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 .

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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 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 .

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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.


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 .

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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 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 .

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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 levels of IFNα were measured in a cohort of 36 patients 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 . 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 .

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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 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 .

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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 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 .

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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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

(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 . 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

(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 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 .

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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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O

(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 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 .

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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 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 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O

(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 and Slh1 / Rqt2 were required for an endonucleolytic cleavage within the 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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-EXP_ASSAY O

(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 : 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O

(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 . 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O O B-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O

(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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O O O O O O B-GENEPROD O O

(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 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 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O 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-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-GENEPROD 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 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 .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O 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 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 .

B-EXP_ASSAY I-EXP_ASSAY O 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-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O 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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD 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 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 - .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD 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 ) 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 ) .

O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O B-GENEPROD 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 assay using the Hel2 - ( 1 - 315 ) mutant and purified ribosomes containing HA - tagged uS10 .

O O O B-GENEPROD O O O O O B-EXP_ASSAY B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O B-GENEPROD 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 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 .

O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-TISSUE O O O B-SUBCELLULAR O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD 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 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 .

O O O O O O O O O O B-TISSUE I-TISSUE O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD 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 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 .

B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O B-CELL_TYPE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD 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 cell extracts of mESCs TDG and TDG - SUMO before and after H2O2 treatment ( 10 mM , 15 min ) .

B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE 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 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 .

B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE B-SUBCELLULAR O O B-CELL_TYPE O O O B-CELL_TYPE O B-GENEPROD O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR 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 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 .

B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-CELL_TYPE O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD 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 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 ) .

B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-CELL_TYPE B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O B-GENEPROD 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 and XRCC1 - SUMO in whole cell extracts of mESCs XRCC1 SUMOylation before and after exposure to H2O2 ( 5 mM , 15 min ) .

B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE B-GENEPROD B-EXP_ASSAY O O O O O B-SMALL_MOLECULE 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 cell extracts of mESCs and MEFs . TET1 and TET1s isoform protein levels in mESCs during RA induced differentiation and in MEFs .

B-EXP_ASSAY O O B-GENEPROD O O O O O O B-CELL_TYPE O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O B-SMALL_MOLECULE O O O O B-CELL_TYPE 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.


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 .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-CELL_TYPE 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 analysis of XRCC1 and XRCC1 - SUMO in GFP - TDGwt and GFP - TDGpip mESCs .

B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD B-CELL_TYPE O

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


" 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD 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 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 .

B-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-CELL_TYPE 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 DNA , 1 µM ) and homoduplex competitor DNA ( G • C DNA , 10 µM ) and equimolar amounts of XRCC1 , POLβ and LIGIII proteins .

O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O 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 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 .

O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD 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 DNA ) and 10 µM homoduplex competitor DNA ( G • C DNA ) were incubated with XRCC1 - SUMO ( 50 % SUMOylated ) and BER proteins as indicated .

O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY 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 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 .

O O O O B-ORGANISM B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD 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 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD 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 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 .

O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O 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 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 .

O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE 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 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 .

O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD 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 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 .

O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD 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 analysis of XRCC1 , TDG , TDG - SUMO in TDGwt ( wt ) , TDGsnm ( snm ) and XRCC1null mESCs ( null ) .

B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD B-CELL_TYPE O O O O

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


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

B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD B-CELL_TYPE O O O O O B-SMALL_MOLECULE 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 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 .

O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O 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-SMALL_MOLECULE 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 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 .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-CELL_TYPE O O O O O O 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 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 .

O O O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O 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 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 .

B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O O O B-SUBCELLULAR O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O 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 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 .

B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O B-SUBCELLULAR O O B-GENEPROD O O B-SMALL_MOLECULE O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O O O O O 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.


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 .

O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-TISSUE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O B-GENEPROD O O O O O O B-GENEPROD 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 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 .

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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 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 ) .

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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 ) 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 ) .

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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 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 .

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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 - 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 .

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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 ) 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 .

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

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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 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 .

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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 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 .

O O O O O O B-GENEPROD B-SUBCELLULAR O O B-ORGANISM I-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O O O 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 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 .

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(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 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 .

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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.


( G ) Top : Graph showing the efficiency of frameshifting INDELs measured using TIDE . N . D . = Not determined . Data shown is from n = 1 biological replicate . Bottom : Graph showing the relative growth of doxycycline treated PLK4Dox cells expressing an sgRNA targeting the indicated genes . Each dot displays measurements from a single experiment . Experiments were performed in polyclonal knockout cells . Data acquired across n ≥ 3 biological replicates . Mean ± s . e . m .

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(G) Top: Graph showing the efficiency of frameshifting INDELs measured using TIDE. N.D. = Not determined. Data shown is from n = 1 biological replicate. Bottom: Graph showing the relative growth of doxycycline treated PLK4Dox cells expressing an sgRNA targeting the indicated genes. Each dot displays measurements from a single experiment. Experiments were performed in polyclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m.


( H ) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes . Experiments were performed in polyclonal knockout cells . Data acquired across n ≥ 3 biological replicates . Mean ± s . e . m .

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(H) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes. Experiments were performed in polyclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m.


( A ) Top , widefield image showing the localization of CEP164 and PIDD1 at the mature - mother centriole . Bottom : Representative 3D STORM image of the same centriole showing PIDD1 localization . STORM image colors correspond to the Z - depth with red being the closest to the coverslip and blue being the most distant .

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(A) Top, widefield image showing the localization of CEP164 and PIDD1 at the mature-mother centriole. Bottom: Representative 3D STORM image of the same centriole showing PIDD1 localization. STORM image colors correspond to the Z-depth with red being the closest to the coverslip and blue being the most distant.


( B ) 3D STORM image of PIDD1 at the mature mother centriole . The overlaid mask represents the inner and outer diameters of the PIDD1 signal .

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(B) 3D STORM image of PIDD1 at the mature mother centriole. The overlaid mask represents the inner and outer diameters of the PIDD1 signal.


( C ) Inner and outer diameter measurements for distal appendage proteins and PIDD1 . Measurements in the shaded region were previously reported in ( Bowler et al . , 2019 ) . Data displayed as box and whisker plots , where the box represents the upper and lower quartile and the whiskers represent the s . d . Scale bars : 1 μm for all wide - field images of centrioles and 500 nm for STORM images . Data acquired across n ≥ 6 cells .

O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O

(C) Inner and outer diameter measurements for distal appendage proteins and PIDD1. Measurements in the shaded region were previously reported in (Bowler et al., 2019). Data displayed as box and whisker plots, where the box represents the upper and lower quartile and the whiskers represent the s.d. Scale bars: 1 μm for all wide-field images of centrioles and 500 nm for STORM images. Data acquired across n ≥6 cells.


( D ) Quantification of the fraction of cells with PIDD1 localized to the mature mother centriole in PLK4Dox cells expressing an sgRNA targeting the indicated genes . A dot displays measurements from each experiment . Experiments were performed in PLK4Dox polyclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

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(D) Quantification of the fraction of cells with PIDD1 localized to the mature mother centriole in PLK4Dox cells expressing an sgRNA targeting the indicated genes. A dot displays measurements from each experiment. Experiments were performed in PLK4Dox polyclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( F ) Graph showing the relative growth of doxycycline treated PLK4Dox cells that were knocked out for the indicated genes . Each dot displays measurements from a single experiment . Experiments were performed in PLK4Dox monoclonal knockout cells . Data acquired across n ≥ 3 biological replicates . Mean ± s . e . m .

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(F) Graph showing the relative growth of doxycycline treated PLK4Dox cells that were knocked out for the indicated genes. Each dot displays measurements from a single experiment. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m.


( G ) Quantification of the fraction of cells with PIDD1 localized at the mature mother centriole . Experiments were performed in PLK4Dox monoclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

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(G) Quantification of the fraction of cells with PIDD1 localized at the mature mother centriole. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( A ) Quantification of the fraction of cells with ANKRD26 localized at the mature mother centriole . Experiments were performed in PLK4Dox monoclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

(A) Quantification of the fraction of cells with ANKRD26 localized at the mature mother centriole. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( B - D ) Representative images of WT or ANKRD26 - / - PLK4Dox cells immunostained with the indicated antibodies . Scale bar = 5 µm .

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(B-D) Representative images of WT or ANKRD26-/- PLK4Dox cells immunostained with the indicated antibodies. Scale bar = 5 µm.


( E ) Quantification of the fraction of WT or ANKRD26 - / - hTERT RPE1 cells with cilia . Each dot displays measurements from a single experiment . Data acquired across n = 5 biological replicates . Mean ± s . e . m .

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(E) Quantification of the fraction of WT or ANKRD26-/- hTERT RPE1 cells with cilia. Each dot displays measurements from a single experiment. Data acquired across n = 5 biological replicates. Mean ± s.e.m.


( F ) Growth assay of the indicated cells with and without doxycycline - inducible overexpression of PLK4 . Experiments were performed in PLK4Dox monoclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

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(F) Growth assay of the indicated cells with and without doxycycline-inducible overexpression of PLK4. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( G ) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes . Experiments were performed in PLK4Dox monoclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

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(G) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( H ) Western blot showing expression of pro - CASP2 and P21 following treatment with dox for the specified number of days . Experiments were performed in PLK4Dox monoclonal knockout cells . ( I ) Quantification of pro - CASP2 levels following treatment with dox for the specified number of days . Experiments were performed in PLK4Dox monoclonal knockout cells . Each dot displays measurements from a single experiment . Data acquired across n = 6 biological replicates . Mean ± s . e . m .

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(H) Western blot showing expression of pro-CASP2 and P21 following treatment with dox for the specified number of days. Experiments were performed in PLK4Dox monoclonal knockout cells. (I) Quantification of pro-CASP2 levels following treatment with dox for the specified number of days. Experiments were performed in PLK4Dox monoclonal knockout cells. Each dot displays measurements from a single experiment. Data acquired across n = 6 biological replicates. Mean ± s.e.m.


( A ) Top : schematic representation of PIDD1 and its various cleavage products . Bottom : HEK293FT cells were transfected with the indicated constructs , subjected to co - immunoprecipitation and immunoblotted with the indicated antibodies .

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(A) Top: schematic representation of PIDD1 and its various cleavage products. Bottom: HEK293FT cells were transfected with the indicated constructs, subjected to co- immunoprecipitation and immunoblotted with the indicated antibodies.


( B ) HEK293FT cells were transfected with the indicated constructs , subjected to co - immunoprecipitation and immunoblotted with the indicated antibodies .

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(B) HEK293FT cells were transfected with the indicated constructs, subjected to co- immunoprecipitation and immunoblotted with the indicated antibodies.


( C ) Western blot showing the expression of ANKRD26 . Experiments were performed in monoclonal ANKRD26 - / - PLK4Dox cells expressing the indicated ANKRD26 transgenes .

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(C) Western blot showing the expression of ANKRD26. Experiments were performed in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated ANKRD26 transgenes.


( D ) Quantification of the fraction of cells with ANKRD26 ( left ) and PIDD1 ( right ) localized to the mature mother centriole in monoclonal ANKRD26 - / - PLK4Dox cells expressing the indicated transgenes . Each dot displays measurements from a single experiment . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

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(D) Quantification of the fraction of cells with ANKRD26 (left) and PIDD1 (right) localized to the mature mother centriole in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated transgenes. Each dot displays measurements from a single experiment. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( E ) Graph showing the relative growth of doxycycline - treated , monoclonal ANKRD26 - / - PLK4Dox cells expressing the indicated ANKRD26 transgenes . Each dot displays measurements from a single experiment . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

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(E) Graph showing the relative growth of doxycycline-treated, monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated ANKRD26 transgenes. Each dot displays measurements from a single experiment. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( F ) Western blot showing expression of pro - CASP2 and P21 following treatment with dox for 4 days . Experiments were performed in monoclonal ANKRD26 - / - PLK4Dox cells expressing the indicated transgenes .

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(F) Western blot showing expression of pro-CASP2 and P21 following treatment with dox for 4 days. Experiments were performed in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated transgenes.


Retrotransposon transcription in WT ( Hu303 ) , fft2Δ ( Hu1955 ) , fft3Δ ( Hu1867 ) , and fft2Δfft3Δ ( Hu2000 ) . A ) Transcription in fft2Δ relative to WT over chromosome 1 . The top and bottom panels represent fold change in tiling array signal from the forward and reverse strands , respectively . Coordinates are shown in the middle panel ; genes more than 2 - fold upregulated are marked : Tf2 retrotransposons ( black ) ; antisense to Tf2 elements ( gray ) ; coding genes ( green ) ; noncoding RNAs ( blue )

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Retrotransposon transcription in WT (Hu303), fft2Δ (Hu1955), fft3Δ (Hu1867), and fft2Δfft3Δ (Hu2000). A) Transcription in fft2Δ relative to WT over chromosome 1. The top and bottom panels represent fold change in tiling array signal from the forward and reverse strands, respectively. Coordinates are shown in the middle panel; genes more than 2-fold upregulated are marked: Tf2 retrotransposons (black); antisense to Tf2 elements (gray); coding genes (green); noncoding RNAs (blue)


Retrotransposon transcription in WT ( Hu303 ) , fft2Δ ( Hu1955 ) , fft3Δ ( Hu1867 ) , and fft2Δfft3Δ ( Hu2000 ) . B ) Northern blot for the Tf2 ORF ; rRNA staining of the same membrane by methylene blue is shown below . Two different exposures of the molecular size marker are shown ( left ) .

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Retrotransposon transcription in WT (Hu303), fft2Δ (Hu1955), fft3Δ (Hu1867), and fft2Δfft3Δ (Hu2000). B) Northern blot for the Tf2 ORF; rRNA staining of the same membrane by methylene blue is shown below. Two different exposures of the molecular size marker are shown (left).


Retrotransposon transcription in WT ( Hu303 ) , fft2Δ ( Hu1955 ) , fft3Δ ( Hu1867 ) , and fft2Δfft3Δ ( Hu2000 ) . C ) qPCR scheme . Amplicon 1 spans the end of the LTR and into the ORF . Amplicon 2 does not include the LTR but covers the translation start site . Amplicon 3 is entirely within the ORF . D ) Bar graph representing RNA levels for the amplicons depicted in ( C ) , relative to a control locus ( SPAC1F8 . 07c ) and to WT levels , as measured by qPCR . Error bars represent standard deviation of duplicate reverse transcriptions of biological triplicates .

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Retrotransposon transcription in WT (Hu303), fft2Δ(Hu1955), fft3Δ (Hu1867), and fft2Δfft3Δ (Hu2000). C) qPCR scheme. Amplicon 1 spans the end of the LTR and into the ORF. Amplicon 2 does not include the LTR but covers the translation start site. Amplicon 3 is entirely within the ORF. D) Bar graph representing RNA levels for the amplicons depicted in (C), relative to a control locus (SPAC1F8.07c) and to WT levels, as measured by qPCR. Error bars represent standard deviation of duplicate reverse transcriptions of biological triplicates.