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 .

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

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 .

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

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 .

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 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

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 .

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 .

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

(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 .

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

(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 .

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

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 ) .

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

(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 .

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

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 .

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

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 .

(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 .

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

(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 .

O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 : 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. (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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR I-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

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 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

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

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 O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O 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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR 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 O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

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 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 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 O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 .

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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 .

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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 .

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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 .

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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 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 ) .

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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 ) 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 ) .

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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 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 .

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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 - 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 .

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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 ) 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 .

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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 BRI125KR - mCit / bri1 / ubp12i / ubp13 . 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 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 .

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 .

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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 .

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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 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.


( G ) Top : Graph showing efficiency of frameshifting INDELs measured using TIDE . N . D . = Not determined . Data shown is from n = 1 biological replicate . Bottom : Graph showing relative growth of doxycycline treated PLK4Dox expressing an sgRNA targeting indicated genes . Each dot displays measurements from a single experiment . Experiments were performed in polyclonal knockout . 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 ) of centrosome number in PLK4Dox expressing an sgRNA targeting indicated genes . Experiments were performed in polyclonal knockout . 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 localization of CEP164 PIDD1 at mature - mother centriole . Bottom : Representative 3D STORM image of same centriole showing PIDD1 localization . STORM image colors correspond to Z - depth with red being closest to coverslip blue being 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 mature mother centriole . overlaid mask represents inner outer diameters of 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 outer diameter measurements for distal appendage PIDD1 . Measurements in shaded region were previously reported in ( Bowler et al . , 2019 ) . Data displayed as box whisker plots , where box represents upper lower quartile whiskers represent s . d . Scale bars : 1 μm for wide - field of centrioles 500 nm for STORM . Data acquired across n ≥ 6 .

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(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 ) of fraction of with PIDD1 localized to mature mother centriole in PLK4Dox expressing an sgRNA targeting indicated genes . A dot displays measurements from each experiment . Experiments were performed in PLK4Dox polyclonal knockout . 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 relative growth of doxycycline treated PLK4Dox that were knocked out for indicated genes . Each dot displays measurements from a single experiment . Experiments were performed in PLK4Dox monoclonal knockout . 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 ) of fraction of with PIDD1 localized at mature mother centriole . Experiments were performed in PLK4Dox monoclonal knockout . 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.


( A ) of fraction of with ANKRD26 localized at mature mother centriole . Experiments were performed in PLK4Dox monoclonal knockout . 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 of WT or ANKRD26 - / - PLK4Dox immunostained with 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 ) of fraction of WT or ANKRD26 - / - hTERT RPE1 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 of indicated with without doxycycline - inducible overexpression of PLK4 . Experiments were performed in PLK4Dox monoclonal knockout . 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 ) of centrosome number in PLK4Dox expressing an sgRNA targeting indicated genes . Experiments were performed in PLK4Dox monoclonal knockout . 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.


( H ) Western blot showing expression of pro - CASP2 P21 following treatment with dox for specified number of days . Experiments were performed in PLK4Dox monoclonal knockout . ( I ) of pro - CASP2 following treatment with dox for specified number of days . Experiments were performed in PLK4Dox monoclonal knockout . 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 its various cleavage products . Bottom : HEK293FT were transfected with indicated constructs , subjected to co - immunoprecipitation immunoblotted with 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 were transfected with indicated constructs , subjected to co - immunoprecipitation immunoblotted with indicated antibodies .

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


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

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


( D ) of fraction of with ANKRD26 ( left ) PIDD1 ( right ) localized to mature mother centriole in monoclonal ANKRD26 - / - PLK4Dox expressing 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 relative growth of doxycycline - treated , monoclonal ANKRD26 - / - PLK4Dox expressing 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 P21 following treatment with dox for 4 days . Experiments were performed in monoclonal ANKRD26 - / - PLK4Dox expressing 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 ) , fft2Δfft3Δ ( Hu2000 ) . A ) Transcription in fft2Δ relative to WT over chromosome 1 . top bottom panels represent fold change in tiling array signal from forward reverse strands , respectively . Coordinates are shown in 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 ) , fft2Δfft3Δ ( Hu2000 ) . B ) Northern blot for Tf2 ORF ; rRNA of same by methylene blue is shown below . Two different exposures of molecular marker are shown ( left ) .

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 ) , fft2Δfft3Δ ( Hu2000 ) . C ) qPCR scheme . Amplicon 1 spans end of LTR into ORF . Amplicon 2 does not include LTR but covers translation start site . Amplicon 3 is entirely within ORF . D ) Bar graph representing for amplicons depicted in ( C ) , relative to a control locus ( SPAC1F8 . 07c ) to WT , 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.


Retrotransposon transcription in WT ( Hu303 ) , fft2Δ ( Hu1955 ) , fft3Δ ( Hu1867 ) , fft2Δfft3Δ ( Hu2000 ) . E ) 5 ' RACE products run on a 1 . 5 % agarose gel .

Retrotransposon transcription in WT (Hu303), fft2Δ (Hu1955), fft3Δ (Hu1867), and fft2Δfft3Δ (Hu2000). E) 5'RACE products run on a 1.5% agarose gel.


Top panel : Y - axis shows CAGE signal ( tag - per - million normalized number of mapped CAGE tags on plus strand ) . X axis shows LTR 5 ' of Tf2 ORF . Three biological replicates are shown for each : fft2Δfft3 WT . Bottom : LTR tf2 ORF are colored : U3 ( green ) , R ( red ) , U5 ( blue ) , self primer primer binding ( PBS ; gray ) , beginning of ORF ( gray striped ) . TSS of Tf2 transcripts from ( Rhind et al , 2011 ) are shown as gray dots above elements . black square indicates 5 ' end of Tf2 mRNA in WT green square indicates 5 ' end in fft2Δ fft3Δ detected by 5 ' RACE .

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Top panel: Y-axis shows the CAGE signal intensity (tag-per-million normalized number of mapped CAGE tags on plus strand). X axis shows LTR and 5' of Tf2 ORF. Three biological replicates are shown for each strain: fft2Δfft3 and WT. Bottom: LTR and tf2 ORF are colored: U3 (green), R (red), U5 (blue), self primer and primer binding sequence (PBS; gray), beginning of ORF (gray striped). TSS of all Tf2 transcripts from (Rhind et al, 2011) are shown as gray dots above the sequence elements. The black square indicates the 5' end of the Tf2 mRNA in WT and the green square indicates the 5' end in fft2Δ fft3Δ detected by 5'RACE.


A ) Fft2 Fft3 are both enriched at LTRs , while Fft2 is also enriched at tf2 coding region . Fft2 LTR enrichment increases in fft3Δ , while Fft3 LTR enrichment decreases slightly in fft2Δ . Top : Fft2 - myc enrichment in WT ( solid ) in fft3Δ ( dashed ) . Bottom : Fft3 - myc enrichment in WT ( solid ) in fft2Δ ( dashed ) . ChIP - chip signal is normalized against no - epitope control arrays .

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A) Fft2 and Fft3 are both enriched at LTRs, while Fft2 is also enriched at the tf2 coding region. Fft2 LTR enrichment increases in fft3Δ cells, while Fft3 LTR enrichment decreases slightly in fft2Δ cells. Top: Fft2 -myc enrichment in WT (solid line) and in fft3Δ (dashed line). Bottom: Fft3 -myc enrichment in WT (solid line) and in fft2Δ (dashed line). ChIP-chip signal is normalized against no-epitope control arrays.


B ) Nucleosome occupancy over LTR U3 is significantly reduced ( average reduction ~ 50 % ; average p - values at each position < 0 . 001 , Poisson ) in fft2Δ fft3Δ . MNase - seq data aligned at start of tf25 ' LTRs is shown for WT ( black ) , fft2Δ fft3Δ ( gray ) . gray indicates significance of difference between WT mutant signals : amplitude reflects significance ( p - value ) sign reflects sign of occupancy change in mutant vs . WT . Error bars show standard deviation of nucleosome occupancy averaged over 13 tf2 elements . LTR tf2ORF are colored : U3 ( green ) , R ( red ) , U5 ( blue ) , self primer primer binding ( PBS ; gray ) , beginning of ORF ( gray striped ) . TSS of tf2 transcripts from ( Rhind et al , 2011 ) are shown as gray dots above elements , while green dot indicates TSS in fft2Δ fft3Δ .

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B) Nucleosome occupancy over the LTR U3 is significantly reduced (average reduction ~50%; average p-values at each position <0.001, Poisson test) in fft2Δ fft3Δ cells. MNase-seq data aligned at the start of tf25'LTRs is shown for WT (black line), and fft2Δ fft3Δ (dark gray line). Light gray line indicates significance of difference between WT and mutant signals: amplitude reflects significance (p-value) and sign reflects the sign of occupancy change in mutant vs. WT cells. Error bars show standard deviation of nucleosome occupancy averaged over 13 tf2 elements. LTR and tf2ORF are colored: U3 (green), R (red), U5 (blue), self primer and primer binding sequence (PBS; gray), beginning of ORF (gray striped). TSS of all tf2 transcripts from (Rhind et al, 2011) are shown as gray dots above the sequence elements, while the green dot indicates the TSS in fft2Δ fft3Δ.


A ) Mean nucleosome occupancy over solo LTRs ( n = 236 ) tf2 - associated LTRs ( n = 25 ) , in WT in fft2Δ fft3Δ .

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A) Mean nucleosome occupancy over solo LTRs (n=236) and tf2-associated LTRs (n=25), in WT and in fft2Δ fft3Δ.


B ) Mean of expression ( fft2Δ fft3Δ / WT ) for each of following categories : non - coding with LTR ( n = 69 ) / without LTR ( n = 1665 ) ( p = 0 . 008 ) ; coding with LTR ( n = 246 ) / without LTR ( n = 4887 ) ( p < 0 . 001 ) ; Tf2s ( n = 13 ) . A B ) Data shown as box plots , significance of difference between categories was assessed by Wilcoxon - Mann - Whitney ( * * p < 0 . 001 ) .

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B) Mean ratio of expression (fft2Δ fft3Δ /WT) for each of the following categories: non-coding with LTR (n=69) / without LTR (n=1665) (p=0.008); protein coding with LTR (n=246) / without LTR (n=4887) (p<0.001); Tf2s (n=13). A and B) Data shown as box plots, significance of difference between categories was assessed by Wilcoxon-Mann-Whitney test (** p<0.001).


A ) Heat oxidative stress cause a Tf2 TSS shift in WT , resulting in production of reverse transcription competent Tf2 transcripts . Transcription fold change as in Figure 1D , but relative to act1 transcription to transcription in same culture immediately prior to stress , as measured by qPCR . Error bars represent standard deviation of reverse transcriptions of biological duplicates .

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A) Heat and oxidative stress cause a Tf2 TSS shift in WT cells, resulting in the production of reverse transcription competent Tf2 transcripts. Transcription fold change as in Figure 1D, but relative to act1 transcription and to transcription in the same culture immediately prior to stress, as measured by qPCR. Error bars represent standard deviation of reverse transcriptions of biological duplicates.


B ) Heat shock - associated TSS shift is accompanied by a reduction in LTR nucleosome occupancy . degree of nucleosome loss is greater in WT than in fft2Δ fft3Δ , where nucleosome occupancy is already very low ( Fig 2B , Fig S3 ) . LTR nucleosome occupancy shown relative to occupancy of Tf2 ORF + 1 nucleosome , measured by MNase - qPCR .

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B) Heat shock-associated TSS shift is accompanied by a reduction in LTR nucleosome occupancy. The degree of nucleosome loss is greater in WT cells than in fft2Δ fft3Δ cells, where nucleosome occupancy is already very low (Fig 2B, Fig S3). LTR nucleosome occupancy shown relative to occupancy of the Tf2 ORF +1 nucleosome, measured by MNase-qPCR.


C ) Exposure to stress conditions does not increase Tf2 transcript further in fft2Δ fft3Δ . Transcription fold change as in Figure 1D , but relative to act1 transcription to transcription in same culture immediately prior to stress , as measured by qPCR . Error bars represent standard deviation of reverse transcriptions of biological

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C) Exposure to stress conditions does not increase Tf2 transcript levels further in fft2Δ fft3Δ cells. Transcription fold change as in Figure 1D, but relative to act1 transcription and to transcription in the same culture immediately prior to stress, as measured by qPCR. Error bars represent standard deviation of reverse transcriptions of biological duplicates


B ) Bar graph representing as in Fig . 1D , relative to a control locus ( act1 ) to WT , as measured by qPCR . Error bars represent standard deviation of nucleosome occupancy of biological duplicates .

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B) Bar graph representing RNA levels as in Fig. 1D, relative to a control locus (act1) and to WT levels, as measured by qPCR. Error bars represent standard deviation of nucleosome occupancy of biological duplicates.


C ) TSS shift in Fft deletion catalytic mutants is accompanied by reduced LTR nucleosome occupancy , measured by MNase - qPCR as in Fig . 5B .

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C) TSS shift in Fft deletion and catalytic mutants is accompanied by reduced LTR nucleosome occupancy, measured by MNase-qPCR as in Fig. 5B.


A ) Fluorescencein situ hybridization with a probe against tf2 elements ( green ) , DAPI ( blue ) in WT fft2Δ fft3Δ . B ) number of tf2 element signal clusters is significantly elevated ( p = 9 . 8x10 - 18 , χ2 - ) in fft2Δ fft3Δ , indicating a loss of retrotransposon clustering . Tf2 clustering was counted in wt ( n = 663 ) , fft2 ∆ ( n = 556 ) , fft3 ∆ ( n = 493 ) , fft2 ∆ fft3 ∆ ( n = 636 ) . Significance of difference between strains was assessed by Chi - square .

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A) Fluorescencein situ hybridization with a probe against tf2 elements (green), and DAPI staining (blue) in WT and fft2Δ fft3Δ cells. B) The number of tf2 element signal clusters is significantly elevated (p=9.8x10-18, χ2-test) in fft2Δ fft3Δ cells, indicating a loss of retrotransposon clustering. Tf2 clustering was counted in wt cells (n=663), fft2∆ (n=556), fft3∆ (n=493), fft2∆ fft3∆ (n=636). Significance of difference between strains was assessed by Chi-square test.


A ) Pictures of YES plates with G418 showing growth of G418 resistant subclones in fft3D ( PP3 ) fft2Dfft3D ( PP4 ) mutant strains with new integrations of Tf2 - 12 - neoAI . After 3 days at 32o C .

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A) Pictures of YES plates with G418 showing growth of G418 resistant subclones in fft3D (PP3) and fft2Dfft3D (PP4) mutant strains with new integrations of Tf2-12-neoAI. After 3 days at 32o C.


C ) Southern blot probed for neoA fft3D ( PP3 ) fft2Dfft3D mutant strains ( PP4 ) derived G418 resistant subclones .

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C) Southern blot probed for neoA fft3D (PP3) and fft2Dfft3D mutant strains (PP4) and derived G418 resistant subclones.


Western blot of nephrin expression in immortalized podocytes cultured in normal ( 5 mM ) or high glucose ( HG ; 30 mM ) or high mannitol ( Mann ; 5 mM glucose plus 25 mM mannitol ) for 24 , 48 72 hours . * P < 0 . 05 versus normal controls for indicated time points ( Wilcoxon two - sample ; n = 3 ) .

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Western blot analysis of nephrin expression in immortalized podocytes cultured in normal (5 mM) or high glucose (HG; 30 mM) or high mannitol (Mann; 5 mM glucose plus 25 mM mannitol) for 24, 48 and 72 hours. *P < 0.05 versus normal controls for the indicated time points (Wilcoxon two-sample test; n = 3).


B of methylation status at nephrin gene promoter in podocytes cultured in normal , high glucose or high mannitol at indicated time points . U , unmethylated - specific primers ; M , methylated - specific primers .

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B Analysis of the methylation status at nephrin gene promoter in podocytes cultured in normal, high glucose or high mannitol at the indicated time points. U, unmethylated-specific primers; M, methylated-specific primers.


C Effect of pargyline hydrochloride , a pan - KDM inhibitor , on high glucose - mediated nephrin expression . Immortalized mouse podocytes were cultured in indicated cultured media in presence or absence of pargyline hydrochloride ( 1 μM ) for 48 hours . lysates from treated podocytes were analyzed for nephrin expression by immunoblotting . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus untreated HG - incubated ( Wilcoxon two - sample ; n = 3 ) .

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C Effect of pargyline hydrochloride, a pan-KDM inhibitor, on high glucose-mediated nephrin expression. Immortalized mouse podocytes were cultured in the indicated cultured media in the presence or absence of pargyline hydrochloride (1 μM) for 48 hours. Protein lysates from the treated podocytes were analyzed for nephrin expression by immunoblotting. *P < 0.05 versus normal controls, #P < 0.05 versus untreated HG-incubated cells (Wilcoxon two-sample test; n = 3).


D Effect of pargyline hydrochloride on high glucose - mediated methylation at nephrin gene promoter . Genomic samples from treated podocytes were analyzed for methylation status of nephrin gene promoter by methylation - specific PCR .

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D Effect of pargyline hydrochloride on high glucose-mediated DNA methylation at nephrin gene promoter. Genomic DNA samples from the treated podocytes were analyzed for methylation status of nephrin gene promoter by methylation-specific PCR.


E Quantitative RT - PCR analyses of different KDMs in immortalized podocytes exposed to high glucose or high mannitol for 48 hours . * P < 0 . 05 versus normal controls ( Wilcoxon two - sample ; n = 3 ) .

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E Quantitative RT-PCR analyses of different KDMs in immortalized podocytes exposed to high glucose or high mannitol for 48 hours. *P < 0.05 versus normal controls (Wilcoxon two-sample test; n = 3).


F Western blot of KDM6A expression in podocytes cultured in normal , high glucose or high mannitol conditions for 24 , 48 ad 72 hours . * P < 0 . 05 versus normal controls for indicated time points ( Wilcoxon two - sample ; n = 3 ) .

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F Western blot analysis of KDM6A expression in podocytes cultured in normal, high glucose or high mannitol conditions for 24, 48 ad 72 hours. *P < 0.05 versus normal controls for the indicated time points (Wilcoxon two-sample test; n = 3).


G Western blot of H3K27me1 , H3K27me2 , H3K27me3 pan - methyl H3K9 in podocytes cultured in normal , high glucose or high mannitol conditions for 9 24 hours . * P < 0 . 05 versus normal controls ( Wilcoxon two - sample ; n = 3 ) .

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G Western blot analysis of H3K27me1, H3K27me2, H3K27me3 and pan-methyl H3K9 in podocytes cultured in normal, high glucose or high mannitol conditions for 9 and 24 hours. *P < 0.05 versus normal controls (Wilcoxon two-sample test; n = 3).


A Effect of KDM6A knockdown on expression of nephrin WT - 1 in immortalized mouse podocytes cultured under high glucose conditions . Western blot analyses were performed using lysates of podocytes that were transfected with scrambled siRNAs or KDM6A - specific siRNAs , cultured in high glucose ( HG ) or high mannitol ( Mann ) for 48 hours . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus control siRNA with HG incubation ( Parametric ANOVA a Bonferroni post hoc ; n = 3 ) .

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A Effect of KDM6A knockdown on the expression of nephrin and WT-1 in immortalized mouse podocytes cultured under high glucose conditions. Western blot analyses were performed using protein lysates of podocytes that were transfected with scrambled siRNAs or KDM6A-specific siRNAs, and cultured in high glucose (HG) or high mannitol (Mann) for 48 hours. *P < 0.05 versus normal controls, #P < 0.05 versus control siRNA with HG incubation (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


B Effect of KDM6A overexpression on expression of nephrin WT - 1 . Immortalized podocytes were transfected with KDM6A expression plasmid or empty vector control for 48 hours , expression of KDM6A , nephrin WT - 1 in these transfected podoytes were analyzed by immunoblotting . * P < 0 . 05 versus vector controls ( Parametric ANOVA a Bonferroni post hoc ; n = 3 ) .

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B Effect of KDM6A overexpression on expression levels of nephrin and WT-1. Immortalized podocytes were transfected with the KDM6A expression plasmid or the empty vector control for 48 hours, and the expression levels of KDM6A, nephrin and WT-1 in these transfected podoytes were analyzed by immunoblotting. *P < 0.05 versus vector controls (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


C Changes in H3K27me3 in podocytes after treatment with high glucose , combination of high glucose KDM6A knockdown , or KDM6A overexpression for 48 hours . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus control siRNA with HG incubation ( Parametric ANOVA a Bonferroni post hoc ; n = 3 ) .

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C Changes in H3K27me3 levels in podocytes after treatment with high glucose, the combination of high glucose and KDM6A knockdown, or KDM6A overexpression for 48 hours. *P < 0.05 versus normal controls, #P < 0.05 versus control siRNA with HG incubation (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


D Changes in methylation at nephrin gene promoter by high glucose , by combination of high glucose plus KDM6A knockdown , or by KDM6A overexpression in immortalized podocytes . U , unmethylated - specific primers ; M , methylated - specific primers . Experiments were repeated three times a representative gel from one experiment is shown .

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D Changes in DNA methylation at nephrin gene promoter by high glucose, by the combination of high glucose plus KDM6A knockdown, or by KDM6A overexpression in immortalized podocytes. U, unmethylated-specific primers; M, methylated-specific primers. Experiments were repeated three times and a representative gel from one experiment is shown.


E Effect of GSK - J4 ( 40 μM ) , a specific KDM6A inhibitor , on high glucose - mediated changes in of nephrin , WT - 1 H3K27me3 . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus untreated HG - incubated ( Parametric ANOVA a Bonferroni post hoc ; n = 3 ) .

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E Effect of GSK-J4 (40 μM), a specific KDM6A inhibitor, on high glucose-mediated changes in levels of nephrin, WT-1 and H3K27me3. *P < 0.05 versus normal controls, #P < 0.05 versus untreated HG-incubated cells (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


F Changes in methylation at nephrin gene promoter by GSK - J4 ( 40 μM ) in immortalized podocytes . U , unmethylated - specific primers ; M , methylated - specific primers .

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F Changes in DNA methylation at nephrin gene promoter by GSK-J4 (40 μM) in immortalized podocytes. U, unmethylated-specific primers; M, methylated-specific primers.


Relative mRNA of KDM6A , nephrin WT - 1 , normalized to β - actin , expressed in kidney glomeruli of normal diabetic mice at 4 , 8 12 after diabetic induction . * Significant differences ( P < 0 . 05 ) compared with normal controls ( Wilcoxon two - sample ; n = 8 each ) .

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Relative mRNA levels of KDM6A, nephrin and WT-1, normalized to β-actin, expressed in kidney glomeruli of normal and diabetic mice at 4, 8 and 12 weeks after diabetic induction. *Significant differences (P < 0.05) compared with normal controls (Wilcoxon two-sample test; n =8 each).