C NSMCE2 levels evaluated by Western blot in 2 independent pairs of Nsmce2 + / + and Nsmce2 + / GT MEFs . β ‐ actin was used as a loading control . Numbers shown bellow indicate the relative amounts of NSMCE2 in each sample .

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C NSMCE2 levels evaluated by Western blot in 2 independent pairs of Nsmce2+/+ and Nsmce2+/GT MEFs. β‐actin was used as a loading control. Numbers shown bellow indicate the relative amounts of NSMCE2 in each sample.


D Sister chromatid exchange ( SCE ) events found per metaphase on Nsmce2 + / + and Nsmce2 + / GT MEFs . * * * P 0 . 001 .

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D Sister chromatid exchange (SCE) events found per metaphase on Nsmce2+/+ and Nsmce2+/GT MEFs. ***P 0.001.


E , F Relative percentages of micronuclei ( E ) and polynucleated cells ( F ) on cultures of Nsmce2 + / + and Nsmce2 + / GT MEFs . Data are representative of three independent experiments . Error bars indicate SD . * * * P 0 . 001 .

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E, F Relative percentages of micronuclei (E) and polynucleated cells (F) on cultures of Nsmce2+/+ and Nsmce2+/GT MEFs. Data are representative of three independent experiments. Error bars indicate SD. ***P 0.001.


NSMCE2 levels assessed by WB in UQ . CreERT2 transgenic Nsmce2 + / + and Nsmce2lox / lox littermate MEFs , 24 h after exposure to 4 ‐ OHT . Tubulin levels are shown as a loading control .

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NSMCE2 levels assessed by WB in UQ.CreERT2 transgenic Nsmce2+/+ and Nsmce2lox/lox littermate MEFs, 24 h after exposure to 4‐OHT. Tubulin levels are shown as a loading control.


Clonogenic assay on immortalized UQ . CreERT2 / Nsmce2lox / lox MEFs . The picture shows the colonies detected after 10 days of growth in the presence or absence of 4 ‐ OHT , upon staining with crystal violet .

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Clonogenic assay on immortalized UQ.CreERT2/Nsmce2lox/lox MEFs. The picture shows the colonies detected after 10 days of growth in the presence or absence of 4‐OHT, upon staining with crystal violet.


NSMCE2 and BRCA1 foci in MMS ‐ treated ( 1 mM for 1 h ; 24 ‐ h recovery ) UQ . CreERT2 / Nsmce2lox / lox MEFs , 48 h after being treated ( or not ) with 4 ‐ OHT . Scale bar , 5 μm .

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NSMCE2 and BRCA1 foci in MMS‐treated (1 mM for 1 h; 24‐h recovery) UQ.CreERT2/Nsmce2lox/lox MEFs, 48 h after being treated (or not) with 4‐OHT. Scale bar, 5 μm.


SCE events per metaphase on UQ . CreERT2 / Nsmce2lox / lox MEFs grown in the presence of 4 ‐ OHT for 72 h . * * * P 0 . 001 .

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SCE events per metaphase on UQ.CreERT2/Nsmce2lox/lox MEFs grown in the presence of 4‐OHT for 72 h. ***P 0.001.


Example of SCE events ( arrows ) found on Nsmce2 ‐ deleted MEFs .

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Example of SCE events (arrows) found on Nsmce2‐deleted MEFs.


SCE events at telomeres on UQ . CreERT2 / Nsmce2lox / lox MEFs grown in the presence of 4 ‐ OHT for 72 h , as detected by CO ‐ FISH ( see ) . Data are representative of three independent experiments . Error bars indicate SD . * P 0 . 05 .

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SCE events at telomeres on UQ.CreERT2/Nsmce2lox/lox MEFs grown in the presence of 4‐OHT for 72 h, as detected by CO‐FISH (see ). Data are representative of three independent experiments. Error bars indicate SD. *P 0.05.


Telomere lengths of UQ . CreERT2 Nsmce2lox / lox MEFs treated or not with 4 ‐ OHT for 72 h .

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Telomere lengths of UQ.CreERT2 Nsmce2lox/lox MEFs treated or not with 4‐OHT for 72 h.


Example of the intensity of telomere signals ( yellow ) used for ( H ) .

Example of the intensity of telomere signals (yellow) used for (H).


Percentage of cells with micronuclei found on cultures of UQ . CreERT2 / Nsmce2lox / lox MEFs treated or not with 4 ‐ OHT for 72 h . Data are representative of three independent experiments . Error bars indicate SD . * * * P 0 . 001 .

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Percentage of cells with micronuclei found on cultures of UQ.CreERT2/Nsmce2lox/lox MEFs treated or not with 4‐OHT for 72 h. Data are representative of three independent experiments. Error bars indicate SD. ***P 0.001.


Intrauterine dwarfism observed upon NSMCE2 deletion on 17 . 5 dpc embryos . Pregnant females were treated at 14 . 5 dpc for 3 days with intraperitoneal injections of 4 ‐ OHT . The image represents the difference in size observed between Nsmce2 + / + and Nsmce2lox / lox embryos carrying the UQ . CreERT2 transgene ( Nsmce2 + / + and Nsmce2lox / lox , from now on ) .

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Intrauterine dwarfism observed upon NSMCE2 deletion on 17.5 dpc embryos. Pregnant females were treated at 14.5 dpc for 3 days with intraperitoneal injections of 4‐OHT. The image represents the difference in size observed between Nsmce2+/+ and Nsmce2lox/lox embryos carrying the UQ.CreERT2 transgene (Nsmce2+/+ and Nsmce2lox/lox, from now on).


Representative picture of Nsmce2 + / + and Nsmce2lox / lox animals fed for 40 weeks with a 4 ‐ OHT ‐ containing diet ( see also Movies EV1 and EV2 ) .

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Representative picture of Nsmce2+/+ and Nsmce2lox/lox animals fed for 40 weeks with a 4‐OHT‐containing diet (see also Movies EV1 and EV2).


Images illustrating the presence of altered pigmentation problems that arise on Nsmce2lox / lox mice .

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Images illustrating the presence of altered pigmentation problems that arise on Nsmce2lox/lox mice.


Percentage of fat against total body mass on Nsmce2 + / + and Nsmce2lox / lox animals . * * * P 0 . 001 .

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Percentage of fat against total body mass on Nsmce2+/+ and Nsmce2lox/lox animals. ***P 0.001.


White bloodcell counts ( WBC ) from Nsmce2 + / + and Nsmce2lox / lox animals . * * * P 0 . 001 .

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White bloodcell counts (WBC) from Nsmce2+/+ and Nsmce2lox/lox animals. ***P 0.001.


Percentage of γH2AX ‐ positive micronuclei found on the intestinal epithelia of Nsmce2 + / + and Nsmce2lox / lox animals ( see Appendix Fig S11 ) . Data are representative of three independent experiments . Error bars indicate SD . * P 0 . 05 .

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Percentage of γH2AX‐positive micronuclei found on the intestinal epithelia of Nsmce2+/+ and Nsmce2lox/lox animals (see Appendix Fig S11). Data are representative of three independent experiments. Error bars indicate SD. *P 0.05.


A WB illustrating the depletion of NSMCE2 in B cells isolated from 2 ‐ month ‐ old CD19 + / Cre animals of the indicated genotypes , 48 h after being stimulated in vitro with lipopolysaccharide ( LPS ) . Tubulin was used as a loading control .

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A WB illustrating the depletion of NSMCE2 in B cells isolated from 2‐month‐old CD19+/Cre animals of the indicated genotypes, 48 h after being stimulated in vitro with lipopolysaccharide (LPS). Tubulin was used as a loading control.


B Representative FACS analysis from the B ‐ cell cultures mentioned in ( A ) . DNA content was measured with propidium iodide ( PI ) . Note the presence of cells with > 4n DNA content in NSMCE2 ‐ deleted cells .

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B Representative FACS analysis from the B‐cell cultures mentioned in (A). DNA content was measured with propidium iodide (PI). Note the presence of cells with > 4n DNA content in NSMCE2‐deleted cells.


C , D Frequency of origin firing ( C ) and fork rate ( D ) were analyzed by DNA fiber analyses ( see ) from fibers prepared from B ‐ cell cultures explained in ( A ) . No significant differences were observed between Nsmce2 + / + and Nsmce2lox / loxB cells . Data are representative of two independent experiments . Error bars indicate SD . Between 200 and 300 tracks were measured to estimate fork rate and 300 - 500 for the calculation of origin firing frequencies .

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C, D Frequency of origin firing (C) and fork rate (D) were analyzed by DNA fiber analyses (see ) from fibers prepared from B‐cell cultures explained in (A). No significant differences were observed between Nsmce2+/+ and Nsmce2lox/loxB cells. Data are representative of two independent experiments. Error bars indicate SD. Between 200 and 300 tracks were measured to estimate fork rate and 300-500 for the calculation of origin firing frequencies.


E Representative images of the DNA fibers used for the analysis explained in ( C , D ) . CldU ( red ) and IdU ( green ) channels are shown .

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E Representative images of the DNA fibers used for the analysis explained in (C, D). CldU (red) and IdU (green) channels are shown.


Representative pictures of the spleens from 2 ‐ month ‐ old CD19 + / Cre animals of the indicated genotypes .

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Representative pictures of the spleens from 2‐month‐old CD19+/Cre animals of the indicated genotypes.


Numbers of B and T cells from 2 ‐ month ‐ old CD19 + / Cre animals of the indicated genotypes evaluated by flow cytometry ( B220 : B ‐ cell marker ; CD3 : T ‐ cell marker ) .

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Numbers of B and T cells from 2‐month‐old CD19+/Cre animals of the indicated genotypes evaluated by flow cytometry (B220: B‐cell marker; CD3: T‐cell marker).


SCE events per metaphase on B cells obtained from CD19 + / Cre animals of the indicated genotypes , 72 h after being stimulated in vitro with LPS . * * P 0 . 01 , * * * P 0 . 001 .

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SCE events per metaphase on B cells obtained from CD19+/Cre animals of the indicated genotypes, 72 h after being stimulated in vitro with LPS. **P 0.01, ***P 0.001.


Analysis of forward ( FSC ) and side ( SSC ) scatter parameters of B cells obtained from CD19 + / Cre animals of the indicated genotypes , 48 h after being stimulated in vitro with LPS . The emergence of bigger cells is noticeable on cells lacking both BLM and NSMCE2 .

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Analysis of forward (FSC) and side (SSC) scatter parameters of B cells obtained from CD19+/Cre animals of the indicated genotypes, 48 h after being stimulated in vitro with LPS. The emergence of bigger cells is noticeable on cells lacking both BLM and NSMCE2.


Representative images of B ‐ cell cultures of the indicated genotypes 48 h after being stimulated in vitro with LPS . Insets provide a magnified view of an individual cell ( arrowhead ) for better comparison of the observed differences in cell size . Scale bar , 100 μm .

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Representative images of B‐cell cultures of the indicated genotypes 48 h after being stimulated in vitro with LPS. Insets provide a magnified view of an individual cell (arrowhead) for better comparison of the observed differences in cell size. Scale bar, 100 μm.


Examples of the types of nuclei found on Blm and Nsmce2 double ‐ mutant B cells ( compared with wild ‐ type B cells ) , in cells obtained from ( E ) . DAPI was used to stain DNA . Whereas wt nuclei are regular in size , double ‐ mutant cells invariably presented enlarged , multilobulated , and irregular nuclei . Scale bar , 2 . 5 μm .

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Examples of the types of nuclei found on Blm and Nsmce2 double‐mutant B cells (compared with wild‐type B cells), in cells obtained from (E). DAPI was used to stain DNA. Whereas wt nuclei are regular in size, double‐mutant cells invariably presented enlarged, multilobulated, and irregular nuclei. Scale bar, 2.5 μm.


d . Real - time reverse transcription quantitative PCR ( RT - qPCR ) showing Fbxw7 , Pten and Trp53 expression in mouse uteri at 4 weeks and 8 weeks postnatal age ( NB Trp53R172H uteri were not analysed at the 4 week timepoint ) . Box extends from 25th to 75th percentiles , with line at median , whiskers indicate minimum and maximum values , and dots individual values ( n = 5 samples per group with exception of Ptendel and Ptendel , Fbxw7mut genotypes at 8 weeks which contain n = 7 samples each ) . Statistical comparison between WT controls and other genotypes was performed by unadjusted , unpaired Mann Whitney test ; * indicates P < 0 . 05 , * * indicates P < 0 .

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d. Real-time reverse transcription quantitative PCR (RT-qPCR) showing Fbxw7, Pten and Trp53 expression in mouse uteri at 4 weeks and 8 weeks postnatal age (NB Trp53R172H uteri were not analysed at the 4 week timepoint). Box extends from 25th to 75th percentiles, with line at median, whiskers indicate minimum and maximum values, and dots individual values (n=5 samples per group with exception of Ptendel and Ptendel, Fbxw7mut genotypes at 8 weeks which contain n=7 samples each). Statistical comparison between WT controls and other genotypes was performed by unadjusted, unpaired Mann Whitney test; * indicates P<0.05, ** indicates P<0.01


e . Western blot of mouse uteri at 8 weeks postnatal age ( NB Trp53R172H uteri were not analysed by Western blotting ) . Values indicate densitometric units ( mean ± SD ) normalised to control group of WT samples in case of Fbxw7 protein and both WT and Fbxw7mut samples in case of Pten and p53 proteins . Image is representative of duplicate technical replicates . Statistical comparison between controls and other groups was performed by unadjusted , unpaired Mann Whitney test ; and are shown next to densitometry results where statistically significant .

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e. Western blot of mouse uteri at 8 weeks postnatal age (NB Trp53R172H uteri were not analysed by Western blotting). Values indicate densitometric units (mean ± SD) normalised to control group of WT samples in case of Fbxw7 protein and both WT and Fbxw7mut samples in case of Pten and p53 proteins. Image is representative of duplicate technical replicates. Statistical comparison between controls and other groups was performed by unadjusted, unpaired Mann Whitney test; and are shown next to densitometry results where statistically significant.


f . Immunohistochemical ( IHC ) analysis of mouse uteri at 8 weeks postnatal age . Images are representative of minimum of 3 biological replicates for each genotype and protein . Scale bar indicates 100μm .

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f. Immunohistochemical (IHC) analysis of mouse uteri at 8 weeks postnatal age. Images are representative of minimum of 3 biological replicates for each genotype and protein. Scale bar indicates 100μm.


a . Kaplan - Meier curves showing survival of genetically engineered mouse models ( GEMM ) of indicated genotypes and group sizes . Statistical comparison between groups was performed by unadjusted log - rank test .

a. Kaplan-Meier curves showing survival of genetically engineered mouse models (GEMM) of indicated genotypes and group sizes. Statistical comparison between groups was performed by unadjusted log-rank test.


c . Low and high power images of GEMM uteri at 8 weeks age and at study endpoint after H & E staining or IHC for cytokeratin 8 ( CK8 ) ( epithelia ) . Scale bars in low and high magnification panels indicate 1000μm and 100μm respectively . Images are representative of a minimum of 5 mice per genotype per timepoint , with exception of wild - type and Fbxw7mut mice at 8 weeks age where 3 mice were analysed .

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c. Low and high power images of GEMM uteri at 8 weeks age and at study endpoint after H&E staining or IHC for cytokeratin 8 (CK8) (epithelia). Scale bars in low and high magnification panels indicate 1000μm and 100μm respectively. Images are representative of a minimum of 5 mice per genotype per timepoint, with exception of wild-type and Fbxw7mut mice at 8 weeks age where 3 mice were analysed.


e . H & E - stained sections of high grade external sarcomas in Trp53del , Trp53del ; Fbxw7mut , Trp53mut and Trp53mut ; Fbxw7mut mice . Images are representative of minimum of 3 mice examined for each genotype . Scale bar indicates 100μm .

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e. H&E-stained sections of high grade external sarcomas in Trp53del, Trp53del; Fbxw7mut, Trp53mut and Trp53mut; Fbxw7mut mice. Images are representative of minimum of 3 mice examined for each genotype. Scale bar indicates 100μm.


a . ( Left panel ) Schematic illustrating gene expression profiling of mouse uterine samples of indicated genotypes and group sizes at 8 weeks age , with groups compared by enrichment analysis . ( Right panel ) Dot plot indicating enrichment of MSigDB C6 oncogenic gene sets in Ptendel ; Fbxw7mut uteri vs Ptendel uteri . Arrows indicate enrichment of LEF1 signatures .

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a. (Left panel) Schematic illustrating gene expression profiling of mouse uterine samples of indicated genotypes and group sizes at 8 weeks age, with groups compared by enrichment analysis. (Right panel) Dot plot indicating enrichment of MSigDB C6 oncogenic gene sets in Ptendel; Fbxw7mut uteri vs Ptendel uteri. Arrows indicate enrichment of LEF1 signatures.


b . Dot plot showing enrichment analysis of 1 , 064 putative FBXW7 substrates against GO biological processes .

b. Dot plot showing enrichment analysis of 1,064 putative FBXW7 substrates against GO biological processes.


a . HEK293T cells were transfected with constructs encoding FLAG - V5 - tagged FBXW7α and Myc - tagged LEF1 . FLAG - V5 - tagged FBXW7 α was immunoprecipitated ( IP ) from cell extracts with anti - FLAG antibody before immunoblotting for tagged proteins as indicated ( upper panels ) . Lower panels show inputs . b . HEK293T cells were transfected with constructs encoding FLAG - V5 - tagged FBXW7α and Myc - tagged TCF7L2 . IP was performed as in ( a ) before immunoblotting for tagged proteins as indicated . Data information : All images shown are representative of a minimum of two independent

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a. HEK293T cells were transfected with constructs encoding FLAG-V5-tagged FBXW7α and Myc-tagged LEF1. FLAG-V5-tagged FBXW7 α was immunoprecipitated (IP) from cell extracts with anti-FLAG antibody before immunoblotting for tagged proteins as indicated (upper panels). Lower panels show inputs. b. HEK293T cells were transfected with constructs encoding FLAG-V5-tagged FBXW7α and Myc-tagged TCF7L2. IP was performed as in (a) before immunoblotting for tagged proteins as indicated. Data information: All images shown are representative of a minimum of two independent experiments


g . HEK293T cells were transfected with Myc - tagged FBXW7α and either FLAG - V5 - tagged full length LEF1 ( FL ) or truncated LEF1 in which the N terminal domain required for β - catenin binding has been deleted . Immunoblotting for indicated proteins was performed following IP by anti - FLAG antibody . Data information : All images shown are representative of a minimum of two independent

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g. HEK293T cells were transfected with Myc-tagged FBXW7α and either FLAG-V5-tagged full length LEF1 (FL) or truncated LEF1 in which the N terminal domain required for β-catenin binding has been deleted. Immunoblotting for indicated proteins was performed following IP by anti-FLAG antibody. Data information: All images shown are representative of a minimum of two independent experiments


a . Dot plot showing log2 - transformed expression of FBXW7 , LEF1 and other wnt pathway genes in endometrial epithelial cell subsets defined by single cell RNAseq ( scRNAseq )

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a. Dot plot showing log2-transformed expression of FBXW7, LEF1 and other wnt pathway genes in endometrial epithelial cell subsets defined by single cell RNAseq (scRNAseq)


b . Representative LEF1 immunohistochemistry in GEMM uteri from mice of indicated genotypes at 8 weeks age . For each of the four columns ( i . e genotypes ) , upper and lower images are taken from littermate females housed in the same cage . Images are representative of 3 - 4 mice as shown in ( c ) . Scale bar indicates 100μm .

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b. Representative LEF1 immunohistochemistry in GEMM uteri from mice of indicated genotypes at 8 weeks age. For each of the four columns (i.e genotypes), upper and lower images are taken from littermate females housed in the same cage. Images are representative of 3-4 mice as shown in (c). Scale bar indicates 100μm.


c . Percentage of mice with epithelial LEF1 expression in endometria at 8 weeks age according to genotype . P value was derived from binomial logistic regression with epithelial LEF1 expression as the dependent variable .

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c. Percentage of mice with epithelial LEF1 expression in endometria at 8 weeks age according to genotype. P value was derived from binomial logistic regression with epithelial LEF1 expression as the dependent variable.


d . Immunohistochemistry for LEF1 and β - catenin from endometrial carcinomas in Ptendel ± Fbxw7mut mice . High power images show representative staining in LEF1 negative and positive glands . Scale bar bar indicates 1000μm .

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d. Immunohistochemistry for LEF1 and β-catenin from endometrial carcinomas in Ptendel ± Fbxw7mut mice. High power images show representative staining in LEF1 negative and positive glands. Scale bar bar indicates 1000μm.


( A - C ) AFM and TEM analysis of unloaded ( u - NPs ) and cholesterol loaded NPs ( NPs - Chol ) . AFM " height " images ( left column ) , 3D reconstruction ( middle column ) and TEM micrograph ( right column ) of u - NPs ( A ) , NPs - Chol1 ( B ) and NPs - Chol3 ( C ) .

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(A-C) AFM and TEM analysis of unloaded (u-NPs) and cholesterol loaded NPs (NPs-Chol). AFM "height" images (left column), 3D reconstruction (middle column) and TEM micrograph (right column) of u-NPs (A), NPs-Chol1 (B) and NPs-Chol3 (C).


( D ) Release profile in water of cholesterol ( continuous line , - ) and NBD - Chol ( dotted line , - - - ) from NPs - Chol1 and NPs - NBD - Chol1 , respectively .

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(D) Release profile in water of cholesterol (continuous line, -) and NBD-Chol (dotted line, - - -) from NPs-Chol1 and NPs-NBD-Chol1, respectively.


( E ) In vitro release of NBD - Chol from NPs at different time - intervals in NS cells . Data in the graph represent mean ( µg ) ± SEM of total NBD - Chol ( embedded into and released from NPs ; red columns ) and NBD - Chol released after NPs degradation ( purple columns ) present in the homogenates of NS cells treated with NPs - NBD -

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(E) In vitro release of NBD-Chol from NPs at different time-intervals in NS cells. Data in the graph represent mean (µg) ± SEM of total NBD-Chol (embedded into and released from NPs; red columns) and NBD-Chol released after NPs degradation (purple columns) present in the homogenates of NS cells treated with NPs-NBD-Chol1


( A - B ) Representative confocal images of liver ( A ) and brain ( B ) slices from R6 / 2 mice ip injected with C - NPs ( left ) or with g7 - NPs ( right ) and sacrificed after 4 hrs . ( C ) Quantification of g7 - NPs localized in the liver , striatum , and cortex of WT ( n = 3 ) and R6 / 2 mice ( n = 3 ) . Data represent mean ( µg ) ± SEM .

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(A-B) Representative confocal images of liver (A) and brain (B) slices from R6/2 mice ip injected with C-NPs (left) or with g7-NPs (right) and sacrificed after 4 hrs. (C) Quantification of g7-NPs localized in the liver, striatum, and cortex of WT (n=3) and R6/2 mice (n=3). Data represent mean (µg) ± SEM.


( D - E ) g7 - NPs in brain slices from R6 / 2 mice administered with a single ip injection and sacrificed after 24 hrs ( D , left ) or 2 weeks ( D , right ) and after multiple ip injections within 1 week ( E ) .

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(D-E) g7-NPs in brain slices from R6/2 mice administered with a single ip injection and sacrificed after 24 hrs (D, left) or 2 weeks (D, right) and after multiple ip injections within 1 week (E).


( F - I ) Representative confocal images of immunostaining against IBA1 ( F ) , GFAP ( G ) , calbindin ( H ) , and DARPP - 32 ( I ) on coronal sections of brains isolated from R6 / 2 mice ip injected with g7 - NPs and sacrificed at the indicated time points . White arrowheads indicate intracellular g7 - NPs .

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(F-I) Representative confocal images of immunostaining against IBA1 (F), GFAP (G), calbindin (H), and DARPP-32 (I) on coronal sections of brains isolated from R6/2 mice ip injected with g7-NPs and sacrificed at the indicated time points. White arrowheads indicate intracellular g7-NPs.


( A ) Representative confocal image ( crop ) of brain slices from R6 / 2 mice ip injected with rhodamine labeled g7 - NPs - NBD - Chol and sacrificed after 12 hrs , showing co - localization of NBD - Chol ( green ) and Rhodamine ( NPs , red ) . Scale bar : 5m .

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(A) Representative confocal image (crop) of brain slices from R6/2 mice ip injected with rhodamine labeled g7-NPs-NBD-Chol and sacrificed after 12 hrs, showing co-localization of NBD-Chol (green) and Rhodamine (NPs, red). Scale bar: 5m.


( B - C ) Representative confocal image ( low magnification ) of brain slices from R6 / 2 mice ip injected with g7 - NPs - NBD - Chol and sacrificed after 24 hrs ( B ) or 2 weeks ( C ) and relative co - localization of NBD - Chol and g7 - NPs . Scale bar : 10 m ( D ) g7 - NPs quantification in brain slices at the same time points in ( B - C ) . DAPI was used to counterstain nuclei . Data in ( D ) are expressed as number of g7 - NPs ( evaluated based their size ) for 100 m2 sem . Statistics : * * P < 0 , 01 ( 48 hrs vs 7 days ; 7 days vs 14 days ) , * * * P < 0 , 001 ( 24 hrs vs 7 days ; 7 days vs 14 days ) determined by one - way ANOVA followed by Newmann - Keuls multiple comparison test .

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(B-C) Representative confocal image (low magnification) of brain slices from R6/2 mice ip injected with g7-NPs-NBD-Chol and sacrificed after 24 hrs (B) or 2 weeks (C) and relative co-localization of NBD-Chol and g7-NPs. Scale bar: 10 m (D) g7-NPs quantification in brain slices at the same time points in (B-C). DAPI was used to counterstain nuclei. Data in (D) are expressed as number of g7-NPs (evaluated based their size) for 100 m2 sem. Statistics: **P<0,01 (48 hrs vs 7 days; 7 days vs 14 days), ***P<0,001 (24 hrs vs 7 days; 7 days vs 14 days) determined by one-way ANOVA followed by Newmann-Keuls multiple comparison test.


( A , D ) Spontaneous IPSCs ( A ) and spontaneous EPSCs ( D ) were recorded from striatal MSNs ( WTs = 52 ; R6 / 2 - untreated = 27 ; R6 / 2 - Chol = 29 ) at a holding potential of + 10 mV and - 70mV respectively . As no differences were found between R6 / 2 mice treated with saline ( R6 / 2 ) or with empty g7 - NPs ( R6 / 2 - emp ) , data were pooled . ( B , E ) Amplitude - frequency histogram and average frequency ( inset ) of IPSCs ( B ) and EPSCs ( E ) from R6 / 2 - Chol , R6 / 2 - untreated and WT MSNs . ( C , F ) Cumulative inter - event histogram showing the release probability of IPSCs ( C ) and EPSCs ( F ) in all groups .

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(A,D) Spontaneous IPSCs (A) and spontaneous EPSCs (D) were recorded from striatal MSNs (WTs=52; R6/2-untreated=27; R6/2-Chol=29) at a holding potential of +10 mV and -70mV respectively. As no differences were found between R6/2 mice treated with saline (R6/2) or with empty g7-NPs (R6/2-emp), data were pooled. (B, E) Amplitude-frequency histogram and average frequency (inset) of IPSCs (B) and EPSCs (E) from R6/2-Chol, R6/2-untreated and WT MSNs. (C,F) Cumulative inter-event histogram showing the release probability of IPSCs (C) and EPSCs (F) in all groups.


( B ) Fall latency from an accelerating rotarod for 5 - 11 - week - old WT and R6 / 2 mice during cholesterol supplementation ( WT = 17 ; R6 / 2 - untreated = 21 ; R6 / 2 - Chol = 13 ) .

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(B) Fall latency from an accelerating rotarod for 5-11-week-old WT and R6/2 mice during cholesterol supplementation (WT=17; R6/2-untreated=21; R6/2-Chol=13).


( C ) Rearing activity in open field at 10 weeks of age ( WT = 14 ; R6 / 2 - untreated = 21 ; R6 / 2 - Chol = 15 ) .

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(C) Rearing activity in open field at 10 weeks of age (WT=14; R6/2-untreated=21; R6/2-Chol=15).


( D ) global activity in the open field test at 10 weeks of age ( WT = 14 ; R6 / 2 - untreated = 21 ; R6 / 2 - Chol = 15 ) .

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(D) global activity in the open field test at 10 weeks of age (WT=14; R6/2-untreated=21; R6/2-Chol=15).


( E ) Index of discrimination ( % ) in WT , R6 / 2 - untreated and R6 / 2 - Chol mice during disease progression , at 8 weeks of age ( WT = 24 ; R6 / 2 - untreated = 36 ; R6 / 2 - Chol = 21 ) , at 10 weeks of age ( WT = 25 ; R6 / 2 - untreated = 35 ; R6 / 2 - Chol = 20 ) and at 12 weeks of age ( WT = 24 ; R6 / 2 - untreated = 30 ; R6 / 2 - Chol = 19 ) ; the index above zero indicates a preference for the novel object ; the index below zero indicates a preference for the familiar object . As no differences were found between R6 / 2 mice treated with saline ( R6 / 2 ) or treated with empty g7 - NPs ( R6 / 2 - emp ) , data were pooled .

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(E) Index of discrimination (%) in WT, R6/2-untreated and R6/2-Chol mice during disease progression, at 8 weeks of age (WT=24; R6/2-untreated=36; R6/2-Chol=21), at 10 weeks of age (WT=25; R6/2-untreated=35; R6/2-Chol=20) and at 12 weeks of age (WT=24; R6/2-untreated=30; R6/2-Chol=19); the index above zero indicates a preference for the novel object; the index below zero indicates a preference for the familiar object. As no differences were found between R6/2 mice treated with saline (R6/2) or treated with empty g7-NPs (R6/2-emp), data were pooled.


( A - B ) protein levels ( A ) and relative densitometry quantification ( B ) of several synaptic proteins in triton - insoluble ( synaptic enriched ) fractions purified from total brains . Levels of PSD95 and NMDA receptor subunits GluN1 and GluN2B are rescued in R6 / 2 mice by cholesterol supplementation .

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(A-B) protein levels (A) and relative densitometry quantification (B) of several synaptic proteins in triton-insoluble (synaptic enriched) fractions purified from total brains. Levels of PSD95 and NMDA receptor subunits GluN1 and GluN2B are rescued in R6/2 mice by cholesterol supplementation.


( C - D - E ) mRNA levels for Bdnf ( C ) , Snap25 ( D ) in cortex and hippocampus ; Complexin II ( E ) in cortex , hippocampus and striatum from a subset of WT ( n = 4 ) , R6 / 2 - untreated ( n = 7 ) and R6 / 2 - chol animals ( n = 3 ) . As no differences were found between R6 / 2 mice treated with saline or treated with empty g7 - NPs , data were pooled .

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(C-D-E) mRNA levels for Bdnf (C), Snap25 (D) in cortex and hippocampus; Complexin II (E) in cortex, hippocampus and striatum from a subset of WT (n=4), R6/2-untreated (n=7) and R6/2-chol animals (n=3). As no differences were found between R6/2 mice treated with saline or treated with empty g7-NPs, data were pooled.


( F - G ) Representative images of Nissl Staining ( F ) and ventricle volume revealed by Neurolucida Analysis at 12 weeks of age in WT ( n = 7 ) , R6 / 2 ( n = 7 ) , R6 / 2 - emp ( n = 6 ) , R6 / 2 - Chol ( n = 8 )

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(F-G) Representative images of Nissl Staining (F) and ventricle volume revealed by Neurolucida Analysis at 12 weeks of age in WT (n= 7), R6/2 (n=7), R6/2-emp (n=6), R6/2-Chol (n=8)


( A - B ) Lathosterol and 24OHC measured by mass spectrometry in the brain of WT ( n = 6 ) , R6 / 2 - untreated ( n = 7 ) and R6 / 2 - Chol ( n = 5 ) mice . Data represent mean ± SEM .

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(A-B) Lathosterol and 24OHC measured by mass spectrometry in the brain of WT (n=6), R6/2-untreated (n=7) and R6/2-Chol (n=5) mice. Data represent mean ± SEM.


( C - D ) mRNA levels of hmgcr and fdft1 in liver and lung of WT ( n = 7 ) , R6 / 2 - untreated ( n = 8 ) and R6 / 2 - Chol ( n = 4 ) mice . As no differences were found between R6 / 2 mice treated with saline or treated with empty g7 - NPs , data were pooled .

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(C-D) mRNA levels of hmgcr and fdft1 in liver and lung of WT (n=7), R6/2-untreated (n=8) and R6/2-Chol (n=4) mice. As no differences were found between R6/2 mice treated with saline or treated with empty g7-NPs, data were pooled.


( E - F ) mRNA levels of inflammatory genes in liver and lung of the of WT ( n = 7 ) , R6 / 2 ( n = 4 ) , R6 / 2 - emp ( n = 4 ) and R6 / 2 - Chol ( n = 5 ) mice .

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(E-F) mRNA levels of inflammatory genes in liver and lung of the of WT (n=7), R6/2 (n=4), R6/2-emp (n=4) and R6/2-Chol (n=5) mice.


C , Quantification of telomeric allelic imbalances ( TAI ) in soft tissue and bone sarcoma .

C, Quantification of telomeric allelic imbalances (TAI) in soft tissue and bone sarcoma.


E - G , Ex vivo treatment of HRDhigh ( NMFH - 1 , OH931 , PM197 , USZ - 21 _ MFS2 and USZ - 21 _ UPS1 ) sarcoma models compared with HRDlow ( USZ - 20 _ REA1 , USZ - 21 _ LG1 , USZ - 22 _ EMC2 , USZ - 20 _ SFT1 and USZ - 21 _ CIC1 ) sarcoma models for 4 days with six doses of the chemotherapy agents oxaliplatin , doxorubicin and trabectedin .

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E-G, Ex vivo treatment of HRDhigh (NMFH-1, OH931, PM197, USZ-21_MFS2 and USZ-21_UPS1) sarcoma models compared with HRDlow (USZ-20_REA1, USZ-21_LG1, USZ-22_EMC2, USZ-20_SFT1 and USZ-21_CIC1) sarcoma models for 4 days with six doses of the chemotherapy agents oxaliplatin, doxorubicin and trabectedin.


M - O , HRDhigh sarcoma cell models and UWB1 . 289 treated for 3 days with five doses olaparib alone and in combination with 1 nM trabectedin . Note that no sensitivity to olaparib in monotherapy was observed at 3

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M-O, HRDhigh sarcoma cell models and UWB1.289 treated for 3 days with five doses olaparib alone and in combination with 1 nM trabectedin. Note that no sensitivity to olaparib in monotherapy was observed at 3 days


Q - S , HRDhigh sarcoma cell models and UWB1 . 289 treated for 3 days with five doses adavosertib alone and in combination with 100 nM doxorubicin .

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Q-S, HRDhigh sarcoma cell models and UWB1.289 treated for 3 days with five doses adavosertib alone and in combination with 100 nM doxorubicin.


A , Immunofluorescence showing nuclear expression of the DNA damage marker γH2A . X ( magenta ) upon 6 h treatment with 10 nM trabectedin and 100 nM olaparib in combination in HRDhigh and HRDlow sarcoma cell models as well as ovarian carcinoma UWB1 . 289 cells . B , Immunofluorescence showing RAD51 nuclear foci ( magenta ) upon 6 h treatment with 10 nM trabectedin and 100 nM olaparib in combination only in the HRDlow sarcoma cell model ( USZ - 21 _ LG1 ) . C , D , Quantification of γH2A . X ( C ) or RAD51 ( D ) nuclear intensity per cell compared to untreated control cells . E ,

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A, Immunofluorescence showing nuclear expression of the DNA damage marker γH2A.X (magenta) upon 6 h treatment with 10 nM trabectedin and 100 nM olaparib in combination in HRDhigh and HRDlow sarcoma cell models as well as ovarian carcinoma UWB1.289 cells. B, Immunofluorescence showing RAD51 nuclear foci (magenta) upon 6 h treatment with 10 nM trabectedin and 100 nM olaparib in combination only in the HRDlow sarcoma cell model (USZ-21_LG1). C, D, Quantification of γH2A.X (C) or RAD51 (D) nuclear intensity per cell compared to untreated control cells. E,


E , Immunohistochemistry ( IHC ) in human tissue sections showing RAD51 ( brown ) expression in HRDlow but not HRDhigh sarcoma patients . Small - nucleated lymphocytes and / or proliferating cells appeared RAD51 - positive in HRDhigh models .

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E, Immunohistochemistry (IHC) in human tissue sections showing RAD51 (brown) expression in HRDlow but not HRDhigh sarcoma patients. Small-nucleated lymphocytes and/or proliferating cells appeared RAD51-positive in HRDhigh models.


F , Immunofluorescence in human tissue sections showing RAD51 ( magenta ) nuclear foci in an HRDlow but not an HRDhigh sarcoma patient .

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F, Immunofluorescence in human tissue sections showing RAD51 (magenta) nuclear foci in an HRDlow but not an HRDhigh sarcoma patient.


H , Magnetic resonance imaging ( MRI ) of LMS patient since primary diagnosis . Open arrowheads point at metastatic lesions .

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H, Magnetic resonance imaging (MRI) of LMS patient since primary diagnosis. Open arrowheads point at metastatic lesions.


J , RAD51 IHC in the metastatic patient ' s tissue . Compare RAD51 nuclear expression in normal tissue ( arrowhead points at gastric gland ) but not in tumorous gastric tissue .

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J, RAD51 IHC in the metastatic patient's tissue. Compare RAD51 nuclear expression in normal tissue (arrowhead points at gastric gland) but not in tumorous gastric tissue.


( A ) Genome - wide ubiquitin binding profiles identify numerous regions of H2B - ubiquitylation in WT cells and distinct sites of non - H2B ubiquitylation ( ' ubiquitin - hotspot ' , ub - hotspot , ub - HS ) which persist in h2b - K123R and rad6 ∆ strains , and increase in cdc48 mutants ( cdc48 - 6 ) . A 90 kb stretch of chromosome XIII ( ChrXIII ) is depicted . Chromatin immunoprecipitation was performed using the FK2 ubiquitin antibody ( see also Appendix Fig . S1A ) and enriched DNA was analyzed on NimbleGen arrays ( Chip - chip ) . DNA from non - specific IgG - ChIP experiments served as background control . Significantly enriched regions are marked by bars above the respective ChIP - chip tracks and are summarized in Dataset EV1 . Data represent means from two independent replicates , except for ubiquitin ( FK2 ) in rad6 ∆ and IgG - ChIP in WT ( n = 1 ) . All experiments , including those using cdc48 - 6 and other temperature - sensitive ( ts ) alleles , were performed at 30 ° C ( semipermissive temperature for ts - alleles ) unless stated otherwise .

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(A) Genome-wide ubiquitin binding profiles identify numerous regions of H2B-ubiquitylation in WT cells and distinct sites of non-H2B ubiquitylation ('ubiquitin-hotspot', ub-hotspot, ub-HS) which persist in h2b-K123R and rad6∆ strains, and increase in cdc48 mutants (cdc48-6). A 90 kb stretch of chromosome XIII (ChrXIII) is depicted. Chromatin immunoprecipitation was performed using the FK2 ubiquitin antibody (see also Appendix Fig. S1A) and enriched DNA was analyzed on NimbleGen arrays (Chip-chip). DNA from non-specific IgG-ChIP experiments served as background control. Significantly enriched regions are marked by bars above the respective ChIP-chip tracks and are summarized in Dataset EV1. Data represent means from two independent replicates, except for ubiquitin (FK2) in rad6∆ and IgG-ChIP in WT (n = 1). All experiments, including those using cdc48-6 and other temperature-sensitive (ts) alleles, were performed at 30°C (semipermissive temperature for ts-alleles) unless stated otherwise.


( B ) Seven ub - HSs show strong correlation between ubiquitin binding and Slx8 enrichment ( Slx8 - 9myc ChIP ) . 16 - kb - windows of the indicated regions centered around the ub - HSs are depicted for ubiquitin and Slx8 binding profiles . Ubiquitin ( FK2 ) data for WT and cdc48 - 6 are from the same experiment as depicted in ( A ) . Data represent means from two independent replicates .

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(B) Seven ub-HSs show strong correlation between ubiquitin binding and Slx8 enrichment (Slx8-9myc ChIP). 16-kb-windows of the indicated regions centered around the ub-HSs are depicted for ubiquitin and Slx8 binding profiles. Ubiquitin (FK2) data for WT and cdc48-6 are from the same experiment as depicted in (A). Data represent means from two independent replicates.


( D ) Slx8 and Slx5 are recruited to ub - HSs . DNA from Slx8 - 9myc ( top ) or 3HA - Slx5 ( bottom ) ChIP experiments of the indicated strains was analyzed by quantitative real - time PCR ( ChIP - qPCR ) at selected ub - HSs . Data represent mean ± standard deviation ( SD , n = 2 ) . Consistent with a previous study , we did not observe any Slx8 enrichment at centromeres ( van de Pasch et al , 2013 ) , however , we note that we could not confirm the reported centromere binding of Slx5 ( see also Appendix Fig . S2E ) .

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(D) Slx8 and Slx5 are recruited to ub-HSs. DNA from Slx8-9myc (top) or 3HA-Slx5 (bottom) ChIP experiments of the indicated strains was analyzed by quantitative real-time PCR (ChIP-qPCR) at selected ub-HSs. Data represent mean ± standard deviation (SD, n = 2). Consistent with a previous study, we did not observe any Slx8 enrichment at centromeres (van de Pasch et al, 2013), however, we note that we could not confirm the reported centromere binding of Slx5 (see also Appendix Fig. S2E).


( E ) K48 - linked ubiquitin chains accumulate in a cdc48 mutant ( cdc48 - 3 ) . Ub - K48 ChIP followed by qPCR for same loci as in ( D ) are depicted . See also Fig . EV1C - D . Data represent means ± SD ( n = 2 ) .

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(E) K48-linked ubiquitin chains accumulate in a cdc48 mutant (cdc48-3). Ub-K48 ChIP followed by qPCR for same loci as in (D) are depicted. See also Fig. EV1C-D. Data represent means ± SD (n = 2).


( F ) Slx5 and Slx8 are required for ub - HS formation . Ub - K48 ChIP - qPCR in strains lacking one subunit of the Slx5 / Slx8 complex ( slx5 ∆ , slx8 ∆ ) . Data represent means ± SD ( n = 3 ) .

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(F) Slx5 and Slx8 are required for ub-HS formation. Ub-K48 ChIP-qPCR in strains lacking one subunit of the Slx5/Slx8 complex (slx5∆, slx8∆). Data represent means ± SD (n = 3).


( G ) Ufd1 and Npl4 act in concert with Cdc48 to remove ubiquitylated proteins from ub - HS sites . Ub - K48 ChIP for the indicated strains grown at the semi - permissive temperature of 30 ° C . Data represent means ± SD ( n = 2 ) .

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(G) Ufd1 and Npl4 act in concert with Cdc48 to remove ubiquitylated proteins from ub-HS sites. Ub-K48 ChIP for the indicated strains grown at the semi-permissive temperature of 30°C. Data represent means ± SD (n = 2).


B ) A 39 bp fragment of ub - HS4 is sufficient to drive ectopic ub - HS formation . Fragments of ub - HS4 were integrated ectopically and ub - K48 ChIP - qPCR was performed for the endogenous ub - HS5 and the ectopic ub - HS4 fragments as depicted in ( A ) . contr . : control , empty YI128 - vector was integrated at LEU2 . Experiments were performed in cdc48 ‑ 6 background . Data represent means ± SD ( n = 2 ) . Experimental mapping and bioinformatic prediction identify a similar ub - HS - motif . Comparison between the experimentally mapped ub - HS4 F7 ( B ) and the consensus motif of all ub - HSs identified by the MEME software .

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B) A 39 bp fragment of ub-HS4 is sufficient to drive ectopic ub-HS formation. Fragments of ub-HS4 were integrated ectopically and ub-K48 ChIP-qPCR was performed for the endogenous ub-HS5 and the ectopic ub-HS4 fragments as depicted in (A). contr.: control, empty YI128-vector was integrated at LEU2. Experiments were performed in cdc48‑6 background. Data represent means ± SD (n = 2). Experimental mapping and bioinformatic prediction identify a similar ub-HS-motif. Comparison between the experimentally mapped ub-HS4 F7 (B) and the consensus motif of all ub-HSs identified by the MEME software.


E ) Euc1 binds to the ub - HS - motif in a Y1H assay . Gal4 - AD - or Gal4 - AD - Euc1 - encoding plasmids were transformed into a Y1H reporter strain as described in ( D ) . Serial dilutions were spotted on control plates and plates lacking histidine with 20mM 3 - amino - triazole ( 3AT ) . Cells were grown at 30 ° C for 3 days .

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E) Euc1 binds to the ub-HS-motif in a Y1H assay. Gal4-AD- or Gal4-AD-Euc1-encoding plasmids were transformed into a Y1H reporter strain as described in (D). Serial dilutions were spotted on control plates and plates lacking histidine with 20mM 3-amino-triazole (3AT). Cells were grown at 30°C for 3 days.


F ) Euc1 is required for formation of ub - HSs . ChIP with ub - K48 specific antibodies was performed for the indicated strains and enriched DNA was analyzed by qPCR . Data represent means ± SD ( n = 4 ) .

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F) Euc1 is required for formation of ub-HSs. ChIP with ub-K48 specific antibodies was performed for the indicated strains and enriched DNA was analyzed by qPCR. Data represent means ± SD (n = 4).


Euc1 binds to endogenous ub - HSs . Genome - wide binding profiles of K48 - linked ubiquitin chains were obtained in ChIP - chip experiments as described in Fig . 1A . Euc1 - ChIP experiments were performed with a polyclonal antibody raised against Euc1 aa 292 - 462 . Data represent means from two independent replicates .

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Euc1 binds to endogenous ub-HSs. Genome-wide binding profiles of K48-linked ubiquitin chains were obtained in ChIP-chip experiments as described in Fig. 1A. Euc1-ChIP experiments were performed with a polyclonal antibody raised against Euc1 aa 292-462. Data represent means from two independent replicates.


Euc1 binds to endogenous ub - HSs . Genome - wide binding profiles of Euc1 were obtained in ChIP - chip experiments as described in Fig . 1A . Euc1 - ChIP experiments were performed with a polyclonal antibody raised against Euc1 aa 292 - 462 . Data represent means from two independent replicates .

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Euc1 binds to endogenous ub-HSs. Genome-wide binding profiles of Euc1 were obtained in ChIP-chip experiments as described in Fig. 1A. Euc1-ChIP experiments were performed with a polyclonal antibody raised against Euc1 aa 292-462. Data represent means from two independent replicates.


( C ) Euc1 can induce transactivation via its N - terminal 30 amino acids . Euc1 constructs under the endogenous EUC1 promoter were transformed in a reporter strain as described for Fig . 2D and serial dilutions were spotted on control or selective media to test HIS3 - activation . Cells were grown at 30 ° C for 3 days .

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(C) Euc1 can induce transactivation via its N-terminal 30 amino acids. Euc1 constructs under the endogenous EUC1 promoter were transformed in a reporter strain as described for Fig. 2D and serial dilutions were spotted on control or selective media to test HIS3-activation. Cells were grown at 30°C for 3 days.


( D ) Quantification of HIS3 mRNA levels from strains used in ( C ) . Cells were grown in liquid media with selection for the transformed plasmids ( SC - Leu ) , harvested in logarithmic growth phase and total mRNA was prepared . After reverse transcription , HIS3 mRNA levels were quantified using qPCR ( RT - qPCR ) , normalized first to ACT1 mRNA and then to the empty vector control strain . Data represent means ± SD ( n = 4 ) . p = 2 . 43 * 10 - 5 ( Student ' s t - test ) .

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(D) Quantification of HIS3 mRNA levels from strains used in (C). Cells were grown in liquid media with selection for the transformed plasmids (SC-Leu), harvested in logarithmic growth phase and total mRNA was prepared. After reverse transcription, HIS3 mRNA levels were quantified using qPCR (RT-qPCR), normalized first to ACT1 mRNA and then to the empty vector control strain. Data represent means ± SD (n = 4). p = 2.43*10-5 (Student's t-test).


( A ) Euc1 interacts with SUMO pathway proteins in a yeast two - hybrid ( Y2H ) assay . A Y2H reporter strain ( PJ69 - 7a ) was transformed with Gal4 DNA - binding domain ( BD ) and Gal4 activation domain ( AD ) fusion constructs in the indicated combinations . AD - SUMO - GG can be conjugated to SUMOylation substrates , while AD - SUMO - AA is conjugation - deficient . Cells were spotted on control media or selective media ( - His ) and grown for 3 and 6 days , respectively .

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(A) Euc1 interacts with SUMO pathway proteins in a yeast two-hybrid (Y2H) assay. A Y2H reporter strain (PJ69-7a) was transformed with Gal4 DNA-binding domain (BD) and Gal4 activation domain (AD) fusion constructs in the indicated combinations. AD-SUMO-GG can be conjugated to SUMOylation substrates, while AD-SUMO-AA is conjugation-deficient. Cells were spotted on control media or selective media (- His) and grown for 3 and 6 days, respectively.


( B ) Euc1 is SUMOylated by Ubc9 , Siz1 and Siz2 on K231 . Euc1 was immunoprecipitated from the indicated strains and eluates were probed by WB against SUMO ( top ) and Euc1 ( bottom ) . An up - shifted band corresponding to Euc1SUMO was detected with both antibodies . Asterisks denote non - specific bands .

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

(B) Euc1 is SUMOylated by Ubc9, Siz1 and Siz2 on K231. Euc1 was immunoprecipitated from the indicated strains and eluates were probed by WB against SUMO (top) and Euc1 (bottom). An up-shifted band corresponding to Euc1SUMO was detected with both antibodies. Asterisks denote non-specific bands.


( C ) Euc1 is predominantly monoSUMOylated on lysine 231 . Denaturing NiNTA - pulldowns ( NiNTA - PD ) with strains expressing His - tagged SUMO ( HisSUMO ) as indicated and 3FLAGEuc1 constructs under the control of an ADH promoter . Covalently SUMO - modified proteins were enriched and eluates probed with a FLAG antibody to visualize SUMOylated Euc1 . Eluates were probed for SUMO to control for equal pull - down , Pgk1 was probed as input control . Euc1 - KR denotes the K231R mutation here and hereafter .

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(C) Euc1 is predominantly monoSUMOylated on lysine 231. Denaturing NiNTA-pulldowns (NiNTA-PD) with strains expressing His-tagged SUMO (HisSUMO) as indicated and 3FLAGEuc1 constructs under the control of an ADH promoter. Covalently SUMO-modified proteins were enriched and eluates probed with a FLAG antibody to visualize SUMOylated Euc1. Eluates were probed for SUMO to control for equal pull-down, Pgk1 was probed as input control. Euc1-KR denotes the K231R mutation here and hereafter.


( D ) Euc1 SUMOylation is required for ub - HS formation . Ub - K48 ChIP quantified by qPCR for the indicated strains . The ubx5 ∆ background was used to enhance the ubiquitin signal , similar results were obtained in a WT background . Data represent means ± SD ( n = 3 ) .

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(D) Euc1 SUMOylation is required for ub-HS formation. Ub-K48 ChIP quantified by qPCR for the indicated strains. The ubx5∆ background was used to enhance the ubiquitin signal, similar results were obtained in a WT background. Data represent means ± SD (n = 3).


( E ) Euc1 ubiquitylation depends on Slx8 and partly on Euc1 SUMOylation . Denaturing NiNTA - PDs as described for ( C ) but with strains expressing His - ubiquitin ( HisUbi ) and 3FLAGEuc1 under the control of the ADH promoter . WBs were developed with a FLAG antibody to probe for 3FLAGEuc1 , a ubiquitin blot served as PD - control and Dpm1 as input control . The slx8 - CD allele carries the C206S , C209S - mutations ( Xie et al , 2007 ) .

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(E) Euc1 ubiquitylation depends on Slx8 and partly on Euc1 SUMOylation. Denaturing NiNTA-PDs as described for (C) but with strains expressing His-ubiquitin (HisUbi) and 3FLAGEuc1 under the control of the ADH promoter. WBs were developed with a FLAG antibody to probe for 3FLAGEuc1, a ubiquitin blot served as PD-control and Dpm1 as input control. The slx8-CD allele carries the C206S, C209S-mutations (Xie et al, 2007).


SUMOylated Euc1 is required to recruit Slx5 and Slx8 to ub - HSs . ChIP against Slx5 in the indicated genetic backgrounds were quantified by qPCR . Note that Slx5 is still recruited in the absence of Slx8 ( 3HA - Slx5 slx8 ∆ ) , but not vice versa ( Slx8 - 9myc slx5 ∆ ) . Data for WT and 3HA - Slx5 in ( F ) and

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SUMOylated Euc1 is required to recruit Slx5 and Slx8 to ub-HSs. ChIP against Slx5 in the indicated genetic backgrounds were quantified by qPCR. Note that Slx5 is still recruited in the absence of Slx8 (3HA-Slx5 slx8∆), but not vice versa (Slx8-9myc slx5∆). Data for WT and 3HA-Slx5 in (F) and WT


SUMOylated Euc1 is required to recruit Slx8 to ub - HSs . ChIP against Slx8 in the indicated genetic backgrounds were quantified by qPCR . Note that Slx5 is still recruited in the absence of Slx8 ( 3HA - Slx5 slx8 ∆ ) , but not vice versa ( Slx8 - 9myc slx5 ∆ ) . Data for WT and Slx8 - 9myc in ( G )

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SUMOylated Euc1 is required to recruit Slx8 to ub-HSs. ChIP against Slx8 in the indicated genetic backgrounds were quantified by qPCR. Note that Slx5 is still recruited in the absence of Slx8 (3HA-Slx5 slx8∆), but not vice versa (Slx8-9myc slx5∆). Data for WT and Slx8-9myc in (G)


( A ) Euc181 - 183 binds to Slx5 in Y2H assays . Y2H assay performed as in Fig . 4A . Serial dilutions were spotted for each plasmid combination and cells were grown at 30 ° C for 2 days . The Slx5 - RING ∆ construct is deleted for the Slx5 C - terminus from beginning of the RING - domain ( ∆ aa 488 - 619 ) . See also Fig . EV3B .

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(A) Euc181-183 binds to Slx5 in Y2H assays. Y2H assay performed as in Fig. 4A. Serial dilutions were spotted for each plasmid combination and cells were grown at 30°C for 2 days. The Slx5-RING∆ construct is deleted for the Slx5 C-terminus from beginning of the RING-domain (∆aa 488-619). See also Fig. EV3B.


( B ) Euc1 binds Slx5 in vitro . Recombinant purified GST or GSTEuc1 were used in GST pull - down assays to co - precipitate recombinant 6HisSlx5 .

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(B) Euc1 binds Slx5 in vitro. Recombinant purified GST or GSTEuc1 were used in GST pull-down assays to co-precipitate recombinant 6HisSlx5.


( C ) Euc1 binds to Slx5 in vivo . Cell lysates from an euc1 ∆ strain expressing the indicated 3FLAGEuc1 constructs from plasmids ( under EUC1 promoter ) were subjected to immunoprecipitation using anti - FLAG beads . IP - eluates were probed by WB with Slx5 , SUMO and Euc1 antibodies , inputs were probed with Slx5 , FLAG and Dpm1 antibodies ( top to bottom ) . Note that the Euc1 - Slx5 interaction is independent of the Slx8 ligase activity ( slx8 - CD , lanes 5 - 8 ) .

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(C) Euc1 binds to Slx5 in vivo. Cell lysates from an euc1∆ strain expressing the indicated 3FLAGEuc1 constructs from plasmids (under EUC1 promoter) were subjected to immunoprecipitation using anti-FLAG beads. IP-eluates were probed by WB with Slx5, SUMO and Euc1 antibodies, inputs were probed with Slx5, FLAG and Dpm1 antibodies (top to bottom). Note that the Euc1-Slx5 interaction is independent of the Slx8 ligase activity (slx8-CD, lanes 5-8).


( D ) Euc1 Slx5 - binding mutants ( SBM1 , SBM2 ) affect Euc1 - Slx5 interaction in Y2H assays . AD - Euc181 - 183 constructs harboring mutations in the region required for Slx5 binding were probed for interaction with BD - Slx5 constructs as described in Fig . 4A . Mutations : SBM1 : aa 139 - 143 ENQKK > ANAAA , SBM2 : aa 162 - 165 KEVF > AAAA . Serial dilutions were spotted and cells were grown at 30 ° C for 2 days . See Appendix Fig . S6A for expression levels .

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(D) Euc1 Slx5-binding mutants (SBM1, SBM2) affect Euc1-Slx5 interaction in Y2H assays. AD-Euc181-183 constructs harboring mutations in the region required for Slx5 binding were probed for interaction with BD-Slx5 constructs as described in Fig. 4A. Mutations: SBM1: aa 139-143 ENQKK>ANAAA, SBM2: aa 162-165 KEVF>AAAA. Serial dilutions were spotted and cells were grown at 30°C for 2 days. See Appendix Fig. S6A for expression levels.


( E ) Euc1 - SBM constructs show defective Slx5 binding in vivo . Mutations described in ( D ) were introduced into full - length Euc1 constructs ( with or without the K231R mutation ) and FLAG IPs were performed as described in ( C ) . WB analysis showed strong Slx5 - binding defects for the SBM1 / SBM2 and SBM1 + 2 constructs ( top panel , immunoprecipitated Slx5 ) . WBs were probed as in ( C ) .

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(E) Euc1-SBM constructs show defective Slx5 binding in vivo. Mutations described in (D) were introduced into full-length Euc1 constructs (with or without the K231R mutation) and FLAG IPs were performed as described in (C). WB analysis showed strong Slx5-binding defects for the SBM1/SBM2 and SBM1+2 constructs (top panel, immunoprecipitated Slx5). WBs were probed as in (C).


( F ) The Slx5 middle domain ( Slx5 - Md ) is required for interaction with Euc1 . Y2H assays with AD - Euc181 - 183 and BD - Slx5 constructs . Slx5 - Md : aa 201 - 335 , Slx5 - Md ∆ : ∆ aa 201 - 338 . Serial dilutions were spotted and cells were grown at 30 ° C for 4 days . See also Fig . EV3C .

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(F) The Slx5 middle domain (Slx5-Md) is required for interaction with Euc1. Y2H assays with AD-Euc181-183 and BD-Slx5 constructs. Slx5-Md: aa 201-335, Slx5-Md∆: ∆aa 201-338. Serial dilutions were spotted and cells were grown at 30°C for 4 days. See also Fig. EV3C.


( G ) Euc1 - SBM constructs show defective binding to the Slx5 - Md . Y2H assay with the indicated constructs as in ( D ) . See Appendix Fig . S6A for expression controls .

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(G) Euc1-SBM constructs show defective binding to the Slx5-Md. Y2H assay with the indicated constructs as in (D). See Appendix Fig. S6A for expression controls.


( A ) Euc1 Slx5 - binding mutations impair Euc1 ubiquitylation . His - ubiquitin - modified proteins were enriched by denaturing NiNTA - PDs as described in Fig . 4E . 3FLAG - tagged Euc1 constructs were expressed from plasmids under the control of the ADH promoter . NiNTA - PD eluates were probed with FLAG and ubiquitin ( P4D1 ) antibodies . Whole cell lysates ( input ) were probed with FLAG and Dpm1 antibodies .

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(A) Euc1 Slx5-binding mutations impair Euc1 ubiquitylation. His-ubiquitin-modified proteins were enriched by denaturing NiNTA-PDs as described in Fig. 4E. 3FLAG-tagged Euc1 constructs were expressed from plasmids under the control of the ADH promoter. NiNTA-PD eluates were probed with FLAG and ubiquitin (P4D1) antibodies. Whole cell lysates (input) were probed with FLAG and Dpm1 antibodies.


( B ) The Slx5 - SIMs and Slx5 - Md are required for Euc1 ubiquitylation . NiNTA - PDs for His - ubiquitin as in ( A ) . All strains expressed wild - type 3FLAGEuc1 from plasmids under the ADH promoter and His - ubiquitin . Slx5 constructs were expressed from plasmids under control of the endogenous promoter . WBs were probed as described in ( A ) , Slx5 - levels were probed using an HA antibody . Asterisk denotes a non - specific band . Slx5 - SIM * : SIMs 1 - 4 were mutated as described ( Xie et al , 2010 ) , for SIM5 , aa 477 - 479 ( IIV ) were mutated to alanines . To avoid possible mislocalization by deletion of the Slx5 - Md , which overlaps with a putative NLS ( Westerbeck et al , 2014 ) , an N - terminal NLS was fused to all constructs . See Appendix Fig . S7C for HU complementation .

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(B) The Slx5-SIMs and Slx5-Md are required for Euc1 ubiquitylation. NiNTA-PDs for His-ubiquitin as in (A). All strains expressed wild-type 3FLAGEuc1 from plasmids under the ADH promoter and His-ubiquitin. Slx5 constructs were expressed from plasmids under control of the endogenous promoter. WBs were probed as described in (A), Slx5-levels were probed using an HA antibody. Asterisk denotes a non-specific band. Slx5-SIM*: SIMs 1-4 were mutated as described (Xie et al, 2010), for SIM5, aa 477-479 (IIV) were mutated to alanines. To avoid possible mislocalization by deletion of the Slx5-Md, which overlaps with a putative NLS (Westerbeck et al, 2014), an N-terminal NLS was fused to all constructs. See Appendix Fig. S7C for HU complementation.


( C ) Euc1 Slx5 - binding mutations ( SBM1 / SBM2 ) reduce / abolish ub - HSs and recruitment of Slx5 to ub - HSs . ChIP - qPCR for ub - K48 ( top ) or Slx5 ( using Slx5 antibody , bottom ) in euc1 ∆ ubx5 ∆ cells expressing the indicated constructs from plasmids under the control of the EUC1 promoter . Data represent means ± SD ( n = 3 ) . See also Appendix Fig . S7E - G .

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(C) Euc1 Slx5-binding mutations (SBM1/SBM2) reduce/abolish ub-HSs and recruitment of Slx5 to ub-HSs. ChIP-qPCR for ub-K48 (top) or Slx5 (using Slx5 antibody, bottom) in euc1∆ ubx5∆ cells expressing the indicated constructs from plasmids under the control of the EUC1 promoter. Data represent means ± SD (n = 3). See also Appendix Fig. S7E-G.


( D ) Slx5 - SIMs and the Slx5 - Md are required for the formation of ub - HSs and recruitment of Slx5 . ChIP - qPCR for ub - K48 ( left ) or Slx5 ( anti - HA ChIP , right ) in ubx5 ∆ cells or slx5 ∆ ubx5 ∆ cells complemented with plasmids expressing the indicated Slx5 - constructs under control of the SLX5 promoter . Data represent means ± SD ( n = 2 ) . See also Appendix Fig . S7H .

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(D) Slx5-SIMs and the Slx5-Md are required for the formation of ub-HSs and recruitment of Slx5. ChIP-qPCR for ub-K48 (left) or Slx5 (anti-HA ChIP, right) in ubx5∆ cells or slx5∆ ubx5∆ cells complemented with plasmids expressing the indicated Slx5-constructs under control of the SLX5 promoter. Data represent means ± SD (n = 2). See also Appendix Fig. S7H.


( A ) Deletion of EUC1 does not lead to ub - HS adjacent gene deregulation , but rather widespread transcriptome changes . Total RNA isolated from WT and euc1 ∆ cells was polyA - enriched and sequenced ( RNAseq , triplicates ) . Significance testing was based on the Wald test , see Materials and Methods for details . See also Appendix Fig . S8A for quantification of selected transcripts by qPCR .

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(A) Deletion of EUC1 does not lead to ub-HS adjacent gene deregulation, but rather widespread transcriptome changes. Total RNA isolated from WT and euc1∆ cells was polyA-enriched and sequenced (RNAseq, triplicates). Significance testing was based on the Wald test, see Materials and Methods for details. See also Appendix Fig. S8A for quantification of selected transcripts by qPCR.


( B ) RNAseq transcriptome analysis of EUC1 overexpression as in ( A ) . ∆ euc1 cells with pGAL - EUC1 or pEUC1 - EUC1 ( control ) integrated at the URA3 - locus ( YIplac211 ) were grown to mid - log phase and 2 % galactose was added for 3h . See also Appendix Fig . S8E .

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(B) RNAseq transcriptome analysis of EUC1 overexpression as in (A). ∆euc1 cells with pGAL-EUC1 or pEUC1-EUC1 (control) integrated at the URA3-locus (YIplac211) were grown to mid-log phase and 2% galactose was added for 3h. See also Appendix Fig. S8E.


( C ) EUC1 overexpression is toxic at elevated temperatures and upon exposure to the membrane fluidizing drug benzylalcohol . For ( C , D , F ) , serial dilutions of the indicated strains were spotted and grown on YPD control ( or selective media ) plates or under conditions as indicated . See also Fig . EV4A - B .

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(C) EUC1 overexpression is toxic at elevated temperatures and upon exposure to the membrane fluidizing drug benzylalcohol. For (C, D, F), serial dilutions of the indicated strains were spotted and grown on YPD control (or selective media) plates or under conditions as indicated. See also Fig. EV4A-B.


( D ) EUC1 overexpression toxicity depends on DNA binding and transactivation . The indicated EUC1 alleles with endogenous or galactose - inducible promoters were integrated at the URA3 locus ( YIplac211 , euc1 ∆ background ) and spotted on glucose control or galactose plates to induce EUC1 overexpression . See also Appendix Fig . S9A - C .

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(D) EUC1 overexpression toxicity depends on DNA binding and transactivation. The indicated EUC1 alleles with endogenous or galactose-inducible promoters were integrated at the URA3 locus (YIplac211, euc1∆ background) and spotted on glucose control or galactose plates to induce EUC1 overexpression. See also Appendix Fig. S9A-C.


( E ) Aberrant binding of overexpressed EUC1 partially depends on Euc1 DNA binding . ChIP - qPCR of Euc1 after 3 hours galactose induction . Note that IP / input ratios of Euc1 signals are shown , also for the control locus ( contr . , TOS1 promoter ) to highlight Euc1 binding at non - ub - HS sites . STI1 - CIN5 : intergenic region . TEC1 - us : upstream ( promoter ) region . Data represent means ± SD ( n = 3 ) . See also Fig . EV4C - D .

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(E) Aberrant binding of overexpressed EUC1 partially depends on Euc1 DNA binding. ChIP-qPCR of Euc1 after 3 hours galactose induction. Note that IP/input ratios of Euc1 signals are shown, also for the control locus (contr., TOS1 promoter) to highlight Euc1 binding at non-ub-HS sites. STI1-CIN5: intergenic region. TEC1-us: upstream (promoter) region. Data represent means ± SD (n = 3). See also Fig. EV4C-D.


( F ) Simultaneous overexpression of SLX5 and SLX8 rescue EUC1 toxicity . Experiment as in ( D ) , but with concomitant plasmid - borne overexpression ( ADH promoter ) of SLX5 and SLX8 ( bottom panels ) and on media selecting for SLX5 / SLX8 plasmids . See also Fig . EV4E and Appendix Fig . S9D .

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(F) Simultaneous overexpression of SLX5 and SLX8 rescue EUC1 toxicity. Experiment as in (D), but with concomitant plasmid-borne overexpression (ADH promoter) of SLX5 and SLX8 (bottom panels) and on media selecting for SLX5/SLX8 plasmids. See also Fig. EV4E and Appendix Fig. S9D.


EUC1 displays negative genetic interactions with genes involved in general and specific transcriptional regulation ( A ) and in particular with members of the Rpd3L histone deacetylase complex ( B ) . For ( A - D ) , serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates , or under conditions as indicated . Note that all strains used in ( A - D ) contained an extra copy of MED11 ,

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EUC1 displays negative genetic interactions with genes involved in general and specific transcriptional regulation (A) and in particular with members of the Rpd3L histone deacetylase complex (B). For (A-D), serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates, or under conditions as indicated. Note that all strains used in (A-D) contained an extra copy of MED11,


EUC1 displays negative genetic interactions with genes involved in particular with members of the Rpd3L histone deacetylase complex For , serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates , or under conditions as indicated . Note that all strains used in ( A - D ) contained an extra copy of MED11 ,

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EUC1 displays negative genetic interactions with genes involved in particular with members of the Rpd3L histone deacetylase complex For , serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates, or under conditions as indicated. Note that all strains used in (A-D) contained an extra copy of MED11,


For serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates , or under conditions as indicated . Note that all strains used contained an extra copy of MED11 ( C ) Plasmid - borne EUC1 complements genetic interactions with Rpd3L subunits . Empty vector ( - ) or a plasmid encoding EUC1 with its endogenous promoter were transformed in the indicated strains and spotted on selective media .

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For serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates, or under conditions as indicated. Note that all strains used contained an extra copy of MED11 (C) Plasmid-borne EUC1 complements genetic interactions with Rpd3L subunits. Empty vector (-) or a plasmid encoding EUC1 with its endogenous promoter were transformed in the indicated strains and spotted on selective media.


For serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates , or under conditions as indicated . Note that all strains used contained an extra copy of MED11 ( D ) EUC1 and Rpd3S act in a common pathway which is redundant with Rpd3L . rco1 ∆ and eaf3 ∆ ( Rpd3S ) show similar phenotypes when paired with sds3 ∆ ( Rpd3L ) as euc1 ∆ and are epistatic with euc1 ∆ .

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For serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates, or under conditions as indicated. Note that all strains used contained an extra copy of MED11 (D) EUC1 and Rpd3S act in a common pathway which is redundant with Rpd3L. rco1∆ and eaf3∆ (Rpd3S) show similar phenotypes when paired with sds3∆ (Rpd3L) as euc1∆ and are epistatic with euc1∆.


( A ) Euc1 is recruited to ub - HSs upon mild heat stress ( 37 ° C , 90 min ) . ChIP - qPCR experiments with strains and conditions as indicated . Data represent means ± SD ( n = 4 ) , see Appendix Fig . S11B for statistical analysis .

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(A) Euc1 is recruited to ub-HSs upon mild heat stress (37°C, 90 min). ChIP-qPCR experiments with strains and conditions as indicated. Data represent means ± SD (n = 4), see Appendix Fig. S11B for statistical analysis.


The ability to form ub - hotspots is crucial for endogenous EUC1 function . The genetic interaction of EUC1 with SDS3 ( Rpd3L complex ) was rescued with plasmid - borne EUC1 - alleles and serial dilutions were spotted on selective media . ( - ) denotes empty vector .

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The ability to form ub-hotspots is crucial for endogenous EUC1 function. The genetic interaction of EUC1 with SDS3 (Rpd3L complex) was rescued with plasmid-borne EUC1-alleles and serial dilutions were spotted on selective media. (-) denotes empty vector.


SLX5 also shows negative genetic interactions with SDS3 upon heat stress . Serial dilutions of the indicated strains were spotted on YPD and incubated as indicated . See also Appendix Fig . S12C - D . Note that all strains used in ( A - C ) contained an extra copy of MED11 ,

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SLX5 also shows negative genetic interactions with SDS3 upon heat stress. Serial dilutions of the indicated strains were spotted on YPD and incubated as indicated. See also Appendix Fig. S12C-D. Note that all strains used in (A-C) contained an extra copy of MED11,


All ORFs for the sense non - AUG - dependent translation of the ( G4C2 ) n ~ 188 , DPR coding for ( GA ) , ( GP ) , ( GR ) can be detected and quantified using a filter - trap binding assay probing for the c - terminal HA tag of the C9 DPR reporter construct . The ( GA ) ORF is the most abundantly detected DPR and GR the lowest in HEK293T cell 24 h post transient transfection of the C9 DPR reporter construct . n = 5 .

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All ORFs for the sense non-AUG-dependent translation of the (G4C2)n~188, DPR coding for (GA), (GP), (GR) can be detected and quantified using a filter-trap binding assay probing for the c-terminal HA tag of the C9 DPR reporter construct. The (GA) ORF is the most abundantly detected DPR and GR the lowest in HEK293T cell 24 h post transient transfection of the C9 DPR reporter construct. n = 5.


The C9orf72 NRE non - AUG - dependent reporter constructs recapitulate localization patterns and pathological features identified in C9orf72 NRE models . The Dendra2 ( green ) c - terminal fusion was used to monitor cellular localization and fluorescence intensity for each DPR translated from the G4C2 strand in HEK293T cells transiently cotransfected with an NES - mIFP - mIFP - NES ( NES - mIFP ) fusion protein cytoplasmic marker .

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The C9orf72 NRE non-AUG-dependent reporter constructs recapitulate localization patterns and pathological features identified in C9orf72 NRE models. The Dendra2 (green) c-terminal fusion was used to monitor cellular localization and fluorescence intensity for each DPR translated from the G4C2 strand in HEK293T cells transiently cotransfected with an NES-mIFP-mIFP-NES (NES-mIFP) fusion protein cytoplasmic marker.


C9 DPR reporter constructs show cell - type variability in the number of cells that are DPR positive and the overall DPR levels . Transient cotransfections of HEK293T cells robustly undergo G4C2 - Dendra2 non - AUG - dependent translation compared to NSC34 or rat primary cortical neurons . DPR positive cells and DPR fluorescent intensity were analyzed and calculated 24 h post - transfection only in cells expressing the co - transfected NES - mIFP construct . DPR fluorescent intensity was normalized to NES - mIFP fluorescent intensity to adjust for cell specific AUG - dependent translation levels ; represented as fold - change relative to HEK293T . HEK293T : n = 5 with m > 500 cells analyzed per n . NSC34 : n = 5 with m > 500 cells per n . Rat primary cortical neuron : n = 5 with m > 40 cells per n . ( * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 ) See also Figure EV1 - 2 .

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C9 DPR reporter constructs show cell-type variability in the number of cells that are DPR positive and the overall DPR levels. Transient cotransfections of HEK293T cells robustly undergo G4C2 -Dendra2 non-AUG-dependent translation compared to NSC34 or rat primary cortical neurons. DPR positive cells and DPR fluorescent intensity were analyzed and calculated 24 h post-transfection only in cells expressing the co-transfected NES-mIFP construct. DPR fluorescent intensity was normalized to NES-mIFP fluorescent intensity to adjust for cell specific AUG-dependent translation levels; represented as fold-change relative to HEK293T. HEK293T: n = 5 with m > 500 cells analyzed per n. NSC34: n = 5 with m > 500 cells per n. Rat primary cortical neuron: n = 5 with m > 40 cells per n. (***P < 0.001, **P < 0.01, *P <0.05) See also Figure EV1-2.


Quantitative fluorescent microscopy imaging shows a dose - dependent increase in the number of C9 DPR reporter positive cells with increasing concentrations of an apoptotic stimulator , staurosporine ( SSP ) . The C9 DPR reporter in the GP frame was utilized for these experiments . n = 4 .

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Quantitative fluorescent microscopy imaging shows a dose-dependent increase in the number of C9 DPR reporter positive cells with increasing concentrations of an apoptotic stimulator, staurosporine (SSP). The C9 DPR reporter in the GP frame was utilized for these experiments. n = 4.


Filter trap binding assays using DPR - specific antibodies show all DPR levels are increased for the sense - coding C9 DPR reporter when NSC34 cells are challenged with stress - inducing compounds . These stress - inducing compounds broadly cover distinct or overlapping cellular pathways ( see Table EV2 ) Antibodies specific for the C9orf72 NRE DPRs , poly - GA , - GP , or - GR , were used to quantify DPR levels normalized to GAPDH 24 h post transient transfection of the C9 DPR reporter constructs . n = 4 .

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Filter trap binding assays using DPR-specific antibodies show all DPR levels are increased for the sense-coding C9 DPR reporter when NSC34 cells are challenged with stress-inducing compounds. These stress-inducing compounds broadly cover distinct or overlapping cellular pathways (see Table EV2) Antibodies specific for the C9orf72 NRE DPRs, poly-GA, -GP, or -GR, were used to quantify DPR levels normalized to GAPDH 24 h post transient transfection of the C9 DPR reporter constructs. n = 4.


Quantitative fluorescent microscopy imaging shows that NSC34 , rat primary cortical neurons have increased relative DPR fluorescent intensity levels following compound induced cellular stress . In NES - mIFP cotransfection positive NSC34 and rat primary cortical neurons there is a significant increase in C9 DPR reporter fluorescent intensity levels 24 post treatment with most stress - inducing compounds . Fluorescent intensity was not normalized to NES - mIFP fluorescent levels . NSC34 : n = 10 with m > 500 cells analyzed per n . Cortical neuron : n = 6 with m > 40 cells per n . Endogenously expression of DPRs in iPS spinal motor neurons ( iPS sMN ) derived from C9orf72 NRE patients increases following induction of cellular stress . DPR fluorescent intensity in iPS sMN was measured using the DPR - specific antibodies described above . IPS sMN : n = 4 with m > 20 cells per n .

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Quantitative fluorescent microscopy imaging shows that NSC34, rat primary cortical neurons have increased relative DPR fluorescent intensity levels following compound induced cellular stress. In NES-mIFP cotransfection positive NSC34 and rat primary cortical neurons there is a significant increase in C9 DPR reporter fluorescent intensity levels 24 post treatment with most stress-inducing compounds. Fluorescent intensity was not normalized to NES-mIFP fluorescent levels. NSC34: n = 10 with m > 500 cells analyzed per n. Cortical neuron: n = 6 with m > 40 cells per n. Endogenously expression of DPRs in iPS spinal motor neurons (iPS sMN) derived from C9orf72 NRE patients increases following induction of cellular stress. DPR fluorescent intensity in iPS sMN was measured using the DPR-specific antibodies described above. IPS sMN: n = 4 with m > 20 cells per n.


Fluorescent microscopy images shows Glutamate - induced ( Glut ) excitotoxic stress increases DPR levels in rat cortical neurons expressing the C9 DPR reporter and in iPS sMN derived from C9orf72 NRE patients . Glutamate - induced DPR levels are reduced by pretreating cells with the AMPA and NMDA specific antagonists NBQX and MK801 , respectively . Relative fluorescent intensity quantification demonstrates there is a significant increase in DPR levels when primary cortical neurons and sMNs are challenged with the excitotoxic stressors : glutamate , AMPA , NMDA , and HC . Blocking either the AMPA or NMDA receptors reduces DPR levels in the presence of excitotoxic stressors . NES - mIFP or MAP2 were used to mark transfection positive rat cortical neurons or iPS sMNs , respectively , for quantification . DPR fluorescent intensity in iPS sMN was measured using DPR - specific antibodies . Changes in DPR levels are relative to non - stressed cells ( CTRL ) Cortical neuron : n = 6 with m > 40 cells per n . IPSC sMN : n = 4 with m > 25 cells per n .

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Fluorescent microscopy images shows Glutamate-induced (Glut) excitotoxic stress increases DPR levels in rat cortical neurons expressing the C9 DPR reporter and in iPS sMN derived from C9orf72 NRE patients. Glutamate-induced DPR levels are reduced by pretreating cells with the AMPA and NMDA specific antagonists NBQX and MK801, respectively. Relative fluorescent intensity quantification demonstrates there is a significant increase in DPR levels when primary cortical neurons and sMNs are challenged with the excitotoxic stressors: glutamate, AMPA, NMDA, and HC. Blocking either the AMPA or NMDA receptors reduces DPR levels in the presence of excitotoxic stressors. NES-mIFP or MAP2 were used to mark transfection positive rat cortical neurons or iPS sMNs, respectively, for quantification. DPR fluorescent intensity in iPS sMN was measured using DPR-specific antibodies. Changes in DPR levels are relative to non-stressed cells (CTRL) Cortical neuron: n = 6 with m > 40 cells per n. IPSC sMN: n = 4 with m > 25 cells per n.


Microscopy images show increased DPR fluorescent intensity levels from medium and high frequency stimulation of ChR2 in primary cortical neurons transfected with the C9 DPR reporter .

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Microscopy images show increased DPR fluorescent intensity levels from medium and high frequency stimulation of ChR2 in primary cortical neurons transfected with the C9 DPR reporter.