B . Limited proteolysis of a purified preparation of the Nse5 / Nse6 complex by trypsin . Samples taken at different time points were analyzed by SDS - Page and Coomassie staining . Selected stable fragments identified by mass spectrometry 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

B. Limited proteolysis of a purified preparation of the Nse5/Nse6 complex by trypsin. Samples taken at different time points were analyzed by SDS-Page and Coomassie staining. Selected stable fragments identified by mass spectrometry are indicated.


A . Pulldown ( ' pd ' ) assays using immobilized Smc5 / Smc6 - Twin - Strep ( ' TS ' ) hexamers and soluble input material ( ' in ' ) of Nse5 / 6 ( left ) , of Nse5 / Nse6 ( 177 - C ) ( middle ) and of Nse6 ( 1 - 179 ) - CPD - His ( right ) . Control pulldowns ( ' - ' ) were performed by omitting the pre - binding of Smc5 / Smc6 - Twin - Strep to the beads . Fractions were analysed by SDS - Page and Coomassie staining .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Pulldown ('pd') assays using immobilized Smc5/Smc6-Twin-Strep ('TS') hexamers and soluble input material ('in') of Nse5/6 (left), of Nse5/Nse6(177-C) (middle) and of Nse6(1-179)-CPD-His (right). Control pulldowns ('-') were performed by omitting the pre-binding of Smc5/Smc6-Twin-Strep to the beads. Fractions were analysed by SDS-Page and Coomassie staining.


B . Salt stability of Smc5 / Smc6 - Twin - Strep interactions with Nse5 / 6 and Nse6 ( 1 - 179 ) . Immobilized Smc5 / Smc6 - Twin - Strep was mixed with Nse5 / 6 or Nse6 ( 1 - 179 ) - CPD - His ( ' in ' ) . Beads were washed with buffers containing the indicated salt concentrations . Bead fractions were analysed by SDS - Page and Coomassie staining . Pulldown efficiencies were estimated from the intensity of Coomassie gel bands .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Salt stability of Smc5/Smc6-Twin-Strep interactions with Nse5/6 and Nse6(1-179). Immobilized Smc5/Smc6-Twin-Strep was mixed with Nse5/6 or Nse6(1-179)-CPD-His ('in'). Beads were washed with buffers containing the indicated salt concentrations. Bead fractions were analysed by SDS-Page and Coomassie staining. Pulldown efficiencies were estimated from the intensity of Coomassie gel bands.


D . Analysis of spore viability by yeast tetrad dissection . Diploid strains heterozygous for alleles of Nse6 ( wt , 86 - C , 177 - C and 179 - C ) were sporulated . Isolated spores were grown on YPD plates . Viable clones were tested for the marker cassette conferring resistance to G418 ( marked by circles in green colours ) . Dead spores were marked by circles in red colours .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Analysis of spore viability by yeast tetrad dissection. Diploid strains heterozygous for alleles of Nse6 (wt, 86-C, 177-C and 179-C) were sporulated. Isolated spores were grown on YPD plates. Viable clones were tested for the marker cassette conferring resistance to G418 (marked by circles in green colours). Dead spores were marked by circles in red colours.


A . ATP hydrolysis rates ( given per Smc5 / 6 complex ) were measured by an enzyme - coupled assay in the absence and presence of plasmidDNA ( conc . 1 . 25 nM ) for the Smc5 / 6 hexamer ( conc . 150 nM ) and the octamer ( conc . 150 nM ) with increasing concentrations of ATP . The curves were fitted to Michaelis Menten equation and Km and kcat values were determined . Please note that the hexamer without plasmidDNA shows cooperative behaviour , thus the Michalis - Menten kinetics is not formally applicable ( marked by asterisk ) . Assays were performed in biological triplicates , mean values are shown with error bars indicating standard deviations . For bar graphs individual data points are also displayed . B . Same as in ( A ) using 40bpDNA ( annealed 40 bp oligo DNA ) ( conc . 1 µM ) instead of plasmidDNA . Note that the data points and curves for samples without DNA ( hexamer - DNA ; octamer - DNA ) are identical to ( A ) .

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

A. ATP hydrolysis rates (given per Smc5/6 complex) were measured by an enzyme-coupled assay in the absence and presence of plasmidDNA (conc. 1.25 nM) for the Smc5/6 hexamer (conc. 150 nM) and the octamer (conc. 150 nM) with increasing concentrations of ATP. The curves were fitted to Michaelis Menten equation and Km and kcat values were determined. Please note that the hexamer without plasmidDNA shows cooperative behaviour, thus the Michalis-Menten kinetics is not formally applicable (marked by asterisk). Assays were performed in biological triplicates, mean values are shown with error bars indicating standard deviations. For bar graphs individual data points are also displayed. B. Same as in (A) using 40bpDNA (annealed 40 bp oligo DNA) (conc. 1 µM) instead of plasmidDNA. Note that the data points and curves for samples without DNA (hexamer -DNA; octamer -DNA) are identical to (A).


D . Pulldown experiments with Smc5 / 6 complexes and circular plasmid ( 2 . 8 kbp ) . DNA is retained after high salt washes ( 1 M NaCl ) only in the presence of both Nse5 / 6 and ATP . The graph on the right shows a quantification ( mean values and standard deviations ) of the amount of co - purified DNA from technical triplicates . Individual data points are also displayed .

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

D. Pulldown experiments with Smc5/6 complexes and circular plasmid (2.8 kbp). DNA is retained after high salt washes (1 M NaCl) only in the presence of both Nse5/6 and ATP. The graph on the right shows a quantification (mean values and standard deviations) of the amount of co-purified DNA from technical triplicates. Individual data points are also displayed.


F . Salt - stable DNA association requires ATP binding and Smc5 / 6 head - engagement , but not ATP hydrolysis . Same experimental setup as in ( D ) using mutant versions of the Smc5 / 6 hexamer as well as the non - hydrolyzable analogue of ATP , ATPγS , as indicated .

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

F. Salt-stable DNA association requires ATP binding and Smc5/6 head-engagement, but not ATP hydrolysis. Same experimental setup as in (D) using mutant versions of the Smc5/6 hexamer as well as the non-hydrolyzable analogue of ATP, ATPγS, as indicated.


B . Cross - linking of engineered variants of the Smc5 / 6 hexamer . As in ( a ) using Smc5 and Smc6 cysteine mutants . Schemes indicate the location of engineered cysteines and their expected ability to cross - link in a rod - like and a ring - like conformation . High - molecular weight species were analysed by SDS - Page and Coomassie staining . Wild - type hexamer ( ' wt ' ) is included as cross - linking control . Species occurring only in the presence of engineered cysteines are labelled by coloured arrowheads . Cross - linking efficiencies were calculated from the intensity of Coomassie - stained bands by comparing the band of the corresponding cross - linked species to the bands of unmodified Smc5 and Smc6 . Numbers below the gel quantify the percentage of cross - linked protein species in the displayed gel . Comparable numbers were obtained in at least one additional independent experiment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Cross-linking of engineered variants of the Smc5/6 hexamer. As in (a) using Smc5 and Smc6 cysteine mutants. Schemes indicate the location of engineered cysteines and their expected ability to cross-link in a rod-like and a ring-like conformation. High-molecular weight species were analysed by SDS-Page and Coomassie staining. Wild-type hexamer ('wt') is included as cross-linking control. Species occurring only in the presence of engineered cysteines are labelled by coloured arrowheads. Cross-linking efficiencies were calculated from the intensity of Coomassie-stained bands by comparing the band of the corresponding cross-linked species to the bands of unmodified Smc5 and Smc6. Numbers below the gel quantify the percentage of cross-linked protein species in the displayed gel. Comparable numbers were obtained in at least one additional independent experiment.


E . Effects of 40bpDNA and plasmidDNA on head engagement ( as judged by E - Cys cross - linking ) of the Smc5 / 6 complex with wildtype ( wt ) or hydrolysis - deficient ( EQ ) heads , in both the presence or absence of the Nse5 / 6 complex . Cross - linking efficiencies were calculated from the intensity of Coomassie - stained bands by comparing the band of the corresponding cross - linked species to the bands of unmodified Smc5 and Smc6 . Numbers below the gel quantify the percentage of cross - linked protein species in the displayed gel .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Effects of 40bpDNA and plasmidDNA on head engagement (as judged by E-Cys cross-linking) of the Smc5/6 complex with wildtype (wt) or hydrolysis-deficient (EQ) heads, in both the presence or absence of the Nse5/6 complex. Cross-linking efficiencies were calculated from the intensity of Coomassie-stained bands by comparing the band of the corresponding cross-linked species to the bands of unmodified Smc5 and Smc6. Numbers below the gel quantify the percentage of cross-linked protein species in the displayed gel.


B . Flow cytometry for Bu - 1a expression in wild type cells with ( GAA ) n tracts of different length knocked into the BU - 1A locus ( in blue ) . DT40 cells are heterozygous and carry one BU - 1A and one BU - 1B allele . All experiments introducing repeats into BU - 1A are carried out in cells in which the + 3 . 5 G4 has been deleted from both A and B alleles , to avoid transvection between the allele Black outline : positive control ( wild type cells ) ; red outline : negative control ( cells carrying a genetic disruption of BU - 1 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Flow cytometry for Bu-1a expression in wild type cells with (GAA)n tracts of different length knocked into the BU-1A locus (in blue). DT40 cells are heterozygous and carry one BU-1A and one BU-1B allele. All experiments introducing repeats into BU-1A are carried out in cells in which the +3.5 G4 has been deleted from both A and B alleles, to avoid transvection between the allele Black outline: positive control (wild type cells); red outline: negative control (cells carrying a genetic disruption of BU-1)


C , D . Bu - 1a fluctuation analysis of wildtype and primpol cells in which the endogenous + 3 . 5 G4 has been deleted ( ΔG4 ) or with ( GAA ) 10 and ( GAA ) 20 sequence orientated such that it is replicated as the leading ( C ) or lagging ( D ) strand template for a fork entering from the 3 ' end of the locus as shown in panel ( A ) . At least two independent fluctuation analyses were performed . Circles represent the percentage of Bu - 1a loss variants in at least 24 individual clones from these experiments , with mean ± SD reported . * * * * p < 0 . 0001 , * p ≤ 0 . 05 , ns = not significant ; one - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D. Bu-1a fluctuation analysis of wildtype and primpol cells in which the endogenous +3.5 G4 has been deleted (ΔG4) or with (GAA)10 and (GAA)20 sequence orientated such that it is replicated as the leading (C) or lagging (D) strand template for a fork entering from the 3' end of the locus as shown in panel (A). At least two independent fluctuation analyses were performed. Circles represent the percentage of Bu-1a loss variants in at least 24 individual clones from these experiments, with mean ± SD reported. ****p < 0.0001, * p ≤ 0.05, ns = not significant; one-way ANOVA.


Human PrimPol , or mutants , tagged with YFP were expressed in primpol cells harbouring ( GAA ) 10 sequence in the BU - 1A locus . Bu - 1a and YFP double positive cells were sorted and expanded for 2 weeks , and then analysed for Bu - 1a expression variants . For each complementation , at least two independently derived clones were subjected to fluctuation analysis . As previously observe expression of hPrimPol [ AxA ] and hPrimPol [ ZfKO ] is deleterious and unstable . Cells expressing these mutations and remaining YFP - positive at the end of the expansion period will have been through fewer divisions the other lines in this analysis . Pooled results from at least three independent fluctuation analyses are represented with mean ± SD indicated with red bar and whiskers . Statistical significance : * * * * p < 0 . 0001 , * * * p < 0 . 001 , * p ≤ 0 . 05 , ns = not significant ; Kruskal - Wallis

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

Human PrimPol, or mutants, tagged with YFP were expressed in primpol cells harbouring (GAA)10 sequence in the BU-1A locus. Bu-1a and YFP double positive cells were sorted and expanded for 2 weeks, and then analysed for Bu-1a expression variants. For each complementation, at least two independently derived clones were subjected to fluctuation analysis. As previously observe expression of hPrimPol[AxA] and hPrimPol[ZfKO] is deleterious and unstable. Cells expressing these mutations and remaining YFP-positive at the end of the expansion period will have been through fewer divisions the other lines in this analysis. Pooled results from at least three independent fluctuation analyses are represented with mean ±SD indicated with red bar and whiskers. Statistical significance: **** p < 0.0001, *** p<0.001, * p ≤ 0.05, ns = not significant; Kruskal-Wallis test


A . DRIP - qPCR analysis reveals accumulation of R - loops across the BU - 1 locus in primpol cells . The DRIP signal was calculated as enrichment over RNase H - treated samples and was normalised to - 0 . 5 kb amplicon . The mean and SD for three biological replicates is presented . An unpaired t - test was used to compare differences between matched amplicons in primpol BU - 1A ( GAA ) 10 and the other cell lines indicated . * * * * p ≤ 0 . 0001 , ns = not

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. DRIP-qPCR analysis reveals accumulation of R-loops across the BU-1 locus in primpol cells. The DRIP signal was calculated as enrichment over RNase H-treated samples and was normalised to -0.5 kb amplicon. The mean and SD for three biological replicates is presented. An unpaired t-test was used to compare differences between matched amplicons in primpol BU-1A(GAA)10 and the other cell lines indicated. **** p ≤ 0.0001, ns = not significant


B . DNA : RNA hybrids in primpol BU - 1A ( GAA ) 10 : GgRNase H and primpol BU - 1A ( GAA ) 10 : hPrimPol . An unpaired t - test on three biological replicates was used to compare differences to primpol BU - 1A ( GAA ) 10 for each matched amplicon . The bar represent the mean and whiskers the SD . * * * p ≤ 0 . 001 , * * p ≤ 0 . 01 , ns = not

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. DNA:RNA hybrids in primpol BU-1A(GAA)10:GgRNase H and primpol BU-1A(GAA)10:hPrimPol. An unpaired t-test on three biological replicates was used to compare differences to primpol BU-1A(GAA)10 for each matched amplicon. The bar represent the mean and whiskers the SD. *** p ≤ 0.001, ** p ≤ 0.01, ns = not significant


C . Overexpression of chicken RNase H1 prevents ( GAA ) 10 induced BU - 1A epigenetic instability in primpol cells . Fluctuation analysis was performed on three primpol BU - 1A ( GAA ) 10 clones . One - way ANOVA was used to calculate the significance of differences in BU - 1 instability between primpol BU - 1A ΔG4 and other cell lines . * * * * p ≤ 0 . 0001 , ns = not

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Overexpression of chicken RNase H1 prevents (GAA)10 induced BU-1A epigenetic instability in primpol cells. Fluctuation analysis was performed on three primpol BU-1A (GAA)10 clones. One-way ANOVA was used to calculate the significance of differences in BU-1 instability between primpol BU-1A ΔG4 and other cell lines. **** p ≤ 0.0001, ns = not significant


D . Diagram of the RNase H1 hybrid binding domain ( HBD ) - mCherry fusion and flow cytometry expression profiles of the construct in four

O O 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. Diagram of the RNase H1 hybrid binding domain (HBD) - mCherry fusion and flow cytometry expression profiles of the construct in four clones


E . R - loop stabilisation induces epigenetic instability of BU - 1 . Bu - 1a fluctuation analysis of wild type cells expressing HBD - mCherry . The scatter plots pool results from at least two different clones with matched HBD - expression . Mean ± SD reported . * * * * p ≤ 0 . 0001 , * * * p ≤ 0 . 001 , ns = not significant ; one - way ANOVA .

E. R-loop stabilisation induces epigenetic instability of BU-1. Bu-1a fluctuation analysis of wild type cells expressing HBD-mCherry. The scatter plots pool results from at least two different clones with matched HBD-expression. Mean ± SD reported. **** p ≤ 0.0001, *** p ≤ 0.001, ns = not significant; one-way ANOVA.


A . Expression of geminin - tagged chicken RNase H1 - YFP . Phases of the cell cycle were determined by staining DNA content in live cells by Hoechst 33342 ( X - axis ) . RNase H1 - YFP with or without the geminin degron protein is detected on the Y - axis . The RNase H1 - YFP - geminin degron is degraded in G1 . In contrast , RNase H1 - YFP levels remain stable irrespective of the phase of the cell cycle . 2n and 4n indicate the chromosome number before and after DNA

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 B-MEASURED_VAR

A . Expression of geminin-tagged chicken RNase H1-YFP. Phases of the cell cycle were determined by staining DNA content in live cells by Hoechst 33342 (X-axis). RNase H1-YFP with or without the geminin degron protein is detected on the Y-axis. The RNase H1-YFP-geminin degron is degraded in G1. In contrast, RNase H1-YFP levels remain stable irrespective of the phase of the cell cycle. 2n and 4n indicate the chromosome number before and after DNA replication


B . Bu - 1a fluctuation analysis of two independently derived primpol BU - 1A ( GAA ) 10 : Gg RNase H1 - YFP - geminin degron clones . Since the expression of the RNase H1 - YFP - geminin degron construct is not stable ( unlike the RNase H1 - YFP construct without the degron ) , Bu - 1a expression was assessed separately in the YFP + ve and YFP - ve cells within each clone . Statistical differences calculated the Kruskal - Wallis test . For all panels , at least 36 individual clones were analysed ; mean ± SD reported . * * * * p ≤ 0 . 0001 , * * * p ≤ 0 . 001 , ns = not

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Bu-1a fluctuation analysis of two independently derived primpol BU-1A(GAA)10: Gg RNase H1-YFP-geminin degron clones. Since the expression of the RNase H1-YFP-geminin degron construct is not stable (unlike the RNase H1-YFP construct without the degron), Bu-1a expression was assessed separately in the YFP +ve and YFP -ve cells within each clone. Statistical differences calculated the Kruskal-Wallis test. For all panels, at least 36 individual clones were analysed; mean ± SD reported. **** p ≤ 0.0001, *** p ≤ 0.001, ns = not significant


C . DRIP - qPCR for R - loops around the engineered + 3 . 5 ( GAA ) 10 repeat in BU - 1 in different phases of the cell cycle . The location of the qPCR amplicons is indicated in the map at the top of the panel . The BU - 1 DRIP signal was normalised to - 0 . 5 kb amplicon in G1 - arrested cells ( t = 0 h ) Black : wild type ; red : primpol . Error bars = SD . * * * * p ≤ 0 . 0001 , * * * p ≤ 0 . 001 , * * p ≤ 0 . 01 , * p ≤ 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. DRIP-qPCR for R-loops around the engineered +3.5 (GAA)10 repeat in BU-1 in different phases of the cell cycle. The location of the qPCR amplicons is indicated in the map at the top of the panel. The BU-1 DRIP signal was normalised to -0.5 kb amplicon in G1-arrested cells (t = 0 h) Black: wild type; red: primpol. Error bars = SD. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05


E . Validation of analysis of nascent DNA : RNA hybrid formation in BU - 1 locus . Enrichment of 4SU labelled RNA moiety of DNA : RNA hybrids was calculated relative to input in three independent asynchronous wild type ( black ) or primpol ( red ) cells , with or without exogenous RNase H treatment . Error bars = SD . * * p ≤ 0 . 01 , * p ≤ 0 . 05 , ns = not significant ; unpaired t -

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Validation of analysis of nascent DNA:RNA hybrid formation in BU-1 locus. Enrichment of 4SU labelled RNA moiety of DNA:RNA hybrids was calculated relative to input in three independent asynchronous wild type (black) or primpol (red) cells, with or without exogenous RNase H treatment. Error bars = SD. ** p ≤ 0.01, * p ≤ 0.05, ns = not significant; unpaired t-test


G . Newly synthesised R - loops in BU - 1 during S phase in wild type ( black ) and primpol ( red ) . Error bars represent 1SD of three biological repeats of the experiment . * * * p ≤ 0 . 001 , * p ≤ 0 . 05 ; unpaired t -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Newly synthesised R-loops in BU-1 during S phase in wild type (black) and primpol (red). Error bars represent 1SD of three biological repeats of the experiment. *** p ≤ 0.001, * p ≤ 0.05; unpaired t-test


A . Representative normalised DRIP - seq data in two genes COL22A1 , spanning over 200 kb , and MYC . The locations of H - DNA and G4 motifs are shown below the gene map . Wild type in blue ; primpol in red . The corresponding RNAse H - treated samples are dashed . See Methods for further details of graphic generation . Data information : P values calculated with Mann - Whitney U test . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper and lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative normalised DRIP-seq data in two genes COL22A1, spanning over 200 kb, and MYC. The locations of H-DNA and G4 motifs are shown below the gene map. Wild type in blue; primpol in red. The corresponding RNAse H-treated samples are dashed. See Methods for further details of graphic generation. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


C . DRIP peak heights in wild type and primpol DT40 normalised to Drosophila S2 spike - in . n ( wild type ) = 41445 ; n ( primpol ) = 48648 . D . Correlation of normalised DRIP peak heights in the overlapping peaks between wild type and primpol . Blue line = 1 : 1 correlation ; red line = linear regression through data . Data information : P values calculated with Mann - Whitney U test . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper and lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. DRIP peak heights in wild type and primpol DT40 normalised to Drosophila S2 spike-in. n (wild type) = 41445; n (primpol) = 48648. D. Correlation of normalised DRIP peak heights in the overlapping peaks between wild type and primpol. Blue line = 1:1 correlation; red line = linear regression through data. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


E . Correlation between H - DNA forming sequences and all genes ( white bar ) , and genes with DRIP peaks in wild type ( blue ) and primpol cells ( red ) . F . Normalised DRIP peak heights in the genes identified as associating with H - DNA . Data information : P values calculated with Mann - Whitney U test . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper and lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Correlation between H-DNA forming sequences and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red). F. Normalised DRIP peak heights in the genes identified as associating with H-DNA. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


G . Correlation between G4 motifs ( [ G3 - 5N1 - 7 ] 4 ) and all genes ( white bar ) , and genes with DRIP peaks in wild type ( blue ) and primpol cells ( red ) . H . Normalised DRIP peak heights in the genes identified as associating with G4 motifs ( [ G3 - 5N1 - 7 ] 4 ) . Data information : P values calculated with Mann - Whitney U test . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper and lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

G. Correlation between G4 motifs ([G3-5N1-7]4) and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red). H. Normalised DRIP peak heights in the genes identified as associating with G4 motifs ([G3-5N1-7]4). Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


A . Representative normalised RNA DIP - seq data in the SKI locus . Wild type in blue ; primpol in red . The locations of H - DNA and G4 motifs are shown below the gene map . The corresponding RNAse H - treated samples are dashed . Since so little material was recovered following RNase H treatment , all samples were pooled prior to library generation . Data information : P values calculated with Mann - Whitney U test . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper and lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative normalised RNA DIP-seq data in the SKI locus. Wild type in blue; primpol in red. The locations of H-DNA and G4 motifs are shown below the gene map. The corresponding RNAse H-treated samples are dashed. Since so little material was recovered following RNase H treatment, all samples were pooled prior to library generation. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


C . RNA DIP - seq peak heights in wild type and primpol BOBSC cells normalised to a DT40 spike - in . n ( wild type ) = 32740 ; n ( primpol ) = 33721 . D . Correlation of normalised RNA DIP - seq peak heights in the overlapping peaks between wild type and primpol . Blue line = 1 : 1 correlation ; red line = linear regression through data . Data information : P values calculated with Mann - Whitney U test . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper and lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RNA DIP-seq peak heights in wild type and primpol BOBSC cells normalised to a DT40 spike-in. n (wild type) = 32740; n (primpol) = 33721. D. Correlation of normalised RNA DIP-seq peak heights in the overlapping peaks between wild type and primpol. Blue line = 1:1 correlation; red line = linear regression through data. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


E . Correlation between H - DNA forming sequences and all genes ( white bar ) , and genes with DRIP peaks in wild type ( blue ) and primpol cells ( red ) . Data information : P values calculated with Mann - Whitney U test . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper and lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Correlation between H-DNA forming sequences and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red). Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


F . Correlation between G4 motifs ( [ G3 - 5N1 - 7 ] 4 ) and all genes ( white bar ) , and genes with DRIP peaks in wild type ( blue ) and primpol cells ( red ) . Data information : P values calculated with Mann - Whitney U test . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper and lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Correlation between G4 motifs ([G3-5N1-7]4) and all genes (white bar), and genes with DRIP peaks in wild type (blue) and primpol cells (red). Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


G . Normalised RNA DIP - seq peak heights in the genes identified as associating with H - DNA . Data information : P values calculated with Mann - Whitney U test . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper and lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Normalised RNA DIP-seq peak heights in the genes identified as associating with H-DNA. Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


H . Normalised RNA DIP - seq peak heights in the genes identified as associating with G4 motifs ( [ G3 - 5N1 - 7 ] 4 ) . Data information : P values calculated with Mann - Whitney U test . In violin plots , bar = median ; box = interquartile range ( IQR ) ; whiskers = upper and lower inner fences ( 1st / 3rd quartile + 1 . 5 * IQR ) .

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

H. Normalised RNA DIP-seq peak heights in the genes identified as associating with G4 motifs ([G3-5N1-7]4). Data information: P values calculated with Mann-Whitney U test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1st / 3rd quartile + 1.5*IQR).


( A ) Consecutive in vivo imaging of the same apical dendrites from layer V pyramidal neurons in the somatosensory cortex over 7 weeks reveals formation and elimination of dendritic spines ( white and empty arrowheads , respectively ) in WT and APP - KO mice . All mice were housed in standard conditions . Scale bar - 10 μm . ( B - E ) Graphical representations of the relative spine density , spine turnover rate ( TOR ) , elimination and formation over the imaging period . Non - linear regression ( F test ) has been used for fitting the data points and comparing the difference between groups . N = 4 mice per group * * p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Consecutive in vivo imaging of the same apical dendrites from layer V pyramidal neurons in the somatosensory cortex over 7 weeks reveals formation and elimination of dendritic spines (white and empty arrowheads, respectively) in WT and APP-KO mice. All mice were housed in standard conditions. Scale bar - 10 μm. (B-E) Graphical representations of the relative spine density, spine turnover rate (TOR), elimination and formation over the imaging period. Non-linear regression (F test) has been used for fitting the data points and comparing the difference between groups. N=4 mice per group ** p<0.01.


( A ) Consecutive in vivo imaging of the same apical dendrites from layer V pyramidal neurons in the somatosensory cortex over 46 days reveals formation and elimination of dendritic spines ( white and empty arrowheads , respectively ) in WT and APP - KO mice . Prior to the exposure to environmental enrichment ( EE ) , all mice were housed in standard conditions . Scale bar - 10 μm . ( B - C ) Graphical representations of the relative spine density and spine elimination over the period of the exposure of mice to EE . Non - linear regression ( F test ) has been used for fitting the data points . Repeated one - way ANOVA was performed followed by Dunnett test . WT n = 5 mice and APP - KO n = 6 mice ; * p < 0 . 05 , * * p < 0 . 01 , NS - no significant difference .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Consecutive in vivo imaging of the same apical dendrites from layer V pyramidal neurons in the somatosensory cortex over 46 days reveals formation and elimination of dendritic spines (white and empty arrowheads, respectively) in WT and APP-KO mice. Prior to the exposure to environmental enrichment (EE), all mice were housed in standard conditions. Scale bar - 10 μm. (B-C) Graphical representations of the relative spine density and spine elimination over the period of the exposure of mice to EE. Non-linear regression (F test) has been used for fitting the data points. Repeated one-way ANOVA was performed followed by Dunnett test. WT n=5 mice and APP-KO n=6 mice; * p<0.05, ** p<0.01, NS - no significant difference.


( A ) Typical confocal images of apical dendrites with spines ( z - projections ) from layer V pyramidal neurons in the somatosensory cortex of WT and APP - KO mice ( top and bottom ) housed in standard and enriched environments ( left and right ) . For classification of spine types , 3D reconstructions by Imaris have been applied . Thin , mushroom and stubby spines are encoded in blue , green and red , respectively . Scale bar represents 2 μm . ( B - C ) Summary plots of thin and mushroom spine fractions in WT and APP - KO mice exposed to standard ( SE ) and enriched environments ( EE ) . . Two - tailed Student t - test was used and n = 6 mice in all experimental groups ; * p < 0 . 05 , * * p < 0 . 01 , NS - no significant difference .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Typical confocal images of apical dendrites with spines (z-projections) from layer V pyramidal neurons in the somatosensory cortex of WT and APP-KO mice (top and bottom) housed in standard and enriched environments (left and right). For classification of spine types, 3D reconstructions by Imaris have been applied. Thin, mushroom and stubby spines are encoded in blue, green and red, respectively. Scale bar represents 2 μm. (B-C) Summary plots of thin and mushroom spine fractions in WT and APP-KO mice exposed to standard (SE) and enriched environments (EE). . Two-tailed Student t-test was used and n=6 mice in all experimental groups; * p<0.05, ** p<0.01, NS - no significant difference.


( A ) Extracellular D - serine concentrations in cortex were detected by enzyme - based microelectrode biosensors .

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(A) Extracellular D-serine concentrations in cortex were detected by enzyme-based microelectrode biosensors.


( B , C ) High performance liquid chromatography was performed to quantify total D - serine and L - serine contents in mouse brain of two genotypes . APP - KO mice were fed with D - serine over one month . One - way ANOVA was used for analysis followed by Bonferroni ' s multiple comparisons test and n > 6 mice per group ; * p < 0 . 05 , * * p < 0 . 01 as compared to WT .

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

(B, C) High performance liquid chromatography was performed to quantify total D-serine and L-serine contents in mouse brain of two genotypes. APP-KO mice were fed with D-serine over one month. One-way ANOVA was used for analysis followed by Bonferroni's multiple comparisons test and n>6 mice per group; * p<0.05, ** p<0.01 as compared to WT.


( A ) Consecutive in vivo imaging of the same apical dendrites from layer V pyramidal neurons in the somatosensory cortex of APP - KO mice housed under standard or enriched environment . Note that both groups of mice received D - serine after the second imaging time point ( 8 d ) ; white and empty arrowheads point to newly formed and eliminated spines , respectively . Scale bar - 10 μm . ( B ) Spine TOR prior and during continuous D - serine treatment . ( C , D ) Summary plots of the fraction of spine elimination and formation in APP - KO mice before and after D - serine treatment ( 8 d and 46 d , respectively ) . ( E ) Relative spine densities in D - serine treated APP - KO mice housed under standard and enriched environments . ( F , G ) Summary plots of the fraction of thin and mushroom spines in control and D - serine treated APP - KO mice . For illustration purpose , the control data from Figure 2 B and C are presented also here . Non - linear regression ( F test ) has been used for fitting the data points . Two - tailed Student t - test was used in ( C - D , F - G ) and repeated one - way ANOVA was performed followed by Dunnett test in ( B and E ) . N = 5 mice in each group ; * p < 0 . 05 , * * p < 0 . 01 , NS - no significant difference .

O O O O O O O O O O O O O O O O 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 O O 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Consecutive in vivo imaging of the same apical dendrites from layer V pyramidal neurons in the somatosensory cortex of APP-KO mice housed under standard or enriched environment. Note that both groups of mice received D-serine after the second imaging time point (8 d); white and empty arrowheads point to newly formed and eliminated spines, respectively. Scale bar - 10 μm. (B) Spine TOR prior and during continuous D-serine treatment. (C, D) Summary plots of the fraction of spine elimination and formation in APP-KO mice before and after D-serine treatment (8 d and 46 d, respectively). (E) Relative spine densities in D-serine treated APP-KO mice housed under standard and enriched environments. (F, G) Summary plots of the fraction of thin and mushroom spines in control and D-serine treated APP-KO mice. For illustration purpose, the control data from Figure 2 B and C are presented also here. Non-linear regression (F test) has been used for fitting the data points. Two-tailed Student t-test was used in (C-D, F-G) and repeated one-way ANOVA was performed followed by Dunnett test in (B and E). N=5 mice in each group; * p<0.05, ** p<0.01, NS - no significant difference.


( A ) Quantification of total distance that mice traveled for 10 min .

(A) Quantification of total distance that mice traveled for 10 min.


( B ) The ratios of mice explored between novel and familiar objects . Lines in boxes indicate mean levels and whiskers represent the data from minimal to maximal range . The box extends from 25 % to 75 % of values . Wilcoxon signed - rank test was performed to compare data to the hypothetical value 1 . N = 9 - 12 mice per group ; * p < 0 . 05 , * * p < 0 . 01 , NS - no significant difference .

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(B) The ratios of mice explored between novel and familiar objects. Lines in boxes indicate mean levels and whiskers represent the data from minimal to maximal range. The box extends from 25% to 75% of values. Wilcoxon signed-rank test was performed to compare data to the hypothetical value 1. N=9-12 mice per group; * p<0.05, ** p<0.01, NS - no significant difference.


( A ) IDH1 acetylation levels upon treatment with NAM or TSA . Flag - tagged IDH1 was ectopically expressed in HEK293T cells treated with NAM ( 5 mM ) and / or TSA ( 0 . 5 mM ) for the indicated time period .

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(A) IDH1 acetylation levels upon treatment with NAM or TSA. Flag-tagged IDH1 was ectopically expressed in HEK293T cells treated with NAM (5 mM) and/or TSA (0.5 mM) for the indicated time period.


( B ) Five putative lysine residues were mutated . Acetylation levels of Flag - bead - purified IDH1 were determined by Western blot analysis using a pan - anti - acetyllysine antibody . Relative IDH1 acetylation ratios were calculated after normalizing against Flag .

O O O O O O O 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

(B) Five putative lysine residues were mutated. Acetylation levels of Flag-bead-purified IDH1 were determined by Western blot analysis using a pan-anti-acetyllysine antibody. Relative IDH1 acetylation ratios were calculated after normalizing against Flag.


( C ) Mutation of IDH1 K224R and K224Q resulted in altered acetylation levels using Western Blot .

(C) Mutation of IDH1 K224R and K224Q resulted in altered acetylation levels using Western Blot.


( D ) IDH1 catalytic activity of IDH1 K224 mutants in vitro . Wild - type IDH1 and K224R , K224Q mutants were expressed in HEK293 cells . Proteins were purified by immunoprecipitation ( IP ) , IDH1 levels were normalized for protein , and activity assays were performed . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For multiple comparisons in ( D ) , a one - way ANOVA with Newman - Keuls post hoc test was used to test for statistical significance .

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(D) IDH1 catalytic activity of IDH1 K224 mutants in vitro. Wild-type IDH1 and K224R, K224Q mutants were expressed in HEK293 cells. Proteins were purified by immunoprecipitation (IP), IDH1 levels were normalized for protein, and activity assays were performed. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For multiple comparisons in (D), a one-way ANOVA with Newman-Keuls post hoc test was used to test for statistical significance.


( E - F ) Steady - state kinetic analysis of IDH1 and variants in HEK293T cells . Comparison of IDH1 WT and K224R and K224Q showed that the K224 site was important in catalysis . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For ( E - F ) , statistical significance was determined using the two - way ANOVA followed by Tukey ' s post hoc test . * P < 0 . 05 and * * P < 0 . 01 .

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(E-F) Steady-state kinetic analysis of IDH1 and variants in HEK293T cells. Comparison of IDH1 WT and K224R and K224Q showed that the K224 site was important in catalysis. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For (E-F), statistical significance was determined using the two-way ANOVA followed by Tukey's post hoc test. *P<0.05 and **P<0.01.


( G ) IDH1 directly interacted with SIRT2 but not SIRT1 . SIRT2 deacetylated IDH1 effectively . Flag tagged IDH1 and HA tagged SIRT1 or SIRT2 were co - transfected into cells . The acetylation level of Flag - bead - purified IDH1 and the protein association between IDH1 and SIRT1 or SIRT2 were determined by Western blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IDH1 directly interacted with SIRT2 but not SIRT1. SIRT2 deacetylated IDH1 effectively. Flag tagged IDH1 and HA tagged SIRT1 or SIRT2 were co-transfected into cells. The acetylation level of Flag-bead-purified IDH1 and the protein association between IDH1 and SIRT1 or SIRT2 were determined by Western blot.


( H ) Co - immunoprecipitation showed endogenous proteins of IDH1 and SIRT2 interacted with each other .

(H) Co-immunoprecipitation showed endogenous proteins of IDH1 and SIRT2 interacted with each other.


( I ) SIRT2 interacted with IDH1 in vitro . GST pull - down assay with GST - fused SIRT2 and Flag - tagged IDH1 proteins .

O O O 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) SIRT2 interacted with IDH1 in vitro. GST pull-down assay with GST-fused SIRT2 and Flag-tagged IDH1 proteins.


( J ) Acetylation level of endogenous IDH1 K224 when treated with SIRT1 or SIRT2 by Western blot analysis .

(J) Acetylation level of endogenous IDH1 K224 when treated with SIRT1 or SIRT2 by Western blot analysis.


( K ) IDH1 K224 deacetylation was dependent on SIRT2 catalytic activity . SIRT2 H187Y was a catalytically inactive mutant .

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

(K) IDH1 K224 deacetylation was dependent on SIRT2 catalytic activity. SIRT2 H187Y was a catalytically inactive mutant.


( A ) Colony formation ability after transfection of SIRT2 plasmids , AGK2 and SIRT2 siRNA . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For A statistical significance was assessed with the one - way ANOVA with Newman - Keuls post hoc test . * P < 0 . 05 and * * P < 0 . 01 .

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(A) Colony formation ability after transfection of SIRT2 plasmids, AGK2 and SIRT2 siRNA. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For A statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01.


( B ) Transwell assay was performed to evaluate the invasion abilities of HCT116 cells after overexpression or downregulation of SIRT2 or treated with AGK2 . Stained cells in the lower chambers were quantified . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For statistical significance was assessed with the one - way ANOVA with Newman - Keuls post hoc test . * P < 0 . 05 and * * P < 0 . 01 .

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

(B) Transwell assay was performed to evaluate the invasion abilities of HCT116 cells after overexpression or downregulation of SIRT2 or treated with AGK2. Stained cells in the lower chambers were quantified. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01.


( C ) Cell migration ability was assessed by wound healing assay in HCT116 cells . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For figure C , statistical significance was assessed with the two - way ANOVA followed by Tukey ' s post hoc test . * P < 0 . 05 and * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell migration ability was assessed by wound healing assay in HCT116 cells. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For figure C, statistical significance was assessed with the two-way ANOVA followed by Tukey's post hoc test. *P<0.05 and **P<0.01.


( D ) Cell cycle distribution was evaluated flow cytometrically after treatment with SIRT2 plasmids , SIRT2 siRNA or AGK2 . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For D , statistical significance was assessed with the one - way ANOVA with Newman - Keuls post hoc test . * P < 0 . 05 and * * P < 0 . 01 .

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 O O O O O O

(D) Cell cycle distribution was evaluated flow cytometrically after treatment with SIRT2 plasmids, SIRT2 siRNA or AGK2. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For D, statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01.


( E ) Immunohistochemical analysis of SIRT2 protein in representative colorectal tissue specimens is shown ( magnification × 100 ) . Weak cytoplasmic staining for SIRT2 was observed in CRC tissues and metastatic tissues , whereas strong cytoplasmic staining was observed in noncancerous tissues .

O O O O O O O O O O O O O O O O O O O O O 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) Immunohistochemical analysis of SIRT2 protein in representative colorectal tissue specimens is shown (magnification ×100). Weak cytoplasmic staining for SIRT2 was observed in CRC tissues and metastatic tissues, whereas strong cytoplasmic staining was observed in noncancerous tissues.


( A - B ) IDH1 K224 acetylation level was enhanced with increasing glucose concentration . Flag - IDH1 was overexpressed in cells treated with increased concentrations of glucose for 6 h . IDH1 proteins were purified by Flag beads , and then IDH1 K224 acetylation level was determined by Western blot and IDH1 catalytic activity was assessed . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For B statistical significance was assessed with the one - way ANOVA with Newman - Keuls post hoc test . * P < 0 . 05 and * * P < 0 . 01 . n . s . = not significant .

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

(A-B) IDH1 K224 acetylation level was enhanced with increasing glucose concentration. Flag-IDH1 was overexpressed in cells treated with increased concentrations of glucose for 6 h. IDH1 proteins were purified by Flag beads, and then IDH1 K224 acetylation level was determined by Western blot and IDH1 catalytic activity was assessed. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For B statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.


( C - D ) IDH1 K224 acetylation level was upregulated and IDH1 activity was weakened with increasing glutamine concentration . Flag - IDH1 was overexpressed in cells treated with increased glutamine for 6 h . IDH1 proteins were purified by Flag beads , and the K224 acetylation level of IDH1 was determined by Western blot analysis and then IDH1 catalytic activity was evaluated . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For , D statistical significance was assessed with the one - way ANOVA with Newman - Keuls post hoc test . P < 0 . 05 and * * P < 0 . 01 . n . s . = not significant .

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

(C-D) IDH1 K224 acetylation level was upregulated and IDH1 activity was weakened with increasing glutamine concentration. Flag-IDH1 was overexpressed in cells treated with increased glutamine for 6 h. IDH1 proteins were purified by Flag beads, and the K224 acetylation level of IDH1 was determined by Western blot analysis and then IDH1 catalytic activity was evaluated. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For , D statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. P<0.05 and **P<0.01. n.s. = not significant.


Downregulation of SIRT2 diminished the effect of glucose on changing IDH1 K224 acetylation . Flag - tagged IDH1 was overexpressed in HCT116 cells with or without transient SIRT2 knockdown . The cells were treated with different concentrations of glucose ( E )

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 B-CONTROLLED_VAR O O O

Downregulation of SIRT2 diminished the effect of glucose on changing IDH1 K224 acetylation. Flag-tagged IDH1 was overexpressed in HCT116 cells with or without transient SIRT2 knockdown. The cells were treated with different concentrations of glucose (E)


Downregulation of SIRT2 diminished the effect of glutamine on changing IDH1 K224 acetylation . Flag - tagged IDH1 was overexpressed in HCT116 cells with or without transient SIRT2 knockdown . The cells were treated with different concentrations of glutamine ( F ) .

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 B-CONTROLLED_VAR O O O O

Downregulation of SIRT2 diminished the effect of glutamine on changing IDH1 K224 acetylation. Flag-tagged IDH1 was overexpressed in HCT116 cells with or without transient SIRT2 knockdown. The cells were treated with different concentrations of glutamine (F).


( G ) IDH1 K224 deacetylation regulated cellular NADPH / NADP + redox in cells . In HCT116 stable cells with IDH1 knockout and re - expressing the indicated proteins , the ratio of NADPH / NADP + in cells was measured as followed by manufacture ' s instruction . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For , G statistical significance was assessed with the one - way ANOVA with Newman - Keuls post hoc test . * P < 0 . 05 and * * P < 0 . 01 . n . s . = not significant .

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

(G) IDH1 K224 deacetylation regulated cellular NADPH/NADP+ redox in cells. In HCT116 stable cells with IDH1 knockout and re-expressing the indicated proteins, the ratio of NADPH/NADP+ in cells was measured as followed by manufacture's instruction. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For , G statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.


( H ) IDH1 K224 deacetylation promoted GSH production in HCT116 cells . In HCT116 stable cells with IDH1 knockout and re - expressing the indicated proteins , the ratio of GSH / GSSG was assessed as followed by manufacture ' s instruction . 1 Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For H , statistical significance was assessed with the one - way ANOVA with Newman - Keuls post hoc test . * P < 0 . 05 and * * P < 0 . 01 . n . s . = not significant .

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

(H) IDH1 K224 deacetylation promoted GSH production in HCT116 cells. In HCT116 stable cells with IDH1 knockout and re-expressing the indicated proteins, the ratio of GSH/GSSG was assessed as followed by manufacture's instruction. 1 Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For H, statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.


( I ) IDH1 K224 deacetylation suppressed cellular ROS levels in HCT116 cells . ROS was determined in cells under non - stressed condition or exposed to menadione . * P < 0 . 05 and * * P < 0 . 01 Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For figure I , statistical significance was assessed with the two - way ANOVA followed by Tukey ' s post hoc test . * P < 0 . 05 and * * P < 0 . 01 . n . s . = not significant .

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

(I) IDH1 K224 deacetylation suppressed cellular ROS levels in HCT116 cells. ROS was determined in cells under non-stressed condition or exposed to menadione. *P<0.05 and **P<0.01 Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For figure I, statistical significance was assessed with the two-way ANOVA followed by Tukey's post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.


( A ) Gene expression profile array analysis in HCT116 cells . Representative genes with higher fold difference ( up or down ) compared IDH1 K224R cells with IDH1 WT cells . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Gene expression profile array analysis in HCT116 cells. Representative genes with higher fold difference (up or down) compared IDH1 K224R cells with IDH1 WT cells. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition).


( B ) ChIP assays for HIF1α and its binding motif . Antibodies anti - IgG and anti - HIF1α were used in the ChIP assays . qRT - PCR was performed to quantify the binding activity . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For B , statistical significance was assessed with a two - tailed unpaired Student ' s t - test . * P < 0 . 05 and * * P < 0 . 01 . n . s . = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ChIP assays for HIF1α and its binding motif. Antibodies anti-IgG and anti- HIF1α were used in the ChIP assays. qRT-PCR was performed to quantify the binding activity. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For B , statistical significance was assessed with a two-tailed unpaired Student's t-test. *P<0.05 and **P<0.01. n.s. = not significant.


( C - D ) The transcriptional activity of the SRC promoter was greatly increased after co - expression of HIF1α and SRC reporter in wild type . SRC promoter constructs containing a potential HIF1α binding motif ( - 1636 ~ - 1629 bp and - 1466 ~ - 1459 bp ) . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For D , statistical significance was assessed with a two - tailed unpaired Student ' s t - test . * P < 0 . 05 and * * P < 0 . 01 . n . s . = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) The transcriptional activity of the SRC promoter was greatly increased after co-expression of HIF1α and SRC reporter in wild type. SRC promoter constructs containing a potential HIF1α binding motif (-1636~-1629 bp and -1466~-1459 bp). Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For D, statistical significance was assessed with a two-tailed unpaired Student's t-test. *P<0.05 and **P<0.01. n.s. = not significant.


( E ) IDH1 K224 deacetylation led to degradation of HIF1α but upregulation of hydroxy - HIF1α . In HCT116 stable cells with IDH1 knockout and re - expressing the indicated proteins , HIF1α and hydroxy - HIF1α was measured by Western blot assay .

(E) IDH1 K224 deacetylation led to degradation of HIF1α but upregulation of hydroxy-HIF1α. In HCT116 stable cells with IDH1 knockout and re-expressing the indicated proteins, HIF1α and hydroxy-HIF1α was measured by Western blot assay.


( F ) HCT116 cells of IDH1 K224Q were cotreated with Octyl - α - KG and NAC , the expression of HIF1α and SRC was further evaluated .

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(F) HCT116 cells of IDH1 K224Q were cotreated with Octyl-α-KG and NAC, the expression of HIF1α and SRC was further evaluated.


HCT116 cells expression IDH1 K224Q with or without treatment with Octyl - α - KG and / or NAC were analyzed for glucose levels . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For G statistical significance was assessed with a one - way ANOVA with Newman - Keuls post hoc test . * P < 0 . 05 and * * P < 0 . 01 . n . s . = not significant .

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

HCT116 cells expression IDH1 K224Q with or without treatment with Octyl-α-KG and/or NAC were analyzed for glucose levels. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For G statistical significance was assessed with a one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.


HCT116 cells expression IDH1 K224Q with or without treatment with Octyl - α - KG and / or NAC were analyzed for lactate levels . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For H , statistical significance was assessed with a one - way ANOVA with Newman - Keuls post hoc test . * P < 0 . 05 and * * P < 0 . 01 . n . s . = not significant .

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

HCT116 cells expression IDH1 K224Q with or without treatment with Octyl-α-KG and/or NAC were analyzed for lactate levels. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For H, statistical significance was assessed with a one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.


( A ) Transwell assay was performed to evaluate the invasion abilities of HCT116 cells after transfection of IDH1 K224R or knockout of IDH1 . Quantification was performed by counting the stained cells that invaded to the lower chamber under a light microscopy . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For A statistical significance was assessed with a one - way ANOVA with Newman - Keuls post hoc test . * P < 0 . 05 and * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Transwell assay was performed to evaluate the invasion abilities of HCT116 cells after transfection of IDH1 K224R or knockout of IDH1. Quantification was performed by counting the stained cells that invaded to the lower chamber under a light microscopy. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For A statistical significance was assessed with a one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01.


( B ) Three - dimension imaging analysis was to assessed velocity of cell movement . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For B , statistical significance was assessed with a one - way ANOVA with Newman - Keuls post hoc test . * P < 0 . 05 and * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Three-dimension imaging analysis was to assessed velocity of cell movement. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For B, statistical significance was assessed with a one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01.


( C ) Cell migration ability was evaluated by wound healing assay in HCT116 cells . Quantification was performed by measuring the smallest clearance distance of the wound . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For figure C , statistical significance was assessed with a two - way ANOVA followed by Tukey ' s post hoc test . * P < 0 . 05 and * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell migration ability was evaluated by wound healing assay in HCT116 cells. Quantification was performed by measuring the smallest clearance distance of the wound. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For figure C, statistical significance was assessed with a two-way ANOVA followed by Tukey's post hoc test. *P<0.05 and **P<0.01.


( D ) Colony formation ability after deacetylation of IDH1 K224 or knockout of IDH1 . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For statistical significance was assessed with a one - way ANOVA with Newman - Keuls post hoc test . * P < 0 . 05 and * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Colony formation ability after deacetylation of IDH1 K224 or knockout of IDH1. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For statistical significance was assessed with a one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01.


( E ) Cell cycle distribution was assessed flow cytometrically after treatment with IDH1 K224R or knockout of IDH1 . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . For E , statistical significance was assessed with a one - way ANOVA with Newman - Keuls post hoc test . * P < 0 . 05 and * * P < 0 . 01 .

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(E) Cell cycle distribution was assessed flow cytometrically after treatment with IDH1 K224R or knockout of IDH1. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For E, statistical significance was assessed with a one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01.


( A ) Pictures of the nude mice and their tumors after inoculation was shown in two subgroups . ( B ) Tumor volumes of each group during the tumor growth process . ( C ) Tumor weight measured from the xenograft model . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . Statistical significance was assessed with a two - tailed unpaired Student ' s t - test . * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pictures of the nude mice and their tumors after inoculation was shown in two subgroups. (B) Tumor volumes of each group during the tumor growth process. (C) Tumor weight measured from the xenograft model. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). Statistical significance was assessed with a two-tailed unpaired Student's t-test. **P<0.01.


( D ) Pictures of livers in nude mice and their respective representative images of the tissues by hematoxylin - eosin ( HE ) staining . Data information : All results were expressed as mean ± SD of three independent experiments ( n ≥ 3 per experimental condition ) . Statistical significance was assessed with a two - tailed unpaired Student ' s t - test . * * P < 0 . 01 .

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(D) Pictures of livers in nude mice and their respective representative images of the tissues by hematoxylin-eosin (HE) staining. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). Statistical significance was assessed with a two-tailed unpaired Student's t-test. **P<0.01.


( A ) Expression levels of IDH1 K224Ac in human CRC ( n = 90 ) and CRN ( n = 90 ) tissue specimens , assessed by immunohistochemistry . CRC , colorectal carcinoma samples ; CRN , matched adjacent non - cancerous colorectal tissue specimens .

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(A) Expression levels of IDH1 K224Ac in human CRC (n=90) and CRN (n=90) tissue specimens, assessed by immunohistochemistry. CRC, colorectal carcinoma samples; CRN, matched adjacent non-cancerous colorectal tissue specimens.


( B ) Kaplan - Meier survival curves of CRC cases with elevated ( n = 49 ) and reduced ( n = 41 ) IDH1 K224Ac . CRC , colorectal carcinoma samples . The overall survival was analyzed by the log - rank test using the Kaplan - Meier method .

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(B) Kaplan-Meier survival curves of CRC cases with elevated (n=49) and reduced (n=41) IDH1 K224Ac. CRC, colorectal carcinoma samples. The overall survival was analyzed by the log-rank test using the Kaplan-Meier method.


Characterization of OAS1 - 3 expression in parental and OAS knockout PC3 cells , obtained using CRISPR - Cas9 technology . Left : Western blot ( WB ) analyses of the expression of OAS1 , 2 , and 3 in clones of OAS single - , double - or triple - knockout PC3 cells ( KO , DKO , TKO ) , treated with IFNβ ( 500 IU / ml ) for 24 h , to increase OAS expression . sgOAS1 - 3 - single guide RNA for OAS1 - 3 . β - actin is shown as a loading control . Right : analysis of the expression of OAS1 , 2 , and 3 in PC3 DKO cells with increased expression of the remaining OAS , without IFNβ treatment . OAS1 expression in DKO2 / 3 cells was measured by qRT - PCR due to insufficient sensitivity of anti - OAS1 ( see Fig EV1A ) , OAS2 and 3 expression was detected by WB . The gene expression data are presented as mean + SD of triplicates . * * , p < 0 . 001 in Student ' s t - test .

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Characterization of OAS1-3 expression in parental and OAS knockout PC3 cells, obtained using CRISPR-Cas9 technology. Left: Western blot (WB) analyses of the expression of OAS1, 2, and 3 in clones of OAS single-, double- or triple-knockout PC3 cells (KO, DKO, TKO), treated with IFNβ (500 IU/ml) for 24 h, to increase OAS expression. sgOAS1-3 - single guide RNA for OAS1-3. β-actin is shown as a loading control. Right: analysis of the expression of OAS1, 2, and 3 in PC3 DKO cells with increased expression of the remaining OAS, without IFNβ treatment. OAS1 expression in DKO2/3 cells was measured by qRT-PCR due to insufficient sensitivity of anti-OAS1 (see Fig EV1A), OAS2 and 3 expression was detected by WB. The gene expression data are presented as mean + SD of triplicates.**, p<0.001 in Student's t-test.


Characterization of OAS1 - 3 expression in parental and OAS knockout HME cells , obtained using CRISPR - Cas9 technology . Left : WB analyses of the expression of OAS1 , 2 , and 3 in selected clones of HME cells in which OAS1 - 3 were knocked out , to produce OAS single - , double - or triple - knockout cells ( KO , DKO , TKO ) , treated with 100 IU / ml IFNβ for 24 h to increase the levels of OAS expression . sgOAS1 - 3 - single guide RNA for OAS1 - 3 . β - actin is shown as a loading control . Right : The expression of OAS1 , 2 , and 3 in HME DKO cells with increased expression of the remaining OAS , without IFNβ treatment . The expression of OAS2 or 3 was determined by WB analysis ( asterisk , non - specific band ) . OAS1 expression in DKO2 / 3 cells was measured by qRT - PCR due to insufficient sensitivity of anti - OAS1 . The gene expression data are presented as mean + SD of triplicates . * * , p < 0 . 001 in Student ' s t - test .

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

Characterization of OAS1-3 expression in parental and OAS knockout HME cells, obtained using CRISPR-Cas9 technology. Left: WB analyses of the expression of OAS1, 2, and 3 in selected clones of HME cells in which OAS1-3 were knocked out, to produce OAS single-, double- or triple-knockout cells (KO, DKO, TKO), treated with 100 IU/ml IFNβ for 24 h to increase the levels of OAS expression. sgOAS1-3 - single guide RNA for OAS1-3. β-actin is shown as a loading control. Right: The expression of OAS1, 2, and 3 in HME DKO cells with increased expression of the remaining OAS, without IFNβ treatment. The expression of OAS2 or 3 was determined by WB analysis (asterisk, non-specific band). OAS1 expression in DKO2/3 cells was measured by qRT-PCR due to insufficient sensitivity of anti-OAS1. The gene expression data are presented as mean + SD of triplicates. **, p<0.001 in Student's t-test.


OAS expression was assessed by WB with anti OAS1 - 3 in HME cells expressing ectopic OAS1 - 3 ( OAS2hi , OAS3hi , OAS1hi ) cells without IFNβ treatment or control HME cells ( vec ) treated with the indicated concentrations of IFNβ for 2 days ( to compare the levels of induced and ectopically expressed OASs ) . β - actin and Ponceau stain are shown as loading controls ( # - transfer artifact ) ; ns , non - specific band .

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OAS expression was assessed by WB with anti OAS1-3 in HME cells expressing ectopic OAS1-3 (OAS2hi, OAS3hi, OAS1hi) cells without IFNβ treatment or control HME cells (vec) treated with the indicated concentrations of IFNβ for 2 days (to compare the levels of induced and ectopically expressed OASs). β-actin and Ponceau stain are shown as loading controls (# - transfer artifact); ns, non-specific band.


Analysis of the percentages of dead PC3 DKO cells or PC3 DKO cells with high expression of the remaining OAS , treated with 75 µM H2O2 , 40 μM p - Benzoquinone ( pBQ ) or 2 mM methyl methane sulfonate ( MMS ) . The number of dead cells ( Cytotox Green - permeable ) was monitored every 3 h using an IncuCyte ZOOM instrument . Means of triplicates ± SD . * * * , p < 0 . 001 ; * , p < 0 . 05 ; ns , no significance in two - way ANOVA . Representative data , which were reproduced more than 3 times , are shown .

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Analysis of the percentages of dead PC3 DKO cells or PC3 DKO cells with high expression of the remaining OAS, treated with 75 µM H2O2, 40 μM p-Benzoquinone (pBQ) or 2 mM methyl methane sulfonate (MMS). The number of dead cells (Cytotox Green-permeable) was monitored every 3 h using an IncuCyte ZOOM instrument. Means of triplicates ± SD. ***, p<0.001; *, p<0.05; ns, no significance in two-way ANOVA. Representative data, which were reproduced more than 3 times, are shown.


Analysis of the percentages of dead HME DKO cells and HME DKO cells with high expression of the remaining OAS , treated with 500 μM H2O2 , and KO1 - 3 and OAS1 - 3hi cells treated with 750 μM H2O2 . Data were obtained and presented as in ( A ) . Means of triplicates ± SD . * * * , p < 0 . 001 ; * * , p < 0 . 01 ; ns , no significance in two - way ANOVA . Representative data , which were reproduced more than 3 times , are shown .

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Analysis of the percentages of dead HME DKO cells and HME DKO cells with high expression of the remaining OAS, treated with 500 μM H2O2, and KO1-3 and OAS1-3hi cells treated with 750 μM H2O2. Data were obtained and presented as in (A). Means of triplicates ± SD. ***, p<0.001; **, p<0.01; ns, no significance in two-way ANOVA. Representative data, which were reproduced more than 3 times, are shown.


Survival of HME cells , measured by the MTT cell survival assay . Left : control ( vec ) and KO1 - 3 cells were treated with 250 , 500 or 750 μM of H2O2 and analyzed after 24 h ; Right : control , OAS1hi and OAS KO were treated with 500 μM of H2O2 and analyzed after 72 h . The data are presented as mean + SD of triplicates . * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 in Student ' s t - test .

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Survival of HME cells, measured by the MTT cell survival assay. Left: control (vec) and KO1-3 cells were treated with 250, 500 or 750 μM of H2O2 and analyzed after 24 h; Right: control, OAS1hi and OAS KO were treated with 500 μM of H2O2 and analyzed after 72 h . The data are presented as mean + SD of triplicates. ***, p<0.001; **, p<0.01; *, p<0.05 in Student's t-test.


An autoradiograph showing radiolabeled PAR2 - 5A bands that were formed in reactions of OAS1 with the activator ( pIC ) and the acceptor PAR ( lanes 11 - 14 ) . Lanes 1 - 2 OAS1 only , lanes 3 - 6 OAS1 with activator pIC ; lanes 7 - 10 OAS1 with substrate PAR ; lane 15 negative control without OAS1 . 32P - α - ATP was added to all reactions .

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An autoradiograph showing radiolabeled PAR2-5A bands that were formed in reactions of OAS1 with the activator (pIC) and the acceptor PAR (lanes 11-14). Lanes 1-2 OAS1 only, lanes 3-6 OAS1 with activator pIC; lanes 7-10 OAS1 with substrate PAR; lane 15 negative control without OAS1. 32P-α-ATP was added to all reactions.


Top : specificity of anti - 2 ' , 5 ' A , as shown by ELISA . Synthetic 2 ' , 5 ' - p3A3 , PAR2 - 5A # 1 , PAR2 - 5A # 2 ( 2 different preparations of PAR2 - 5A ) . PAR and 3 ' , 5 ' - pA3 were used as competitors . Bottom : Dot - blot of synthetic PAR and 2 - 5A - PAR . In vitro synthesized PAR and PAR - 2 - 5 - A were applied in 1 : 2 dilutions ( left to right ) to nylon membrane and probed with anti - 2 - 5A or anti - PAR .

Top: specificity of anti-2',5'A, as shown by ELISA. Synthetic 2',5'-p3A3, PAR2-5A #1, PAR2-5A #2 (2 different preparations of PAR2-5A). PAR and 3',5'-pA3 were used as competitors. Bottom: Dot-blot of synthetic PAR and 2-5A-PAR. In vitro synthesized PAR and PAR-2-5-A were applied in 1:2 dilutions (left to right) to nylon membrane and probed with anti-2-5A or anti-PAR.


Immunostaining of PAR - 2 - 5A synthesized in vitro . Reaction mixtures were resolved by electrophoresis in a polyacrylamide - 8 M urea denaturing gel , transferred to a charged nylon membrane , and probed with mouse anti - PAR 10H ( left panel ) or rabbit anti - 2 - 5A after stripping ( right panel ) . XC : Xylene Cyanol ( apparent size 150 nucleotides ) , BPB : Bromophenol Blue ( apparent size 35 nucleotides ) . Lane 1 , purified PARP1 alone ; lane 2 , PAR reaction : PARP1 , activated DNA , NAD + , and histone ; lane 3 , 2 - 5A reaction : OAS1 , pIC , ATP ; lane 4 , 2 - 5A and PAR reactions : OAS1 , pIC , ATP and PARP1 , activated DNA , NAD , histone . After incubation at 37oC for 60 min , the proteins were removed by digestion with proteinase K .

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Immunostaining of PAR-2-5A synthesized in vitro. Reaction mixtures were resolved by electrophoresis in a polyacrylamide-8 M urea denaturing gel, transferred to a charged nylon membrane, and probed with mouse anti-PAR 10H (left panel) or rabbit anti-2-5A after stripping (right panel). XC: Xylene Cyanol (apparent size 150 nucleotides), BPB: Bromophenol Blue (apparent size 35 nucleotides). Lane 1, purified PARP1 alone; lane 2, PAR reaction: PARP1, activated DNA, NAD+, and histone; lane 3, 2-5A reaction: OAS1, pIC, ATP; lane 4, 2-5A and PAR reactions: OAS1, pIC, ATP and PARP1, activated DNA, NAD, histone. After incubation at 37oC for 60 min, the proteins were removed by digestion with proteinase K.


Western analysis of PARP1 auto - modified with PAR in vitro in the absence or presence of OAS1 . Reaction mixtures were resolved by SDS - PAGE , transferred to a charged nylon membrane , and probed with mouse anti - PARP1 . Reactions were set up as in ( C ) , but without proteinase K digestion . Lane 1 , PARP1 alone ; lane 2 , PAR reaction : PARP1 , activated DNA and NAD ; lane 3 , 2 - 5A reaction : OAS1 , pIC , and ATP ; lane 4 , 2 - 5A and PAR reactions : OAS1 , pIC , and ATP with PARP1 , activated DNA , NAD and histone ; lane 5 , 2 - 5A and PAR reactions without ATP . The large smeary signals are due to PAR chains attached to PARP1 and the smaller sharp bands are free PARP1 . The same blot was probed with anti - PAR , anti - 2 - 5A and anti - OAS1 .

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Western analysis of PARP1 auto-modified with PAR in vitro in the absence or presence of OAS1. Reaction mixtures were resolved by SDS-PAGE, transferred to a charged nylon membrane, and probed with mouse anti-PARP1. Reactions were set up as in (C), but without proteinase K digestion. Lane 1, PARP1 alone; lane 2, PAR reaction: PARP1, activated DNA and NAD; lane 3, 2-5A reaction: OAS1, pIC, and ATP; lane 4, 2-5A and PAR reactions: OAS1, pIC, and ATP with PARP1, activated DNA, NAD and histone; lane 5, 2-5A and PAR reactions without ATP. The large smeary signals are due to PAR chains attached to PARP1 and the smaller sharp bands are free PARP1. The same blot was probed with anti-PAR, anti-2-5A and anti-OAS1.


Western analysis of 2 - 5A precipitated with anti - PAR antibodies from lysates of H2O2 - treated HME cells . Cells were treated with 500 M H2O2 for 12 min , and lysates were incubated with control ( mouse IgG3 ) or mouse anti - PAR . Samples were probed with anti - PAR , anti - 2 - 5A or anti - PARP1 antibodies . Flow - thr . : flow - through , WCL : whole cell lysate , IP : immuno - precipitate . Right panels ( IP ) show exposures for 10 x longer than left panels ( WCL , flow - thr ) . Apparent MW ( kDa ) is indicated on the right . Experiments were repeated two times with similar results .

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Western analysis of 2-5A precipitated with anti-PAR antibodies from lysates of H2O2-treated HME cells. Cells were treated with 500 M H2O2 for 12 min, and lysates were incubated with control (mouse IgG3) or mouse anti-PAR. Samples were probed with anti-PAR, anti-2-5A or anti-PARP1 antibodies. Flow-thr.: flow-through, WCL: whole cell lysate, IP: immuno-precipitate. Right panels (IP) show exposures for 10 x longer than left panels (WCL, flow-thr). Apparent MW (kDa) is indicated on the right. Experiments were repeated two times with similar results.


Western analysis of 2 - 5A precipitated with anti - PAR antibodies from lysates of HME control , KO1 and TKO cells , and control cells pre - incubated with olaparib , treated with H2O2 for 12 min . Samples were probed with anti - PAR or anti - 2 - 5A antibodies . Ponceau staining of the membrane is shown as loading control . Experiments were repeated four times with similar results .

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Western analysis of 2-5A precipitated with anti-PAR antibodies from lysates of HME control, KO1 and TKO cells, and control cells pre-incubated with olaparib, treated with H2O2 for 12 min. Samples were probed with anti-PAR or anti-2-5A antibodies. Ponceau staining of the membrane is shown as loading control. Experiments were repeated four times with similar results.


HME cells were treated with H2O2 for the indicated times and stained with anti - PAR and anti - 2 - 5A . Nuclei were visualized with DAPI . The densities of the 2 - 5A and PAR fluorescence signals in the nuclei are plotted . Signals in 40 + cells per sample were measured . Data are presented as distribution plot with mean value + / - SD ; * * * , p < 0 . 001 ; * , p < 0 . 5 ; ns , non - significant ; as determined by unpaired Kolmogorov - Smirnov t - test . Scale bar , 10μm .

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HME cells were treated with H2O2 for the indicated times and stained with anti-PAR and anti-2-5A. Nuclei were visualized with DAPI. The densities of the 2-5A and PAR fluorescence signals in the nuclei are plotted. Signals in 40+ cells per sample were measured. Data are presented as distribution plot with mean value +/- SD; ***, p<0.001; *, p<0.5; ns, non-significant; as determined by unpaired Kolmogorov-Smirnov t-test. Scale bar, 10μm.


HME control ( vector ) and TKO cells were treated with H2O2 for 12 min , incubated on ice for 7 min with PBS or detergent - containing Buffer 1 , to extract unbound proteins , fixed with methanol / acetone , and stained with anti - 2 - 5A and anti - PAR . Scale bar , 10μm .

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HME control (vector) and TKO cells were treated with H2O2 for 12 min, incubated on ice for 7 min with PBS or detergent-containing Buffer 1, to extract unbound proteins, fixed with methanol/acetone, and stained with anti-2-5A and anti-PAR. Scale bar, 10μm.


HME vector control ( vec ) , DKO , and TKO cells were treated with H2O2 for 12 min . In untreated cells , no PAR staining was detected . The densities of fluorescence signals in the nuclei relative to the untreated vector control are plotted . Signals in 40 or more cells per sample were measured . Data are presented as distribution plot with mean value + / - SD ; * * * , p < 0 . 001 ; * , p < 0 . 5 ; ns , non - significant as determined by unpaired Kolmogorov - Smirnov t - test . Scale bar , 10μm .

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HME vector control (vec), DKO, and TKO cells were treated with H2O2 for 12 min. In untreated cells, no PAR staining was detected. The densities of fluorescence signals in the nuclei relative to the untreated vector control are plotted. Signals in 40 or more cells per sample were measured. Data are presented as distribution plot with mean value +/- SD; ***, p<0.001; *, p<0.5; ns, non-significant as determined by unpaired Kolmogorov-Smirnov t-test. Scale bar, 10μm.


HME control ( vector ) and KO1 cells were treated with H2O2 for the indicated times and stained with anti - 2 - 5A and anti - PAR . Scale bar , 10μm .

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HME control (vector) and KO1 cells were treated with H2O2 for the indicated times and stained with anti-2-5A and anti-PAR. Scale bar, 10μm.