ( SD ) , P ≤ 0 . 05 , * * : P < 0 . 01 , by one - way ANOVA , Dunnett ' s multiple comparisons .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cell proliferation assay. Relative viability of Talazoparib treated to untreated cells. n = 3. Means with standard deviation (SD), P≤0.05, **: P<0.01, by one-way ANOVA, Dunnett's multiple comparisons test.


ChIP - qPCR of TET1 , TDG XRCC1 at gene promoters in mESCs ( 24 h 5 µM RA ) . Relative enrichments to an unbound control region ( Chr2neg ) . n ≥ 3 . Means with standard deviation ( SD ) . * : P ≤ 0 . 05 ; * * : P < 0 . 001 ; * * * : P ≤ 0 . 0001 , by two - Mann - Whitney .

O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ChIP-qPCR analysis of TET1, TDG and XRCC1 at gene promoters in mESCs (24 h 5 µM RA). Relative enrichments to an unbound control region (Chr2neg). n ≥ 3. Means with standard deviation (SD). *: P≤0.05; **: P<0.001; ***: P≤0.0001, by two-tailed Mann-Whitney test.


Representative of mESC neuronal differentiation as in Fig 4B . Shown are XRCC1wt XRCC1null ( XRCC1null1 , XRCC1null2 ) mESCs in 2i medium supplemented with LIF ( 2i LIF ) in ESC medium supplemented with LIF ( ESCM LIF ) , EBs in ESCM LIF 5µM RA ( EB 4d ESCM RA ) dissociated neuron - like in B27 ( B27 ) . Scale bars 25µM , 50µM , 500µM as indicated . Neurite grids were observed for XRCC1wt ; heterogeneous populations formed with XRCC1null ( XRCC1null1 , null2 ) clones . ( below ) of neural with neurites of total differentiating XRCC1wt , XRCC1null mESCs . n = 5 . Means with standard deviation ( SD ) . * * : P < 0 . 01 . Non parametric , Mann - Whitney .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of mESC neuronal differentiation as in Fig 4B. Shown are XRCC1wt and XRCC1null (XRCC1null1, XRCC1null2) mESCs in 2i medium supplemented with LIF (2i LIF) and in ESC medium supplemented with LIF (ESCM LIF), EBs in ESCM LIF and 5µM RA (EB 4d ESCM RA) and dissociated neuron-like cells in B27 (B27). Scale bars 25µM, 50µM, 500µM as indicated. Neurite grids were observed for XRCC1wt; heterogeneous cell populations formed with the XRCC1null (XRCC1null1, null2) clones. Quantification (below) of neural cells with neurites of total differentiating XRCC1wt, XRCC1null mESCs. n = 5. Means with standard deviation (SD). **: P<0.01. Non parametric, Mann-Whitney test.


of mRNA expression of pluripotency ectodermal / neurogenesis marker genes by RT - qPCR . n ≥ 3 . Mean values standard deviations ( SD ) of EBs relative to their mESC ( ESCM LIF , 16h ) , normalised to Gapdh .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of mRNA expression of pluripotency and ectodermal/neurogenesis marker genes by RT-qPCR. n ≥ 3. Mean values and standard deviations (SD) of EBs relative to their mESC (ESCM LIF, 16h), normalised to Gapdh.


Growth of wild - type ( WT , Col - 0 ) , bri1 - null mutant , two independent ubp12i / ubp13 double mutant lines grown on 1 / 2MS medium supplemented with 10 μM DEX ( DEX medium ) for 10 days transferred to DEX medium containing 1 μM BL or mock solution ( 0 . 1 % [ v / v ] EtOH ) for additionally 8 days . Scale bars : 10 mm .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

Growth phenotypes of the wild-type (WT, Col-0), bri1-null mutant, and two independent ubp12i/ubp13 double mutant lines grown on 1/2MS medium supplemented with 10 μM DEX (DEX medium) for 10 days and transferred to DEX medium containing 1 μM BL or mock solution (0.1% [v/v] EtOH) for additionally 8 days. Scale bars: 10 mm.


Phenotype of WT ubp12i / ubp13 double mutant grown on DEX medium in presence of increasing concentrations of BL in for 6 days . Scale bars : 10 mm . Hypocotyl relative to mock control of seedlings ( B ) . Experiments were done in triplicate ( n > 15 seedlings for each ) .

O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Phenotype of the WT and ubp12i/ubp13 double mutant grown on DEX medium in the presence of increasing concentrations of BL in the light for 6 days. Scale bars: 10 mm.   Hypocotyl length relative to the mock control of seedlings (B). Experiments were done in triplicate (n >15 seedlings for each line).


Western blot ( WB ) of BES1 dephosphorylation after BL treatment with α - BES1 antibody . Total were isolated from 6 - day - old seedlings grown on DEX medium in , either in presence of 100 nM BL or mock . PEPC was used as a loading control . of dephosphorylated BES1 relative to total BES1 from ( D ) ( n = 6 biological replicates ) .

O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O

Western blot (WB) analysis of BES1 dephosphorylation after BL treatment with the α-BES1 antibody. Total proteins were isolated from 6-day-old seedlings grown on DEX medium in the light, either in the presence of 100 nM BL or mock. PEPC was used as a loading control.   Percentage of dephosphorylated BES1 relative to the total BES1 from (D) (n = 6 biological replicates).


Western blot of BRI1 amount in ubp12i / ubp13 with α - BRI1 antibody . Total were isolated from 18 - day - old grown on 1 / 2MS medium supplemented with 10 μM DEX ( + ) or not ( - ) . Western blot of BRI1 level in concanamycin A ( ConcA ) - treated . Eighteen - day - old were grown on DEX medium exposed to 1 μM ConcA for 4 h before total isolation .

O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O

Western blot analysis of BRI1 protein amount in ubp12i/ubp13 plants with the α-BRI1 antibody. Total proteins were isolated from 18-day-old plants grown on 1/2MS medium supplemented with 10 μM DEX (+) or not (-).   Western blot analysis of BRI1 protein level in concanamycin A (ConcA)-treated plants. Eighteen-day-old plants were grown on DEX medium and exposed to 1 μM ConcA for 4 h before total protein isolation.


Deubiquitinating activity of UBP13 against K48 - K63 - linked ubiquitination . Diubiquitins linked through K48 or K63 ( K48 Di - Ub or K63 Di - Ub ) were incubated alone or with GST - UBP13 or GST - UBP13C207S for 18 h . Deubiquitination of diubiquitins was analyzed by western blot with α - ubiquitin ( α - Ub , P4D1 ) antibody . presence of GST - UBP13 / UBP13C207S recombinant was confirmed by α - GST antibody .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O

Deubiquitinating activity of UBP13 against K48- and K63-linked ubiquitination. Diubiquitins linked through K48 or K63 (K48 Di-Ub or K63 Di-Ub) were incubated alone or with GST-UBP13 or GST-UBP13C207S for 18 h. Deubiquitination of the diubiquitins was analyzed by western blot with the α-ubiquitin (α-Ub, P4D1) antibody. The presence of GST-UBP13/UBP13C207S recombinant proteins was confirmed by the α-GST antibody.


UBP13 deubiquitination of K63 - ubiquitinated BRI1 in vitro . Polyubiquitinated MBP - BRI1CD was generated by incubation with GST - PUB13 ( Ub reaction ) then with GST - UBP13 or GST - UBP13C207S ( DUB reaction - MBP pull - down , input ) . After in vitro deubiquitination , MBP - BRI1CD was purified by an MBP pull - down to analyze BRI1CD ubiquitination ( DUB reaction - MBP pull - down , SE ) . ubiquitinated were detected by western blot with an α - ubiquitin antibody ( FK2 ) . Ubiquitin chain specificity was detected with an α - K63Ub antibody ( Apu3 ) . presence of recombinant was confirmed by α - GST α - MBP antibodies .

B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

UBP13 deubiquitination of K63-ubiquitinated BRI1 in vitro. Polyubiquitinated MBP-BRI1CD was generated by incubation with GST-PUB13 (Ub reaction) and then with GST-UBP13 or GST-UBP13C207S (DUB reaction-MBP pull-down, input). After in vitro deubiquitination, MBP-BRI1CD was purified by an MBP pull-down to analyze the BRI1CD ubiquitination (DUB reaction-MBP pull-down, SE). The ubiquitinated proteins were detected by western blot with an α-ubiquitin antibody (FK2). Ubiquitin chain specificity was detected with an α-K63Ub antibody (Apu3). The presence of recombinant proteins was confirmed by the α-GST and α-MBP antibodies.


Growth phenotype comparison of BRI1 - mCit / bri1 / ubp12i / ubp13 BRI125KR - mCit / bri1 / ubp12i / ubp13 . indicated transgenic lines were grown on DEX medium for 18 days . Scale bars : 10 mm .

O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

Growth phenotype comparison of BRI1-mCit/bri1/ubp12i/ubp13 and BRI125KR-mCit/bri1/ubp12i/ubp13. The indicated transgenic lines were grown on DEX medium for 18 days. Scale bars: 10 mm.


Western blot monitoring BRI1 accumulation in BRI125KR - mCit / bri1 / ubp12i / ubp13 with α - BRI1 antibody . Total were isolated from roots of lines indicated ( A ) . PEPC was used as a loading control .

O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis monitoring the BRI1 protein accumulation in BRI125KR-mCit/bri1/ubp12i/ubp13 plants with the α-BRI1 antibody. Total proteins were isolated from the roots of the lines indicated (A). PEPC was used as a loading control.


A - D of BRI1 vacuolar targeting on Arabidopsis seedlings expressing BRI1 ( A ) or BRI125KR ( C ) tagged with mCitrine ( mCit ) in either bri1 / UBP12 / UBP13 or bri1 / ubp12i / ubp13 background . Six - day - old seedlings were grown on DEX medium , kept in for 2 h in presence of 50 μM CHX before imaging . Confocal of epidermal from root meristem are shown . Scale bars : 10 μm . Measurements of relative PM / intracellular BRI1 - mCit . For each , at least 55 25 from over 25 15 seedlings were measured in ( B ) ( D ) , respectively .

O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-D Analysis of the BRI1 vacuolar targeting on Arabidopsis seedlings expressing BRI1 (A) or BRI125KR (C) tagged with mCitrine (mCit) in either bri1/UBP12/UBP13 or bri1/ubp12i/ubp13 background. Six-day-old seedlings were grown on DEX medium, and kept in the dark for 2 h in the presence of 50 μM CHX before imaging. Confocal images of epidermal cells from root meristem are shown. Scale bars: 10 μm. Measurements of the relative PM/intracellular BRI1-mCit fluorescence intensity. For each line, at least 55 and 25 cells from over 25 and 15 seedlings were measured in (B) and (D), respectively.


( A ) Growth of indicated with without doxycycline - inducible overexpression of PLK4 . Experiments were performed in wild - type or SAS6 monoclonal knockout . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

(A) Growth assay of the indicated cells with and without doxycycline-inducible overexpression of PLK4. Experiments were performed in wild-type or SAS6 monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( D ) Top hits that emerged from screens ranked by MaGeCK FDR value . Blue hits are genes required for centriole duplication or stability . Green hits are genes predicted to be required to arrest growth of with extra centrosomes .

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(D) Top hits that emerged from the screens ranked by MaGeCK FDR value. Blue hits are genes required for centriole duplication or stability. Green hits are genes predicted to be required to arrest the growth of cells with extra centrosomes.