( g ) Flow cytometry of untreated macrophages ( control ) or of macrophages stimulated with LPS ATP , stained with MitoTracker Deep Red . Numbers above bracketed lines indicate with loss of mitochondrial potential .

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(g) Flow cytometry of untreated macrophages (control) or of macrophages stimulated with LPS and ATP, stained with MitoTracker Deep Red. Numbers above bracketed lines indicate percent cells with loss of mitochondrial membrane potential.


( h ) Flow cytometry of LPS - primed wild - type , ASC - deficient NALP3 - deficient peritoneal macrophages stimulated for 30 min with ATP stained with annexin V . Data represent three experiments ( mean s . d . in a - e ) .

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(h) Flow cytometry of LPS-primed wild-type, ASC-deficient and NALP3-deficient peritoneal macrophages stimulated for 30 min with ATP and stained with annexin V. Data represent three experiments (mean and s.d. in a-e).


( a ) ELISA of serum IL - 1β IL - 18 at 24 h after intraperitoneal injection of LPS ( 12 mg per kg ) into Map1lc3b + / + Map1lc3b − / − mice .

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(a) ELISA of serum IL-1β and IL-18 at 24 h after intraperitoneal injection of LPS (12 mg per kg body weight) into male Map1lc3b+/+ and Map1lc3b−/− mice.


( b ) Survival of Map1lc3b + / + mice ( n = 11 ) Map1lc3b − / − mice ( n = 10 ) after LPS challenge ( 100 mg per kg ) , assessed over a period of 2 .

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(b) Survival of Map1lc3b+/+ mice (n = 11) and Map1lc3b−/− mice (n = 10) after LPS challenge (100 mg per kg body weight), assessed over a period of 2 weeks.


( c , d ) ELISA of serum IL - 1β IL - 18 in Map1lc3b + / + Map1lc3b − / − mice ( c ) Becn1 + / + Becn1 + / − mice ( d ) at 24 h after CLP or sham laparotomy . In a , c , d , each symbol represents an mouse ; small horizontal lines indicate mean . P values , unpaired two - Student ' s t - ( a , c , d ) or log - rank ( b ) . Data are representative of two experiments .

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(c,d) ELISA of serum IL-1β and IL-18 in male Map1lc3b+/+ and Map1lc3b−/− mice (c) and Becn1+/+ and Becn1+/− mice (d) at 24 h after CLP or sham laparotomy. In a,c,d, each symbol represents an individual mouse; small horizontal lines indicate the mean. P values, unpaired two-tailed Student's t-test (a,c,d) or log-rank test (b). Data are representative of two experiments.


( A ) features mapped here displayed differences between CLL ( CLL1 ) an NBC donor ( H7 ) as shown for TCF4 locus , which encodes for a transcription factor from E family . on increased H3K4me1 , H3K27ac ATAC signal , two predicted enhancer loci were marked that became active in CLL . Note that y - axis for - seq is scaled differently for CLL ( 8000 ) NBCs ( 100 ) to visualize that TCF4 gene was not completely silenced but lowly expressed also in NBCs as evident also from H3K36me3 mark . grey depicts active region grey confined enhancer locus coinciding with an open region .

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(A) Chromatin features mapped here displayed differences between CLL (patient CLL1) and an NBC donor (H7) as shown for the TCF4 locus, which encodes for a transcription factor from the E protein family. Based on the increased H3K4me1, H3K27ac and ATAC signal, two predicted enhancer loci were marked that became active in CLL. Note that the y-axis for RNA-seq is scaled differently for CLL (8000) and NBCs (100) to visualize that the TCF4 gene was not completely silenced but lowly expressed also in NBCs as evident also from the H3K36me3 mark. Light grey depicts active chromatin region and dark grey the confined enhancer locus coinciding with an open chromatin region.


( D ) Distribution of ~ 24 , 400 annotated differentially accessible regions ( ATAC - seq ) in NBC CLL samples according to state annotation . In total 7 , 605 annotated regions gained an ATAC - seq signal in CLL , while it was lost at 16 , 790 loci .

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(D) Distribution of ~24,400 annotated differentially accessible regions (ATAC-seq) in NBC and CLL samples according to the chromatin state annotation. In total 7,605 annotated regions gained an ATAC-seq signal in CLL, while it was lost at 16,790 loci.


( A ) Left , example of a large PMD on chromosome 2 derived from a consensus of CLL samples ( n = 11 ) . Right , genome - wide of PMDs across CLL samples ( n = 11 ) NBCs ( n = 6 ) . Red , methylated ; blue , unmethylated .

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(A) Left, example of a large PMD on chromosome 2 derived from a consensus of CLL samples (n=11). Right, genome-wide quantification of PMDs across CLL samples (n=11) and NBCs (n=6). Red, methylated DNA; blue, unmethylated DNA.


( B ) Expression of genes located inside ( blue ) outside ( grey ) PMD regions . ( C ) Fraction of differentially expressed genes inside or outside PMD regions . Up - downregulated genes are shown in red green , respectively .

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(B) Expression of genes located inside (blue) and outside (grey) the PMD regions. (C) Fraction of differentially expressed genes inside or outside PMD regions. Up- and downregulated genes are shown in red and green, respectively.


( D ) Upper panel : Average signal of histone modification marks normalized to H3 standard deviation in 5 - kb windows around ± 50 kb flanking regions of PMD boundaries . Normalized fold changes were calculated by dividing to average signal flanking outside PMD boundaries . Blue box , within PMDs ; thin , outside PMDs , nor . - normalized . Lower panel : Distribution of bound CTCF sites in CLL as determined by ChIP - seq ( blue ) around ± 50 kb flanking regions of PMD boundaries in 5 kb windows . height of curves gives sum of next nearest CTCF peak at given to PMD boundary .

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(D) Upper panel: Average signal of histone modification marks normalized to H3 and standard deviation in 5-kb windows around the ±50 kb flanking regions of PMD boundaries. Normalized fold changes were calculated by dividing to the average signal flanking outside the PMD boundaries. Blue box, within PMDs; thin line, outside PMDs, nor. - normalized. Lower panel: Distribution of bound CTCF sites in CLL cells as determined by ChIP-seq (blue line) around the ±50 kb flanking regions of PMD boundaries in 5 kb windows. The height of the curves gives the sum of the next nearest CTCF peak at the given distance to the PMD boundary.


( A ) Correlation function of H3K4me3 ChIP - seq reads . A broadening of H3K4me3 domains in CLL by 1 - 2 nucleosomes was detected . Error bars represent .

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(A) Correlation function of H3K4me3 ChIP-seq reads. A broadening of H3K4me3 domains in CLL by 1-2 nucleosomes was detected. Error bars represent the SEM.


( B ) H3K4me3 peak width distribution at common promoters in CLL NBC controls .

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(B) H3K4me3 peak width distribution at common promoters in CLL and NBC controls.


( C ) Distribution of nucleosome occupancy calculated from histone H3 ChIP - seq averaged over a 1000 bp window within promoters . H3K4me3 regions displayed higher nucleosome density for CLL as compared to NBC samples .

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(C) Distribution of nucleosome occupancy calculated from histone H3 ChIP-seq averaged over a 1000 bp window within promoters. H3K4me3 regions displayed higher nucleosome density for CLL as compared to NBC samples.


( D ) Exemplary region at TAF13 promoter showing higher H3K4me3 upstream of TSS with lost ATAC signal ( grey bar ) as compared to NBC control .

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(D) Exemplary region at the TAF13 promoter showing higher H3K4me3 levels upstream of the TSS with lost ATAC signal (grey bar) as compared to the NBC control.


( E ) TF motif of ATAC signal lost at CLL promoters with broadened H3K4me3 regions . An enrichment for NFY TF motif was detected .

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(E) TF motif analysis of ATAC signal lost at CLL promoters with broadened H3K4me3 regions. An enrichment for the NFY TF motif was detected.


( F ) of genome - wide histone modification patterns at promoters within - 2 to 5 kb around TSS ( x - axis ) for an NBC ( H3 ) a ( CLL1 ) sample . Each on y - axis corresponds to one promoter . clustering revealed one cluster with loss of H3K4me3 at bivalent promoters in CLL .

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(F) Heatmap of genome-wide histone modification patterns at promoters within -2 to 5 kb around the TSS (x-axis) for an NBC (H3) and a patient (CLL1) sample. Each line on the y-axis corresponds to one promoter. The clustering revealed one cluster with loss of H3K4me3 at bivalent promoters in CLL.


( D ) Clustering of samples via expression of bidirectionally regions that are differential between NBCs CLL quantified using DESeq2 .

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(D) Clustering of samples via expression of bidirectionally regions that are differential between NBCs and CLL and quantified using DESeq2.


( E ) Volcano plot of differential super - enhancers targeting known leukemia cancer genes . Examples include SE loss at CDKN1A , PI3KC2B KMT2B ( MLL2 ) SE gain at FMOD , CREB3L2 , CTLA4 , TCF4 , LEF1 BCL2 . Points represent non - differential SEs ( grey ) , differential SEs ( FDR < 0 . 01 ) with fold change greater than 1 ( orange ) .

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(E) Volcano plot of differential super-enhancers targeting known leukemia and cancer genes. Examples include SE loss at CDKN1A, PI3KC2B and KMT2B (MLL2) and SE gain at FMOD, CREB3L2, CTLA4, TCF4, LEF1 and BCL2. Points represent non-differential SEs (grey), and differential SEs (FDR < 0.01) with fold change greater than 1 (orange).


( F ) expression changes of selected genes associated with differential SEs . Top : genes significantly ( FDR < 0 . 05 ) up - regulated by SEs in CLL . Bottom : Genes significantly down - regulated by SEs in CLL .

(F) RNA expression changes of selected genes associated with differential SEs. Top: genes significantly (FDR < 0.05) up-regulated by SEs in CLL. Bottom: Genes significantly down-regulated by SEs in CLL.


( G ) Comparison of normalized gene expression of histone deacetylases between CLL NBCs . Histone deacetylases significantly up - regulated in CLL are shown in bold .

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(G) Comparison of normalized gene expression of histone deacetylases between CLL and NBCs. Histone deacetylases significantly up-regulated in CLL are shown in bold.


( H ) HDAC activity its inhibition by panobinostat in B from CLL ( red ) in comparison to healthy donors ( grey ) . Error bars indicate standard deviation measured in four biological replicates .

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(H) HDAC activity and its inhibition by panobinostat in B cells from CLL patients (red) in comparison to healthy donors (grey). Error bars indicate standard deviation measured in four biological replicates.


( I ) Genome browser view of H3K27ac tracks ( in grey ) at exemplary genes for NBCs CLL 24 hours after mock after panobinostat treatment . At genes such as CDKN1A ( cycle control ) KLF13 , reduced H3K27ac signal in CLL was increased upon HDAC inhibition to level found in NBCs . WNT11 is shown as an example of a de novo gain of an active enhancer due to treatment with panobinostat .

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(I) Genome browser view of H3K27ac tracks (in grey) at exemplary genes for NBCs and CLL cells 24 hours after mock and after panobinostat treatment. At genes such as CDKN1A (cell cycle control) and KLF13, reduced H3K27ac signal in CLL was increased upon HDAC inhibition to the level found in NBCs. WNT11 is shown as an example of a de novo gain of an active enhancer due to treatment with panobinostat.


( J ) displaying changes in H3K27ac read occupancy in CLL upon panobinostat treatment for 24 h . A general gain of H3K27ac in enhancers upon panobinostat treatment was observed .

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(J) Heatmap displaying changes in H3K27ac read occupancy in CLL upon panobinostat treatment for 24 h. A general gain of H3K27ac in enhancers upon panobinostat treatment was observed.


( B ) H3K27ac ( left ) methylation ( right ) at NFAT binding sites . CLL showed both an H3K27ac enrichment - hypomethylation at NFAT target sites , suggesting a higher activity of TFs from NFAT family in CLL .

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(B) H3K27ac (left) and DNA methylation (right) at NFAT binding sites. CLL cells showed both an H3K27ac enrichment and DNA-hypomethylation at NFAT target sites, suggesting a higher activity of TFs from the NFAT family in CLL.


( D ) ATAC footprints for E2A , EBF CTCF motifs from Homer . E2A motif footprint ( binding site of E family TFs like TCF4 ) displayed an increased binding signal in CLL while sites with EBF CTCF motif lost ATAC signal .

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(D) ATAC footprints for E2A, EBF and CTCF motifs from the Homer analysis. The E2A motif footprint (binding site of E protein family TFs like TCF4) displayed an increased binding signal in CLL while sites with the EBF and CTCF motif lost the ATAC signal.


( E ) Expression of genes nearest to target enhancers with constitutively bound ( " stable " ) CTCF vs . enhancers that lost CTCF in CLL . Loss of CTCF binding correlated with reduced gene expression .

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(E) Expression of the genes nearest to target enhancers with constitutively bound ("stable") CTCF vs. enhancers that lost CTCF in CLL. Loss of CTCF binding correlated with reduced gene expression.


( G ) Enhancer - promoter rewiring at NFKB2 locus . A switch of interactions between NFKB2 promoter two different enhancers in CLL ( red ) vs . NBCs ( grey ) was observed . on CTCF ChIP - seq both intronic enhancers at NFKB2 FBXL15 gene show ( nearby ) constitutively bound CTCF , even though targets of two enhancers are switched .

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(G) Enhancer-promoter rewiring at the NFKB2 locus. A switch of interactions between the NFKB2 promoter and two different enhancers in CLL (red line) vs. NBCs (grey line) was observed. Based on the CTCF ChIP-seq analysis both intronic enhancers at the NFKB2 and the FBXL15 gene show (nearby) constitutively bound CTCF, even though the targets of the two enhancers are switched.


( E ) of regulation of SNX22 by intronic H464 enhancer shown in panel B . This enhancer lost its ATAC signal at predicted EBF1 binding site . EBF1 ChIP - seq validated that EBF1 is indeed lost at this site . Although H3K27ac at locus was only slightly reduced , transcription of SNX22 was largely reduced . These findings are consistent with a mechanism where EBF1 binding drives gene expression of SNX22 by binding to H464 .

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(E) Analysis of regulation of SNX22 by the intronic H464 enhancer shown in panel B. This enhancer lost its ATAC signal at the predicted EBF1 binding site. EBF1 ChIP-seq analysis validated that EBF1 is indeed lost at this site. Although H3K27ac at the locus was only slightly reduced, transcription of SNX22 was largely reduced. These findings are consistent with a mechanism where EBF1 binding drives gene expression of SNX22 by binding to H464.


B Silver stain of GST ‐ PTB RRM2pull ‐ down of wild ‐ type RRM2 Y247Q mutant . Five microlitre of pull ‐ down was run on a 15 % SDS - PAGE silver ‐ stained . Three strong bands can be seen which are due to recombinant beads used for pull ‐ down . region encompassing 50 kDa to top of gel was sliced subjected to in ‐ gel digestion mass spectrometry . two strongest bands visible in this region are labelled as Matrin3 Raver1 .

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B Silver stain of the GST‐PTB RRM2pull‐down of the wild‐type RRM2 and the Y247Q mutant. Five microlitre of the pull‐down was run on a 15% SDS-PAGE and silver‐stained. Three strong bands can be seen which are due to the recombinant protein and the beads used for the pull‐down. The region encompassing 50 kDa to the top of the gel was sliced and subjected to in‐gel digestion and mass spectrometry. The two strongest bands visible in this region are labelled as Matrin3 and Raver1.


C identified in GST ‐ PTB RRM2 pull ‐ down ranked by their unique peptide number . table shows different we found binding to RRM2 , as well as when present , of PRI motif . indicated function is only a guideline as many have more functions than shown .

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C Proteins identified in the GST‐PTB RRM2 pull‐down ranked by their unique peptide number. The table shows the different proteins we found binding to RRM2, as well as when present, the sequence of the PRI motif. The indicated function is only a guideline as many have more functions than shown.


D Western blot of GSTpull ‐ down using antibodies against Matrin3 Raver1 . Lanes 1 2 show 5 10 % of input , respectively , lanes 3 4 show GST ‐ PTB full ‐ pull ‐ down of wild ‐ type Y247Q mutant , respectively , lanes 5 6 show GST ‐ PTB RRM2 pull ‐ down of wild ‐ type Y247Q mutant , respectively , lane 7 with pull ‐ down using GST alone .

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D Western blot of the GSTpull‐down using antibodies against Matrin3 and Raver1. Lanes 1 and 2 show 5 and 10% of input, respectively, lanes 3 and 4 show GST‐PTB full‐length pull‐down of wild‐type and Y247Q mutant, respectively, and lanes 5 and 6 show GST‐PTB RRM2 pull‐down of wild‐type and Y247Q mutant, respectively, and lane 7 with pull‐down using GST alone.


B FLAGimmunoprecipitation of Matrin3 Raver1 , both with wild ‐ type with PRI mutated , FLAG ‐ MS2 as a negative control . immunoprecipitated was separated in a SDS - PAGE subjected to Western blot using antibody against PTB which showed interaction to wild ‐ type Matrin3 ( lane 2 ) Raver1 ( lane 4 ) . input was also analysed by Western blot with antibodies against PTB as a loading control against FLAG tag to ensure equal expression of .

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B FLAGimmunoprecipitation of Matrin3 and Raver1, both with wild‐type and with PRI mutated, and FLAG‐MS2 as a negative control. The immunoprecipitated complex was separated in a SDS-PAGE and subjected to Western blot using antibody against PTB which showed interaction to wild‐type Matrin3 (lane 2) and Raver1 (lane 4). The input was also analysed by Western blot with antibodies against PTB as a loading control and against the FLAG tag to ensure equal expression of proteins.


C 20 ‐ residue Raver1 491 - 511 ( lane 1 ) Matrin3 346 - 365 ( lane 3 ) peptide fused to MS2 were transcribed translated in vitro ( Input ) then pulled down with GST ‐ PTB or with GST ‐ SXL as a control . Effects of single mutation of Matrin3 PRI GILPPP to GIAPPP were also tested ( lane 4 ) .

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C The 20‐residue Raver1 491-511 (lane 1) and the Matrin3 346-365 (lane 3) peptide fused to MS2 were transcribed and translated in vitro (Input) and then pulled down with GST‐PTB or with GST‐SXL as a control. Effects of single mutation of the Matrin3 PRI GILPPP to GIAPPP were also tested (lane 4).


A Western blot probed for Matrin3 ( top panel ) , PTB ( middle panel ) actin ( lower panel ) . Lanes 1 - 4 contain a twofold dilution of control C2 sample ( lane 1 - 12 . 5 % , lane 2 - 25 % , lane 3 - 50 % , lane 4 - 100 % ) . Lanes 4 - 7 contain equal amount of , as can be confirmed by anti ‐ actin ( lower panel ) , of control sample ( lane 4 ) , double ‐ PTB nPTB siRNA ‐ treated sample ( lane 5 ) , Matrin3 siRNA ‐ treated sample ( lane 6 ) triple knockdown of Matrin3 , PTB nPTB ( lane 7 ) . Lane 7 is from same gel exposure , but some lanes present in original gel were cropped for clarity , a black to indicate cropping was placed .

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A Western blot probed for Matrin3 (top panel), PTB (middle panel) and actin (lower panel). Lanes 1-4 contain a twofold dilution of the control C2 sample (lane 1-12.5%, lane 2-25%, lane 3-50%, lane 4-100%). Lanes 4-7 contain equal amount of proteins, as can be confirmed by the anti‐actin (lower panel), of control sample (lane 4), double‐PTB and nPTB siRNA‐treated sample (lane 5), Matrin3 siRNA‐treated sample (lane 6) and triple knockdown of Matrin3, PTB and nPTB (lane 7). Lane 7 is from the same gel and exposure, but some lanes present in the original gel were cropped for clarity, and a black line to indicate cropping was placed.


F RT - PCR validation of Matrin3 ‐ regulated alternative spicing events in ST7 , ACSL3 , PLEKHA3 , TCF12 , VWA5A , PTBP2 , PTBP3 , C3orf17 , ZMYND8 , VEZT , PIGX DMD genes . In each case , triplicates for each condition ( C - control , M - Matrin3 , PTB / nPTB Matrin3 / PTB / nPTB siRNA transfection samples ) were analysed exon inclusion ( EI ) is shown beneath corresponding lane , along with standard deviation ( s . d . ) .

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F RT-PCR validation of Matrin3‐regulated alternative spicing events in the ST7, ACSL3, PLEKHA3, TCF12, VWA5A, PTBP2, PTBP3, C3orf17, ZMYND8, VEZT, PIGX and DMD genes. In each case, triplicates for each condition (C-control, M-Matrin3, PTB/nPTB and Matrin3/PTB/nPTB siRNA transfection samples) were analysed and exon inclusion (EI) percentage is shown beneath the corresponding lane, along with the standard deviation (s.d.).


A Matrin3 crosslinking in Matrin3 ‐ regulated pre ‐ mRNAs where position of crosslinked nucleotides was mapped onto regulated exon 500 nucleotides upstream downstream of its 3 ′ ss 5 ′ ss , respectively , upstream flanking exon 500 nucleotides downstream of its 5 ′ ss downstream flanking exon with 500 nucleotides upstream of its 5 ′ ss . iCLIP tags were mapped onto silenced ASE ( blue , n = 421 ) , enhanced ASE ( red , n = 187 ) to control ASE ( grey , n = 16 , 242 ) , of occupancy is plotted .

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A Matrin3 crosslinking in Matrin3‐regulated pre‐mRNAs where position of crosslinked nucleotides was mapped onto the regulated exon and the 500 nucleotides upstream and downstream of its 3′ss and 5′ss, respectively, the upstream flanking exon and 500 nucleotides downstream of its 5′ss and the downstream flanking exon with 500 nucleotides upstream of its 5′ss. The iCLIP tags were mapped onto silenced ASE (blue, n = 421), enhanced ASE (red, n = 187) and to control ASE (grey, n = 16,242), and percentage of occupancy is plotted.


B Same as in ( A ) but using iCLIP tags obtained from PTBiCLIP mapped onto PTB / nPTB ‐ regulated ASE , silenced ASE ( blue , n = 729 ) , enhanced ( red , n = 820 ) control ASE ( grey , n = 14 , 599 ) .

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B Same as in (A) but using iCLIP tags obtained from PTBiCLIP and mapped onto PTB/nPTB‐regulated ASE, silenced ASE (blue, n = 729), enhanced (red, n = 820) and control ASE (grey, n = 14,599).


C Matrin3 crosslinked nucleotides mapped onto PTB ‐ regulated ASE .

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C Matrin3 crosslinked nucleotides mapped onto PTB‐regulated ASE.


D PTB crosslinked nucleotides mapped onto Matrin3 ‐ regulated ASE .

D PTB crosslinked nucleotides mapped onto Matrin3‐regulated ASE.


B Western blot of overexpressed Matrin3 its mutants probed with anti ‐ FLAG anti ‐ actin antibodies .

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B Western blot of the overexpressed Matrin3 and its mutants probed with anti‐FLAG and anti‐actin antibodies.


C , D RT - PCR using primers specific to ST7 exon 11 ( C ) ABI2 exon 8 ( D ) splicing reporters , respectively , of samples where FLAG ‐ tagged Matrin3 its mutants have been overexpressed . of at least three replicates for each condition is shown as a histogram of of exon inclusion . * P 0 . 01 compared with control sample ( − ) .

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C, D RT-PCR analysis using primers specific to the ST7 exon 11 (C) and ABI2 exon 8 (D) splicing reporters, respectively, of samples where FLAG‐tagged Matrin3 and its mutants have been overexpressed. Quantification of at least three replicates for each condition is shown as a histogram of the percentage of exon inclusion. *P 0.01 compared with control sample (−).


E Western blot of overexpressed FLAG ‐ tagged Matrin3 PTB , in control siRNA PTB / nPTB siRNA ‐ transfected , probed with anti ‐ FLAG to detect overexpressed , anti ‐ PTB to detect knockdown anti ‐ actin to confirm loading . A titration of control sample ( lane 1 - 12 . 5 % , lane 2 - 25 % , lane 3 - 50 % lane 4 - 100 % ) is also included to assess of knockdown . band present above PTB doublet in lane 6 is FLAG ‐ tagged PTB .

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E Western blot of overexpressed FLAG‐tagged Matrin3 and PTB, in control siRNA and PTB/nPTB siRNA‐transfected cells, probed with anti‐FLAG to detect overexpressed proteins, anti‐PTB to detect the knockdown levels and anti‐actin to confirm protein loading. A titration of control sample (lane 1-12.5%, lane 2-25%, lane 3-50% and lane 4-100%) is also included to assess the levels of knockdown. The band present above the PTB doublet in lane 6 is FLAG‐tagged PTB.


F , G RT - PCR using primers specific to ST7 exon 11 ( F ) ABI2 exon 8 ( G ) splicing reporters , respectively , of samples where Matrin3 or PTB expression vectors were co ‐ transfected , in control siRNA PTB / nPTB siRNA ‐ transfected . of at least three replicates for each condition is shown as a histogram of of exon inclusion . * P 0 . 01 when compared with control sample in each condition ( ‐ ) .

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

F, G RT-PCR analysis using primers specific to the ST7 exon 11 (F) and ABI2 exon 8 (G) splicing reporters, respectively, of samples where Matrin3 or PTB expression vectors were co‐transfected, in control siRNA and PTB/nPTB siRNA‐transfected cells. Quantification of at least three replicates for each condition is shown as a histogram of the percentage of exon inclusion. *P 0.01 when compared with control sample in each condition (‐).


Western blot of whole lysates from HEK 293 WT NEDP1 KO reveals a loss of free NEDD8 ( indicated by asterisk ) an accumulation of NEDD8 reactive species in NEDP1 KO lysate . predicted molecular sizes of putative , unanchored , poly - NEDD8 chains are denoted by N2 through to N5 . Unconjugated NEDD8 is denoted by N1 .

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Western blot analysis of whole cell lysates from HEK 293 WT and NEDP1 KO cells reveals a loss of free NEDD8 (indicated by asterisk) and an accumulation of NEDD8 reactive species in the NEDP1 KO lysate. The predicted molecular weight sizes of putative, unanchored, poly-NEDD8 chains are denoted by N2 through to N5. Unconjugated NEDD8 is denoted by N1.


NEDD8 affinity resin shows enrichment of endogenous neddylated in WT NEDP1 KO . Recombinant HALO - NEDP1 C163A ( CA ) conjugated to HALO - Link beads was used as an affinity resin to enrich for neddylated in lysates from HEK 293 WT NEDP1 KO . Enriched were resolved by SDS - PAGE processed for Western blot with NEDD8 or Ubiquitin antibodies . HALO - NEDP1 CA specifically enriches for NEDD8 - reactive in both WT NEDP1 KO , but does not enrich for Ubiquitin - modified in either .

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NEDD8 affinity resin shows enrichment of endogenous neddylated proteins in WT and NEDP1 KO cells. Recombinant HALO-NEDP1 C163A (CA) conjugated to HALO-Link beads was used as an affinity resin to enrich for neddylated proteins in lysates from HEK 293 WT and NEDP1 KO cells. Enriched proteins were resolved by SDS-PAGE and processed for Western blot analysis with NEDD8 or Ubiquitin antibodies. HALO-NEDP1 CA specifically enriches for NEDD8-reactive proteins in both WT and NEDP1 KO cells, but does not enrich for Ubiquitin-modified proteins in either cell line.


Components of NEDD8 conjugation machinery are enriched in HALO - NEDP1 pulldowns from NEDP1 KO lysates . Neddylated from HEK293 KO were enriched by HALO - NEDP1 CA pulldown , as in ( B ) but not by NEDD8 nonbinder mutant , HALO - NEDP1 DAGC ( D29W A98K G99K C163A ) . NEDD8 E1s , UBA3 ULA1 , are modified in NEDP1 KO , as well as E2 UBE2M , co - E3s DCNL1 DCNL2 . Cul2 Cul3 are hyper - neddylated in NEDP1 KO . CSN components , CSN5 CSN8 , also co - precipitate in HALO - NEDP1 CA pulldowns .

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Components of the NEDD8 conjugation machinery are enriched in HALO-NEDP1 pulldowns from NEDP1 KO lysates. Neddylated proteins from HEK293 KO cells were enriched by HALO-NEDP1 CA pulldown, as in (B) but not by the NEDD8 nonbinder mutant, HALO-NEDP1 DAGC (D29W A98K G99K C163A). The NEDD8 E1s, UBA3 and ULA1, are modified in NEDP1 KO cells, as well as E2 UBE2M, and co-E3s DCNL1 and DCNL2. Cul2 and Cul3 are hyper-neddylated in NEDP1 KO cells. CSN components, CSN5 and CSN8, also co-precipitate in HALO-NEDP1 CA pulldowns.


Western blot from HEK 293 WT NEDP1 KO of components of NEDD8 conjugation / de - conjugation pathway shows that similar of NEDD8 pathway components are present in both WT NEDP1 KO . Apart from UBA3 , there is no detectable amount of NEDD8 - modified enzymes in whole - lysates from NEDP1 KO .

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Western blot analysis from HEK 293 WT and NEDP1 KO cells of the components of the NEDD8 conjugation/de-conjugation pathway shows that similar levels of NEDD8 pathway components are present in both WT and NEDP1 KO cells. Apart from UBA3, there is no detectable amount of NEDD8-modified enzymes in whole-cell lysates from NEDP1 KO cells.


Poly - NEDD8 chains can be generated by in vitro reactions ( Rxn ) . NAE ( 0 . 15 μM ) , UBE2M , NEDD8 ( 20 μM ) were incubated on ice or incubated at 30 ° C for 3 hours reactions were stopped by addition of LDS loading buffer . Reactions were resolved by SDS - PAGE stained with colloidal Coomaisse . Indicated bands were excised from gel processed for in gel trypsin digestion mass spectrometry . predicted molecular sizes for a theoretical unanchored NEDD8 chain are denoted by N2 - N4 . Unconjugated NEDD8 is indicated by N1 . UBE2M modified by NEDD8 is indicated with an asterisk .

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Poly-NEDD8 chains can be generated by in vitro reactions (Rxn). NAE (0.15 μM), UBE2M, and NEDD8 (20 μM) were incubated on ice or incubated at 30°C for 3 hours and reactions were stopped by addition of LDS loading buffer. Reactions were resolved by SDS-PAGE and stained with colloidal Coomaisse. Indicated bands were excised from the gel and processed for in gel trypsin digestion and mass spectrometry analysis. The predicted molecular weight sizes for a theoretical unanchored NEDD8 chain are denoted by N2-N4. Unconjugated NEDD8 is indicated by N1. UBE2M modified by NEDD8 is indicated with an asterisk.


Neddylated species are NEDD8 E1 dependent . WT NEDP1 KO HEK 293 were treated with NAE inhibitor MLN4924 at 3 μM for indicated time . Lysed were then processed for Western blot . NEDD8 E1 inhibition results in a time - dependent decrease in amount of Cullin non - Cullin NEDD8 reactive bands .

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Neddylated species are NEDD8 E1 dependent. WT and NEDP1 KO HEK 293 cells were treated with NAE inhibitor MLN4924 at 3 μM for the indicated time. Lysed cells were then processed for Western blot analysis. NEDD8 E1 inhibition results in a time-dependent decrease in the amount of Cullin and non-Cullin NEDD8 reactive bands.


Neddylated species are UBE2M dependent . WT NEDP1 KO HEK 293 were left untreated or treated with indicated siRNA for 48 hours . Lysed were then processed for Western blot . Neddylated species are reduced when NEDD8 E2 UBE2M was depleted after siRNA treatment ( si2M ) , but not with control siRNA ( siCTRL ) or when NEDD8 E2 UBE2F was depleted ( si2F ) . double knockdown ( si2M / 2F ) does not further reduce NEDD8 modified .

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Neddylated species are UBE2M dependent. WT and NEDP1 KO HEK 293 cells were left untreated or treated with the indicated siRNA for 48 hours. Lysed cells were then processed for Western blot analysis. Neddylated species are reduced when NEDD8 E2 UBE2M was depleted after siRNA treatment (si2M), but not with control siRNA (siCTRL) or when NEDD8 E2 UBE2F was depleted (si2F). The double knockdown (si2M/2F) does not further reduce NEDD8 modified proteins.


( Left ) Western blot of large scale HALO - NEDP1 pulldowns from HEK 293 NEDP1 KO lysates . ( Right ) Diagram of mass spectrometry sample preparation .

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(Left) Western blot analysis of large scale HALO-NEDP1 pulldowns from HEK 293 NEDP1 KO lysates. (Right) Diagram of mass spectrometry sample preparation.


Scatterplot of identified by mass spectrometry identifies NEDD8 components of NEDD8 conjugation pathway as being most abundant in HEK 293 NEDP1 KO lysates following NEDP1 - CA pulldown . iBAQ of identified in A ) are plotted as log2 value of enrichment ( mass spectrometry of HALO - NEDP1 CA pulldown over HALO - NEDP1 DAGC pulldown ) versus log10 value of iBAQ from HALO - NEDP1 CA pulldown . Blue markers indicate known components of NEDD8 pathway red markers indicate that have been identified as substrates of PARP - 1 in database of ADP - ribosylated , ADPriboDB ( Vivelo et al . , 2017 ) .

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Scatterplot of proteins identified by mass spectrometry analysis identifies NEDD8 and components of the NEDD8 conjugation pathway as being the most abundant proteins in HEK 293 NEDP1 KO lysates following NEDP1-CA pulldown. iBAQ analysis of proteins identified in A) are plotted as the log2 value of the enrichment ratio (mass spectrometry intensity of the HALO-NEDP1 CA pulldown over HALO-NEDP1 DAGC pulldown) versus the log10 value of the iBAQ intensity from the HALO-NEDP1 CA pulldown. Blue markers indicate known components of the NEDD8 pathway and red markers indicate proteins that have been identified as substrates of PARP-1 in the database of ADP-ribosylated proteins, ADPriboDB (Vivelo et al., 2017).


Non - cullin neddylated species accumulate after oxidative stress in WT U2OS with most highly induced species detected at 25 kDa ( as indicated by arrow ) . WT U2OS were treated with indicated of H2O2 for 30 minutes then lysed in LDS sample loading buffer processed for Western blot .

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Non-cullin neddylated species accumulate after oxidative stress in WT U2OS cells with the most highly induced species detected at 25 kDa (as indicated by the arrow). WT U2OS cells were treated with the indicated concentration of H2O2 for 30 minutes and then lysed in LDS sample loading buffer and processed for Western blot analysis.


( Left ) 25 - kDa neddylated species induced by oxidative stress increases with time in WT U2OS . U2OS WT or NEDP1 KO were treated with 600 μM H2O2 for indicated time then lysed in LDS sample loading buffer processed for Western blot . ( Right ) same 25 kDa NEDD8 species , which is strongly induced in WT , is present in untreated NEDP1 KO does not further increase after oxidative stress induced by H2O2 ( as indicated by arrow ) .

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(Left) The 25-kDa neddylated species induced by oxidative stress increases with time in WT U2OS cells. U2OS WT or NEDP1 KO cells were treated with 600 μM H2O2 for the indicated time and then lysed in LDS sample loading buffer and processed for Western blot analysis. (Right) The same 25 kDa NEDD8 species, which is strongly induced in WT cells, is present in untreated NEDP1 KO cells and does not further increase after oxidative stress induced by H2O2 (as indicated by the arrow).


U2OS NEDP1 KO are resistant to PARP - 1 inducer , H2O2 . WT NEDP1 U2OS were plated in 96 - well plates after 24 hours were treated with indicated of H2O2 . They were assessed for viability 24 hours later by CellTiter - Glo . Graphs represent mean ± S . E . M . of survival compared to untreated .

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U2OS NEDP1 KO cells are resistant to the PARP-1 inducer, H2O2. WT and NEDP1 U2OS cells were plated in 96-well plates and after 24 hours were treated with the indicated concentration of H2O2. They were assessed for viability 24 hours later by the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.


NEDP1 KO are re - sensitized to H2O2 , following re - expression of NEDP1 by transient transfection . U2OS were plated in 96 well plates reversed transfected with NEDP1 or an empty vector . 48 hours after plating , were challenged with indicated amount of H2O2 viability was assessed 24 hours later by CellTiter - Glo . Graphs represent mean ± S . E . M . of survival compared to untreated .

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NEDP1 KO cells are re-sensitized to H2O2, following re-expression of NEDP1 by transient transfection. U2OS cells were plated in 96 well plates and reversed transfected with NEDP1 or an empty vector. 48 hours after plating, cells were challenged with the indicated amount of H2O2 and cell viability was assessed 24 hours later by the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.


Neddylation of cullins is decreased in NEDP1 KO is rescued by re - expression of NEDP1 by transient transfection . Western blot from HEK 293 WT , NEDP1 KO NEDP1 KO rescued with transient transfection of NEDP1 reveals that upon NEDP1 re - expression , of NEDD8 reactive bands is reduced of free NEDD8 are increased . NEDP1 re - expression also increased neddylation of each Cullin . neddylated band of each Cullin is denoted with a star . graph of mean ± S . E . M . of neddylation of each Cullin in ( E ) .

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Neddylation of cullins is decreased in NEDP1 KO cells and is rescued by re-expression of NEDP1 by transient transfection. Western blot analysis from HEK 293 WT, NEDP1 KO cells and NEDP1 KO cells rescued with transient transfection of NEDP1 reveals that upon NEDP1 re-expression, the intensity of the NEDD8 reactive bands is reduced and the levels of free NEDD8 are increased. NEDP1 re-expression also increased the neddylation of each Cullin. The neddylated band of each Cullin is denoted with a star. Quantification and graph of the mean ± S.E.M. of the percentage neddylation of each Cullin in (E).


Deletion of Nedp1 does not lead to induction Nrf2 response genes . was harvested from WT NEDP1 KO U2OS , reversed transcribed to cDNA , analysed by qPCR for Nrf2 NQO1 expression .

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Deletion of Nedp1 does not lead to induction Nrf2 response genes. RNA was harvested from WT and NEDP1 KO U2OS cells, reversed transcribed to cDNA, and analysed by qPCR for Nrf2 and NQO1 expression.


Deletion of Nedp1 but not of Cand1 in HEK 293 results in resistance to from H2O2 . WT , NEDP1 KO , CAND1 KO were plated in 96 wells plates treated with indicated amount of H2O2 . 24 hours after treatment , viability was measured using CellTiter - Glo . Graphs represent mean ± S . E . M . of survival compared to untreated .

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Deletion of Nedp1 but not of Cand1 in HEK 293 cells results in resistance to cell death from H2O2. WT, NEDP1 KO, and CAND1 KO cells were plated in 96 wells plates and treated with the indicated amount of H2O2. 24 hours after treatment, cell viability was measured using the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.


PARP - 1 activity is reduced in NEDP1 KO . Western blot of whole lysates from WT NEDP1 KO U2OS after treatment with 600 μM H2O2 for indicated amount of time . PAR polymer generation is induced in WT after H2O2 treatment but induction is reduced in NEDP1 KO .

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PARP-1 activity is reduced in NEDP1 KO cells. Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells after treatment with 600 μM H2O2 for the indicated amount of time. PAR polymer generation is induced in WT cells after H2O2 treatment but induction is reduced in NEDP1 KO cells.


PARP - 1 activity is rescued by re - expression of NEDP1 in NEDP1 KO . Western blot of whole lysates from WT NEDP1 KO U2OS transfected with indicated constructs . 72 hours after transfection , were treated with indicated amount of H2O2 processed for Western blot . PAR polymer generation is reduced in NEDP1 KO but can be rescued by transient re - expression of NEDP1 .

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PARP-1 activity is rescued by re-expression of NEDP1 in NEDP1 KO cells. Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells transfected with the indicated constructs. 72 hours after transfection, cells were treated with the indicated amount of H2O2 and processed for Western blot analysis. PAR polymer generation is reduced in NEDP1 KO cells but can be rescued by the transient re-expression of NEDP1.


PARP - 1 inhibitor Olaparib protects WT from H2O2 treatment to same extent as NEDP1 KO . U2OS were plated in 96 well plates 24 hours later pre - treated with Olaparib ( 10 μM ) for 1 hour before treatment with indicated of H2O2 . 24 hours later , viability was measured using CellTiterGlo . NEDP1 deletion protects from H2O2 treatment to same extent as PARP - 1 inhibition in WT . Olaparib does not provide additional protection to NEDP1 from H2O2 treatment .

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PARP-1 inhibitor Olaparib protects WT cells from H2O2 treatment to the same extent as NEDP1 KO. U2OS cells were plated in 96 well plates and 24 hours later pre-treated with Olaparib (10 μM) for 1 hour before treatment with the indicated concentration of H2O2. 24 hours later, cell viability was measured using the CellTiterGlo assay. NEDP1 deletion protects cells from H2O2 treatment to the same extent as PARP-1 inhibition in WT cells. Olaparib does not provide additional protection to NEDP1 cells from H2O2 treatment.


AIF translocation to nucleus is impaired in NEDP1 KO . WT NEDP1 KO were plated on coverslips 24 hours later were treated with H2O2 ( 600 μM ) for indicated amount of time then processed for immunofluorescence using α - AIF antibodies DAPI . PARP - 1 dependent is induced in WT U2OS , as indicated by translocation of AIF from mitochondria to nucleus ( Scale bar 10 μm ) .

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AIF translocation to the nucleus is impaired in NEDP1 KO cells. WT and NEDP1 KO cells were plated on coverslips and 24 hours later were treated with H2O2 (600 μM) for the indicated amount of time and then processed for immunofluorescence analysis using α-AIF antibodies and DAPI staining. PARP-1 dependent cell death is induced in WT U2OS cells, as indicated by translocation of AIF from the mitochondria to the nucleus (Scale bar 10 μm).


Total AIF from ( D ) was measured with ImageJ plotted as a vertical scatter plot with group mean indicated with a black bar . AIF has translocated to nucleus after WT were exposed to H2O2 but not in NEDP1 KO .

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Total nuclear AIF intensity from (D) was measured with ImageJ and plotted as a vertical scatter plot with the group mean intensity indicated with a black bar. AIF has translocated to the nucleus after WT cells were exposed to H2O2 but not in NEDP1 KO cells.


NEDP1 KO express normal of PARP - 1 AIF . Western blot of whole lysates from WT NEDP1 KO U2OS shows that necessary for induction of PARP - 1 dependent are present at equal in both lines .

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NEDP1 KO cells express normal levels of PARP-1 and AIF. Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells shows that the proteins necessary for the induction of PARP-1 dependent cell death are present at equal levels in both cell lines.


NEDP1 KO U2OS are not resistant to induction of apoptosis from combined treatment with TNF - α cycloheximide . U2OS were plated in 96 well plates incubated with cycloheximide ( 10 μg / mL ) before addition of indicated amount of TNF - α . survival was determined 24 hours later using CellTiter - Glo . Graphs represent mean ± S . E . M . of survival compared to untreated .

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NEDP1 KO U2OS cells are not resistant to induction of apoptosis from combined treatment with TNF-α and cycloheximide. U2OS cells were plated in 96 well plates and incubated with cycloheximide (10 μg/mL) before addition of the indicated amount of TNF-α. Cell survival was determined 24 hours later using the CellTiter-Glo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.


NEDP1 KO U2OS are not resistant to treatment with camptothecin ( CPT ) . WT U2OS NEDP1 KO were plated in 96 - well plates treated with indicated amount of CPT . 48 hours after exposure survival was measured using CellTiterGlo . Graphs represent mean ± S . E . M . of survival compared to untreated .

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NEDP1 KO U2OS cells are not resistant to treatment with camptothecin (CPT). WT U2OS and NEDP1 KO cells were plated in 96-well plates and treated with the indicated amount of CPT. 48 hours after exposure cell survival was measured using the CellTiterGlo assay. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells.


NEDD8 trimers co - immunoprecipitate with endogenous PARP - 1 in NEDP1 KO . Lysates were prepared from U2OS NEDP1 KO immunoprecipitation was performed with PARP - 1 - Trap or GFP - Trap as a negative control . Bound were resolved by SDS - PAGE processed for Western blot with indicated antibodies .

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NEDD8 trimers co-immunoprecipitate with endogenous PARP-1 in NEDP1 KO cells. Lysates were prepared from U2OS NEDP1 KO cells and immunoprecipitation was performed with PARP-1-Trap or GFP-Trap as a negative control. Bound proteins were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies.


NEDD8 trimers bind to GFP - PARP - 1 . NEDP1 U2OS KO were transfected with GFP or with GFP - PARP - 1 24 hours later lysates were collected . Immunoprecipitation was then performed with GFP - Trap , bound were resolved by SDS - PAGE processed for Western blot with indicated antibodies . GFP - PARP - 1 but not GFP alone can co - immunoprecipitate NEDD8 trimers from NEDP1 KO .

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NEDD8 trimers bind to GFP-PARP-1. NEDP1 U2OS KO cells were transfected with GFP or with GFP-PARP-1 and 24 hours later cell lysates were collected. Immunoprecipitation was then performed with GFP-Trap, and bound proteins were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies. GFP-PARP-1 but not GFP alone can co-immunoprecipitate NEDD8 trimers from NEDP1 KO cells.


NEDD8 trimers form in WT U2OS after treatment with H2O2 co - immunoprecipitate with endogenous PARP - 1 but not with immunoglobulin control ( IGG ) . WT U2OS were left untreated of treated with H2O2 ( 600 μM ) for 30 min were harvested for immunoprecipitation Western blot

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NEDD8 trimers form in WT U2OS cells after treatment with H2O2 and co-immunoprecipitate with endogenous PARP-1 but not with immunoglobulin control (IGG). WT U2OS cells were left untreated of treated with H2O2 (600 μM) for 30 min and were harvested for immunoprecipitation and Western blot analysis


NEDD8 trimers bind to GFP - PARP - 1 . WT or NEDP1 U2OS KO were transfected with GFP or with full - GFP - PARP - 1 24 hours later treated as indicated with H2O2 ( 600 μM ) for 30 min before lysates were collected . Immunoprecipitation was then performed with GFP - Trap , bound were resolved by SDS - PAGE processed for Western blot with indicated antibodies . GFP - PARP - 1 but not GFP alone can co - immunoprecipitate NEDD8 trimers from WT only after H2O2 treatment or from both treated or untreated NEDP1 KO .

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NEDD8 trimers bind to GFP-PARP-1. WT or NEDP1 U2OS KO cells were transfected with GFP or with full-length GFP-PARP-1 and 24 hours later treated as indicated with H2O2 (600 μM) for 30 min before cell lysates were collected. Immunoprecipitation was then performed with GFP-Trap, and bound proteins were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies. GFP-PARP-1 but not GFP alone can co-immunoprecipitate NEDD8 trimers from WT cells only after H2O2 treatment or from both treated or untreated NEDP1 KO cells.


NEDD8 trimers bind to - binding domain of PARP - 1 ( Zn1 - 3 ) but not to its automodification catalytic domain ( CAT ) . NEDP1 KO U2OS were transfected with GFP , GFP - PARP - 1 , GFP fused to - binding domain of PARP - 1 ( AA 1 - 336 ) or with GFP fused to PARP - 1 automodification catalytic domain ( AA 336 - 1014 ) . 24 hours post transfection , lysates were collected immunoprecipitation with GFP - Trap was performed . Bound were eluted resolved by SDS - PAGE , followed by Western blot with indicated antibodies .

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NEDD8 trimers bind to the DNA-binding domain of PARP-1 (Zn1-3) but not to its automodification and catalytic domain (CAT). NEDP1 KO U2OS cells were transfected with GFP, GFP-PARP-1, GFP fused to the DNA-binding domain of PARP-1 (AA 1-336) or with GFP fused to the PARP-1 automodification and catalytic domain (AA 336-1014). 24 hours post transfection, cell lysates were collected and immunoprecipitation with GFP-Trap was performed. Bound proteins were eluted and resolved by SDS-PAGE analysis, followed by Western blot analysis with the indicated antibodies.


NEDD8 trimers specifically bind to second zinc finger of PARP - 1 . NEDP1 KO U2OS were transfected with GFP , GFP - PARP - 1 , or with GFP fused to Zn1 domain ( AA 1 - 96 ) , Zn2 domain ( AA 97 - 215 ) , Zn3 domain ( AA 216 - 336 ) or Zn1 + 2 domains ( AA 1 - 215 ) of PARP - 1 . 24 hours post transfection , lysates were collected immunoprecipitation with GFP - Trap was performed . Bound were eluted resolved by SDS - PAGE , followed by Western blot with indicated antibodies .

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NEDD8 trimers specifically bind to the second zinc finger of PARP-1. NEDP1 KO U2OS cells were transfected with GFP, GFP-PARP-1, or with GFP fused to the Zn1 domain (AA 1-96), the Zn2 domain (AA 97-215), the Zn3 domain (AA 216-336) or the Zn1+2 domains (AA 1-215) of PARP-1. 24 hours post transfection, cell lysates were collected and immunoprecipitation with GFP-Trap was performed. Bound proteins were eluted and resolved by SDS-PAGE analysis, followed by Western blot analysis with the indicated antibodies.


2D gel electrophoresis of lysate from WT followed by Western blot with α - NEDD8 antibody indicates free NEDD8 migrates near its predicted isoelectic point ( pI ) of 6 .

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2D gel electrophoresis of lysate from WT cells followed by Western blot analysis with α-NEDD8 antibody indicates free NEDD8 migrates near its predicted isoelectic point (pI) of 6.59


2D Gel electrophoresis of pulldowns from NEDP1 KO lysate with GST - Zn1 + 2 followed by Western blot with α - NEDD8 antibody . Some of 25 - kDa NEDD8 band migrates to same isoelectric point as unconjugated NEDD8 ( denoted by triangle ) , indicating species is most likely an unanchored NEDD8 trimer . However , majority of NEDD8 trimer migrates to multiple spots of lower pI ( indicated in brackets ) , which suggests that NEDD8 trimers undergo a post - translational modification that either adds a negative charge or blocks a positive charge on NEDD8 trimer .

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2D Gel electrophoresis of pulldowns from NEDP1 KO lysate with GST-Zn1+2 followed by Western blot analysis with α-NEDD8 antibody. Some of the 25-kDa NEDD8 band migrates to the same isoelectric point as unconjugated NEDD8 (denoted by white triangle), indicating the species is most likely an unanchored NEDD8 trimer. However, the majority of NEDD8 trimer migrates to multiple spots of lower pI (indicated in the brackets), which suggests that NEDD8 trimers undergo a post-translational modification that either adds a negative charge or blocks a positive charge on the NEDD8 trimer.


Overexpression of HDAC1 or HDAC2 decreases PARP - 1 - NEDD8 trimer binding . NEDP1 KO U2OS were transfected with GFP or GFP fused to first two zinc finger domains of PARP - 1 ( GFP - Zn1 + 2 ) . were also co - transfected , as indicated , with empty FLAG vector , HDAC1 - FLAG , or HDAC2 - FLAG . 24 hours post transfection , were harvested immunoprecipitation was performed on lysates with GFP - Trap . Immunoprecipitated were resolved by SDS - PAGE followed by Western blot with indicated antibodies .

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Overexpression of HDAC1 or HDAC2 decreases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP-1 (GFP-Zn1+2). Cells were also co-transfected, as indicated, with empty FLAG vector, HDAC1-FLAG, or HDAC2-FLAG. 24 hours post transfection, cells were harvested and immunoprecipitation was performed on the lysates with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies.


HDAC inhibition increases PARP - 1 - NEDD8 trimer binding . NEDP1 KO U2OS were transfected with GFP or GFP fused to first two zinc finger domains of PARP - 1 ( GFP - Zn1 + 2 ) . 24 hours later , were left untreated or treated with HDAC inhibitor sodium butyrate ( NaB ) ( 10 mM ) for 4 hours . were harvested immunoprecipitation was performed on lysates with GFP - Trap . Immunoprecipitated were resolved by SDS - PAGE followed by Western blot with indicated antibodies .

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HDAC inhibition increases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP-1 (GFP-Zn1+2). 24 hours later, cells were left untreated or treated with the HDAC inhibitor sodium butyrate (NaB) (10 mM) for 4 hours. Cells were harvested and immunoprecipitation was performed on the lysates with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies.


HDAC inhibition increases PARP - 1 - NEDD8 trimer binding . NEDP1 KO U2OS were left untreated or treated with HDAC inhibitor sodium butyrate ( NaB ) ( 10 mM ) for 4 hours . were harvested lysates were incubated with recombinant Zn1 - GFP or Zn2 - GFP ( 25 nM ) for 1 hour followed by immunoprecipitation with GFP - Trap . Immunoprecipitated were resolved by SDS - PAGE followed by Western blot with indicated antibodies .

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HDAC inhibition increases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were left untreated or treated with the HDAC inhibitor sodium butyrate (NaB) (10 mM) for 4 hours. Cells were harvested and lysates were incubated with recombinant Zn1-GFP or Zn2-GFP (25 nM) for 1 hour followed by immunoprecipitation with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies.


Overexpression of HDAC1 or HDAC2 in NEDP1 KO can rescue induction of PAR polymer following H2O2 treatment . U2OS WT or NEDP1 KO were transfected with indicated FLAG vectors . 48 post - transfection , were treated with H2O2 ( 600 μM ) for indicated amount of time harvested directly in sample loading buffer . Lysates were resolved by SDS - PAGE processed for Western blot with indicated antibodies .

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Overexpression of HDAC1 or HDAC2 in NEDP1 KO cells can rescue the induction of PAR polymer following H2O2 treatment. U2OS WT or NEDP1 KO cells were transfected with the indicated FLAG vectors. 48 post-transfection cells, were treated with H2O2 (600 μM) for the indicated amount of time and harvested directly in sample loading buffer. Lysates were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies.


In vitro de - neddylation by NEDP1 is sensitive to oxidation . Poly - NEDD8 reactions were prepared with NAE , UBE2M , NEDD8 as in Figure 1E . NEDD8 reactions were stopped by addition of NAE inhibitor MLN4924 ( 3 μM ) . NEDD8 reactions were incubated with GST - NEDP1 at 30 ° C for indicated amount of time under reducing ( 50 μM DTT ) or oxidizing conditions ( 50 μM DTT + 150 μM H2O2 ) . Reactions were stopped by addition of LDS sample loading buffer resolved by SDS - PAGE before processing for Western blot with indicated antibodies .

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In vitro de-neddylation by NEDP1 is sensitive to oxidation. Poly-NEDD8 reactions were prepared with NAE, UBE2M, and NEDD8 as in Figure 1E. NEDD8 reactions were stopped by the addition of NAE inhibitor MLN4924 (3 μM). NEDD8 reactions were incubated with GST-NEDP1 at 30°C for the indicated amount of time under reducing (50 μM DTT) or oxidizing conditions (50 μM DTT + 150 μM H2O2). Reactions were stopped by the addition of LDS sample loading buffer and resolved by SDS-PAGE before processing for Western blot analysis with the indicated antibodies.


In vitro neddylation reactions with UBE2M are insensitive to H2O2 . Poly - NEDD8 reactions were prepared with NAE , UBE2M , NEDD8 as in Figure 1E . Before reactions were started , increasing concentrations of H2O2 ( as indicated ) were added to NEDD8 reactions . Subsequently reactions were incubated at 30 ° C for indicated amount of time . Reactions were stopped by addition of LDS sample loading buffer resolved by SDS - PAGE before processing for Western blot with indicated antibodies .

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In vitro neddylation reactions with UBE2M are insensitive to H2O2. Poly-NEDD8 reactions were prepared with NAE, UBE2M, and NEDD8 as in Figure 1E. Before reactions were started, increasing concentrations of H2O2 (as indicated) were added to the NEDD8 reactions. Subsequently the reactions were incubated at 30°C for the indicated amount of time. Reactions were stopped by the addition of LDS sample loading buffer and resolved by SDS-PAGE before processing for Western blot analysis with the indicated antibodies.


A Intracellular localization of total ( HA ) polymeric ATZ ( 2C1 ) in WT MEF mock - treated , Confocal Scanning Microscopy ( CLSM ) . B Same as A for MEF exposed to 50 nM BafA1 for 12 h . C Same as A , 4 h after BafA1 wash - out . D of ATZ - positive , LAMP1 - positive endolysosomes ( EL ) ( n = 13 , 10 , 11 , respectively ) . One - way ANOVA Dunnett ' s multiple comparisons , ns P > 0 . 05 , * * * * P < 0 . 0001 . Data information : Scale bars : 10 μm .

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A Intracellular localization of total (HA) and polymeric ATZ (2C1) in WT MEF mock-treated, Confocal Laser Scanning Microscopy (CLSM). B Same as A for MEF exposed to 50 nM BafA1 for 12 h. C Same as A, 4 h after BafA1 wash-out. D Quantification of ATZ-positive, LAMP1-positive endolysosomes (EL) (n=13, 10, 11 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001. Data information: Scale bars: 10 μm.


E ATZ polymers immunoisolated from lysates of WT MEF mock treated ( lane 1 ) , incubated for 12 h with BafA1 ( lane 2 ) 4 h after BafA1 wash - out ( lane 3 ) . Immunoprecipitation ( IP ) of ATZ polymers with polymer - specific 2C1 antibody , transfer on PVDF , revealed with anti - HA antibody on western blot ( WB ) . F of E , n = 3 , mean ± . Unpaired two - t - , ns P > 0 . 05 , * P < 0 . 05 . Data information : Scale bars : 10 μm .

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E ATZ polymers immunoisolated from lysates of WT MEF mock treated (lane 1), incubated for 12 h with BafA1 (lane 2) and 4 h after BafA1 wash-out (lane 3). Immunoprecipitation (IP) of ATZ polymers with polymer-specific 2C1 antibody, transfer on PVDF membrane, revealed with anti-HA antibody on western blot (WB). F Quantification of E, n=3, mean ± SEM. Unpaired two-tailed t-test, ns P>0.05, * P<0.05. Data information: Scale bars: 10 μm.


G - K Same as B in WT MEF , in exposed to 20 mM CST in Cnx - , Crt - ERp57 - KO MEF . L of ATZ - positive EL ( n = 10 , 9 , 11 , 10 , 11 , respectively ) . One - way ANOVA Dunnett ' s multiple comparisons , ns P > 0 . 05 , * * * * P < 0 . 0001 . Data information : Scale bars : 10 μm .

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G-K Same as B in WT MEF, in cells exposed to 20 mM CST and in Cnx-, Crt- and ERp57-KO MEF. L Quantification of ATZ-positive EL (n=10, 9, 11, 10, 11 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001. Data information: Scale bars: 10 μm.


A HEK293 transfected with empty vector ( lanes 1 , 6 ) , ATZ - HA ( 2 , 7 ) , FAM134B - V5 ( 3 8 ) , FAM134B - V5 ATZ - HA ( 4 , 9 ) , or FAM134BLIR - V5 ATZ - HA ( 5 , 10 ) , incubated for 12 h with 50 nM BafA1 then treated with cross - linker DSP before lysis as described in Methods . Lanes 1 - 5 , WB of total extract ( TCE ) ; lanes 6 - 10 , WB of antiV5 immunoprecipites to isolate complexes containing ectopically expressed FAM134B or FAM134BLIR . membranes were probed with antiV5 ( upper panels ) , antiCNX , antiHA antiLC3 antibodies . B of LC3 co - precipitating with FAM134B - V5 ( A , lanes 8 9 ) . Mean ± , n = 3 , unpaired two - t - , * P < 0 . 05 .

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A HEK293 cells transfected with empty vector (lanes 1, 6), ATZ-HA (2, 7), FAM134B-V5 (3 and 8), FAM134B-V5 and ATZ-HA (4, 9), or FAM134BLIR-V5 and ATZ-HA (5, 10), incubated for 12 h with 50 nM BafA1 and then treated with the cross-linker DSP before lysis as described in Methods. Lanes 1-5, WB of the total cell extract (TCE); lanes 6-10, WB of antiV5 immunoprecipites to isolate complexes containing ectopically expressed FAM134B or FAM134BLIR. The membranes were probed with antiV5 (upper panels), antiCNX, antiHA and antiLC3 antibodies. B Quantification of LC3 co-precipitating with FAM134B-V5 (A, lanes 8 and 9). Mean ± SEM, n=3, unpaired two-tailed t-test, * P<0.05.


WT MEF . Scale bar : 10 μm . D Ectopic expression of FAM134BLIR in WT MEF inhibits ATZ delivery to endolysosomes . Scale bar : 10 μm .

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WT MEF. Scale bar: 10 μm. D Ectopic expression of FAM134BLIR in WT MEF inhibits ATZ delivery to endolysosomes. Scale bar: 10 μm.


E , F Distribution of gold - labeled ATZ - HA by IEM in BafA1 treated WT MEF WT MEF overexpressing FAM134BLIR , respectively . EV , ER - derived vesicles , red arrowheads ; EL , endolysosome . G of ATZ - gold density of E , F ( n = 75 79 EL , respectively ) . Unpaired two - t - , * * * * P < 0 . 0001 .

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E, F Distribution of gold-labeled ATZ-HA by IEM in BafA1 treated WT MEF and WT MEF overexpressing FAM134BLIR, respectively. EV, ER-derived vesicles, red arrowheads; EL, endolysosome. G Quantification of ATZ-gold density of E, F (n=75 and 79 EL, respectively). Unpaired two-tailed t-test, **** P <0.0001.


H Max projection of same as in D after deconvolution . Insets show orthogonal section of select regions . Scale bar : 10 μm .

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H Max projection of the same cell as in D after deconvolution. Insets show orthogonal section of select regions. Scale bar: 10 μm.


I Decay of ATZ polymers ( CHX chase , upper panel ) immunoisolated with polymer - specific 2C1 antibody ( visualized with antiHA in WB ) in HEK293 mock - transfected ( lanes 1 - 4 ) or expressing FAM134BLIR - V5 ( lanes 5 - 8 ) . Middle panel , expression of FAM134BLIR - V5 assessed by WB ; lower panel , loading control . ( J ) of ( I ) ( Mean ± , n = 3 , unpaired two - t - , * P < 0 . 05 ) .

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I Decay of ATZ polymers (CHX chase, upper panel) immunoisolated with the polymer-specific 2C1 antibody (visualized with antiHA in WB) in HEK293 cells mock-transfected (lanes 1-4) or expressing FAM134BLIR-V5 (lanes 5-8). Middle panel, expression of FAM134BLIR-V5 assessed by WB; lower panel, loading control. (J) Quantification of (I) (Mean ± SEM, n=3, unpaired two-tailed t-test, * P<0.05).


K Flow cytometry of ATZ - HA polymer in MEFs mock treated , exposed to BafA1 , co - expressing FAM134LIR . MFI : mean ( Mean ± , n = 5 , unpaired two - t - , ns P > 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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K Flow cytometry analysis of ATZ-HA polymer levels in MEFs mock treated, exposed to BafA1, and co-expressing FAM134LIR. MFI: mean fluorescence intensity (Mean ± SEM, n=5, unpaired two-tailed t-test, ns P>0.05, ** P<0.01, *** P <0.001).


A WB showing KO efficiency for CRISPR134B MEF .

A WB analysis showing KO efficiency for CRISPR134B MEF.


WB showing KO efficiency for CRISPR62 MEF .

WB analysis showing KO efficiency for CRISPR62 MEF.


C - E CRISPR WT MEF , CRISPR134B MEF , CRISPR62 MEF , respectively . Scale bars : 10 μm . F of C - E ( n = 10 , 8 , 7 , respectively ) . One - way ANOVA Dunnett ' s multiple comparisons , ns P > 0 . 05 , * * * * P < 0 . 0001 .

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C-E CRISPR WT MEF, CRISPR134B MEF, CRISPR62 MEF, respectively. Scale bars: 10 μm. F Quantification of C-E (n=10, 8, 7 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001.


A - H Intracellular localization of LAMP1 - positive EL , total ( HA ) polymeric ( 2C1 ) ATZ in WT MEF exposed to BafA1 for 12 h ( A ) ; in Atg4BKO MEF ( B ) ; in Atg7KO MEF ( C ) ; in Fip200KO MEF ( D ) ; in Ulk1 / 2 double - KO MEF ( E ) ; in Atg13KO MEF ( F ) ; in Atg9KO MEF ( G ) ; in RubiconKO MEF ( H ) . Scale bars : 10 μm . I Quantifications of A - H ( n = 10 , 9 , 9 , 10 , 7 , 15 , 9 , 12 ) . One - way ANOVA Dunnett ' s multiple comparisons , ns P > 0 . 05 , * * * * P < 0 . 0001 .

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A-H Intracellular localization of LAMP1-positive EL, total (HA) and polymeric (2C1) ATZ in WT MEF exposed to BafA1 for 12 h (A); in Atg4BKO MEF (B); in Atg7KO MEF (C); in Fip200KO MEF (D); in Ulk1/2 double-KO MEF (E); in Atg13KO MEF (F); in Atg9KO MEF (G); in RubiconKO MEF (H). Scale bars: 10 μm. I Quantifications of A-H (n=10, 9, 9, 10, 7, 15, 9, 12 cells). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001.


A Distribution of gold - labeled ATZ - HA by IEM in WT MEF exposed to BafA1 ( on right is not transfected , as specificity control for gold labeling ) . ER , endoplasmic reticulum ; EV , ER - derived vesicle ; EL , endolysosomes . Scale bar : 1 μm . B Inset of A . Red asterisks show site of EV : EL membranes fusion

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A Distribution of gold-labeled ATZ-HA by IEM in WT MEF exposed to BafA1 (the cell on the right is not transfected, as specificity control for gold labeling). ER, endoplasmic reticulum; EV, ER-derived vesicle; EL, endolysosomes. Scale bar: 1 μm. B Inset of A. Red asterisks show site of EV:EL membranes fusion


C - E Distribution of gold - labeled ATZ - HA by IEM in WT MEF . ES , ER subdomains . Red asterisk shows site of EV : EL membranes fusion . Scale bar : 1 μm .

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C-E Distribution of gold-labeled ATZ-HA by IEM in WT MEF. ES, ER subdomains. Red asterisk shows site of EV:EL membranes fusion. Scale bar: 1 μm.


F 3D visualization by means of electron tomography of organelles containing gold labeled ATZ - HA ( visualized as dots ES , ER subdomains . Scale bar : 500 nm .

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F 3D visualization by means of electron tomography of organelles containing gold labeled ATZ-HA (visualized as white dots ES, ER subdomains. Scale bar: 500 nm.


G CLEM showing ATZ ( red ) , LAMP1 - positive EL ( cyan ) GFP - derived ( green ) in correspondence to heavily labeled vesicles , which were identified as autophagosomes for their double ( insets 1 - 3 ) . GFP - LC3 was also stained by immunogold . Scale bar : 1 μm .

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G CLEM showing ATZ (red), LAMP1-positive EL (cyan) and GFP-derived fluorescence (green) in correspondence to heavily labeled vesicles, which were identified as autophagosomes for their double membrane (insets 1-3). GFP-LC3 was also stained by immunogold. Scale bar: 1 μm.


A , B Triple of with LAMP1 ( cyan ) , HaloTag - ATZ ( red ) luminal ER marker sfGFP - KDEL ( green ) .

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A, B Triple staining of cells with LAMP1 (cyan), HaloTag-ATZ (red) and the luminal ER marker sfGFP-KDEL (green).


Triple of with LAMP1 ( cyan ) , HaloTag - ATZ ( red ) with CNX as ER membranes marker .

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Triple staining of cells with LAMP1 (cyan), HaloTag-ATZ (red) and with CNX as ER membranes marker.


Triple of with LAMP1 ( cyan ) , HaloTag - ATZ ( red ) with CLIMP63 as ER membranes marker .

Triple staining of cells with LAMP1 (cyan), HaloTag-ATZ (red) and with CLIMP63 as ER membranes marker.


G of ER markers HaloTag - ATZ co - localization in time ( sfGFP - KDEL n = 10 , 10 , 12 , 13 , CNX n = 8 , 10 , 9 , 8 , CLIMP63 n = 8 , 10 , 6 , 10 for corresponding time points ) . Mean ± , unpaired two - t - , * * * * P < 0 . 0001 . Data information : Scale bars 10 μm .

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G Quantification of ER markers and HaloTag-ATZ co-localization in time (sfGFP-KDEL n=10, 10, 12, 13, CNX n=8, 10, 9, 8, CLIMP63 n=8, 10, 6, 10 cells for corresponding time points). Mean ± SEM, unpaired two-tailed t-test, **** P<0.0001. Data information: Scale bars 10 μm.


H of double - positive endolysosomes at 300 ' ( ATZ : sfGFP - KDEL n = 14 , ATZ : CNX n = 8 , ATZ : CLIMP63 n = 10 . One - way ANOVA Dunnett ' s multiple comparisons , * * P < 0 . 01 * * * * P < 0 . 0001 ) . Data information : Scale bars 10 μm .

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H Quantification of double-positive endolysosomes at 300' (ATZ:sfGFP-KDEL n=14, ATZ:CNX n=8, ATZ:CLIMP63 n=10 cells. One-way ANOVA and Dunnett's multiple comparisons test, ** P<0.01 **** P<0.0001). Data information: Scale bars 10 μm.


C - E CLSM of ATZ delivery to LAMP1 - positive endolysosomes in BafA1 - treated CRISPRWT MEF ( C ) ; in CRISPR17 MEF ( D ) ; in CRISPR8 MEF ( E ) . Scale bars : 10 μm .

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C-E CLSM analysis of ATZ delivery to LAMP1-positive endolysosomes in BafA1-treated CRISPRWT MEF (C); in CRISPR17 MEF(D); in CRISPR8 MEF (E). Scale bars: 10 μm.


( n = 10 , 10 , 11 , respectively ) . One - way ANOVA Dunnett ' s multiple comparisons , * * * * P < 0 . 0001 .

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Quantification (n=10, 10, 11 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, **** P<0.0001.


G - I Distribution of gold - labelled ATZ - HA by IEM in CRISPRWT MEF exposed to BafA1 ( G ) ; in CRISPR17 ( H ) ; in CRISPR8 . ( I ) . ER , endoplasmic reticulum ; EV , ER - derived vesicles ; EL , endolysosomes . Scale bars : 1 μm .

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G-I Distribution of gold-labelled ATZ-HA by IEM in CRISPRWT MEF exposed to BafA1 (G); in CRISPR17 (H); in CRISPR8. (I). ER, endoplasmic reticulum; EV, ER-derived vesicles; EL, endolysosomes. Scale bars: 1 μm.


GFP reconstitution in ATZ - HA transfected CRISPR17 MEF expressing FAM134B - T11 GFP1 - 10 - LC3 . Scale bar : 10 μm . K Same as J by CLEM . ATZ - HA in red , LAMP1 - positive endolysosomes in cyan . FAM134B : LC - induce GFP reconstitution in green . FAM134B in gold is shown . Scale bar : 1 μm .

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GFP reconstitution in ATZ-HA transfected CRISPR17 MEF expressing FAM134B-T11 and GFP1-10-LC3. Scale bar: 10 μm. K Same as J by CLEM. ATZ-HA staining in red, LAMP1-positive endolysosomes in cyan. FAM134B:LC-induce GFP reconstitution in green. FAM134B in gold is shown. Scale bar: 1 μm.


A . production of Sox1 - GFP + neural - lineage during 7 days of neural differentiation , as shown by microscopy . Scale bars , 100 µm .

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A. The production of Sox1-GFP+ neural-lineage cells during 7 days of neural differentiation, as shown by microscopy. Scale bars, 100 µm.


B . qRT - PCR of NDIME expression during 7 days of neural differentiation ( D3 , P = 0 . 068 ; D5 , P = 0 . 0049 ; D7 , P = 0 . 0003 ) .

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B. qRT-PCR analysis of NDIME expression during 7 days of neural differentiation (D3, P = 0.068; D5, P = 0.0049; D7, P = 0.0003).


C . Relative expression level of NDIME mRNA in Sox1 - GFP - population Sox1 - GFP + population at day 7 of neural differentiation ( P = 0 . 0118 ) .

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C. Relative expression level of NDIME mRNA in the Sox1-GFP- population and Sox1-GFP+ population at day 7 of neural differentiation (P = 0.0118).


D . mESCs were cultured in differentiation medium alone , or with 10 % FBS , at day 5 of neural differentiation as shown by microscopy . Scale bars , 100 µm . E . Temporal expression profile of NDIME by qRT - PCR . mESCs were cultured in differentiation medium alone or with 10 % FBS ( D3 , P = 0 . 074 ; D5 , P = 0 . 0048 ; D7 , P = 0 . 0034 ) .

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D. mESCs were cultured in differentiation medium alone, or with 10% FBS, at day 5 of neural differentiation as shown by microscopy. Scale bars, 100 µm. E. Temporal expression profile of NDIME by qRT-PCR. mESCs were cultured in differentiation medium alone or with 10% FBS (D3, P = 0.074; D5, P = 0.0048; D7, P = 0.0034).


D . mESCs were cultured in differentiation medium alone , or with 10 % FBS , at day 5 of neural differentiation as shown by microscopy . Scale bars , 100 µm .

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D. mESCs were cultured in differentiation medium alone, or with 10% FBS, at day 5 of neural differentiation as shown by microscopy. Scale bars, 100 µm.


F . Distribution of NDIME in cytoplasm nucleus at day 7 of neural differentiation . XIST GAPDH mRNA were considered as positive controls for fractions , respectively . Data are represented as mean , error bars represent in panels . n = 3 .

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F. Distribution of NDIME in the cytoplasm and nucleus at day 7 of neural differentiation. The XIST RNA and GAPDH mRNA were considered as the positive controls for the nuclear and cytosolic fractions, respectively. Data are represented as mean, and error bars represent SEM in all panels. n= 3.


G . Verification of efficiency of knockout of exon 3 of NDIME in mESCs via qRT - PCR .

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G. Verification of the efficiency of knockout of exon 3 of NDIME in mESCs via qRT-PCR.


Knockout of NDIME strongly inhibited neural differentiation , as shown by microscopy analyses during 7 days of neural differentiation . Scale bars , 100 µm .

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Knockout of NDIME strongly inhibited neural differentiation, as shown by microscopy analyses during 7 days of neural differentiation. Scale bars, 100 µm.


I . Knockout of NDIME strongly inhibited neural differentiation , as shown by FACS analyses during 7 days of neural differentiation . Scale bars , 100 µm .

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I. Knockout of NDIME strongly inhibited neural differentiation, as shown by FACS analyses during 7 days of neural differentiation. Scale bars, 100 µm.


J . Knockout of NDIME inhibits expression of neural - lineage genes at day 5 of neural differentiation .

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J. Knockout of NDIME inhibits the expression of neural-lineage genes at day 5 of neural differentiation.


K . NESTIN GFP immunostaining of in ( H ) at day 5 of neural differentiation . Nuclei were stained with Hoechst 33342 ( blue ) . Scale bars , 100 µm .

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K. NESTIN and GFP immunostaining of the cells in (H) at day 5 of neural differentiation. Nuclei were stained with Hoechst 33342 (blue). Scale bars, 100 µm.


L . NESTIN GFP immunostaining of Ctrl , NDIME - / - , NDIME - / - + Rs26 - NDIME at day 5 of neural differentiation . Scale bars , 100 µm .

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L. NESTIN and GFP immunostaining of Ctrl, NDIME-/-, and NDIME-/-+Rs26-NDIME cells at day 5 of neural differentiation. Scale bars, 100 µm.


M . Expression of neural - lineage genes of Ctrl , NDIME - / - , NDIME - / - + Rs26 - NDIME at day 5 of neural differentiation .

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M. Expression of neural-lineage genes of Ctrl, NDIME-/-, and NDIME-/-+Rs26-NDIME cells at day 5 of neural differentiation.


N . Relative expression of Emx1 , Brn2 , Sox5 , Zeb2 mRNA in different differentiation stages of Ctrl NDIME - knockout ( compared with control ) .

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N. Relative expression of Emx1, Brn2, Sox5, and Zeb2 mRNA in different differentiation stages of Ctrl and NDIME-knockout cells (compared with control cells).


A , Expression of MEF2C ( D0 , P = 0 . 0020 ; D3 , P = 0 . 0025 ; D5 , P = 0 . 0003 ; D7 , P = 0 . 0142 ) of Ctrl NDIME - / - during 7 days of neural differentiation , following knockout of NDIME at day 5 of neural differentiation .

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A, Expression levels of MEF2C (D0, P = 0.0020; D3, P = 0.0025; D5, P = 0.0003; D7, P = 0.0142) of Ctrl and NDIME-/- cells during 7 days of neural differentiation, following knockout of NDIME at day 5 of neural differentiation.


B . ( D0 , P = 0 . 0020 ; D3 , P = 0 . 0025 ; D5 , P = 0 . 0003 ; D7 , P = 0 . 0142 ) of Ctrl NDIME - / - during 7 days of neural differentiation , level of MEF2C following knockout of NDIME at day 5 of neural differentiation .

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B. (D0, P = 0.0020; D3, P = 0.0025; D5, P = 0.0003; D7, P = 0.0142) of Ctrl and NDIME-/- cells during 7 days of neural differentiation, and protein level of MEF2C following knockout of NDIME at day 5 of neural differentiation.


C . Western blot of effects of MEF2C overexpression in NDIME - knockout at day 5 of neural differentiation .

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C. Western blot analysis of the effects of MEF2C overexpression in NDIME-knockout cells at day 5 of neural differentiation.


Overexpression of MEF2C in NDIME - knockout rescued neural differentiation defects at day 5 of neural differentiation , as shown by microscopy

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Overexpression of MEF2C in the NDIME-knockout cells rescued neural differentiation defects at day 5 of neural differentiation, as shown by microscopy


Overexpression of MEF2C in NDIME - knockout rescued neural differentiation defects at day 5 of neural differentiation , as shown by

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E . Overexpression of MEF2C in NDIME - knockout rescued neural differentiation defects at day 5 of neural differentiation , as shown by FACS

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E. Overexpression of MEF2C in the NDIME-knockout cells rescued neural differentiation defects at day 5 of neural differentiation, as shown by FACS


E . Overexpression of MEF2C in NDIME - knockout rescued neural differentiation defects at day 5 of neural differentiation , as shown by

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F . NESTIN GFP immunostaining of in ( D ) at day 5 of neural differentiation . Scale bars , 100 µm .

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F. NESTIN and GFP immunostaining of the cells in (D) at day 5 of neural differentiation. Scale bars, 100 µm.


G . Expression of neural - lineage genes in Ctrl , NDIME - / - , NDIME - / - + Mef2c during 7 days of neural differentiation .

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G. Expression of neural-lineage genes in Ctrl, NDIME-/-, and NDIME-/-+Mef2c cells during 7 days of neural differentiation.


Enrichment of H3K27me3 at Mef2c promoter in NDIME - knockout at day 5 of neural differentiation compared with control .

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Enrichment of H3K27me3 at the Mef2c promoter in the NDIME-knockout cells at day 5 of neural differentiation compared with control cells.


B . Enrichment of polymerase II at Mef2c promoter in NDIME - knockout at day 5 of neural differentiation compared with control .

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B. Enrichment of RNA polymerase II at the Mef2c promoter in the NDIME-knockout cells at day 5 of neural differentiation compared with control cells.