Cells were cultured under normal or hypoxic condition . Relative levels of the HIF1A - AS transcripts in total vs . poly ( A ) + RNA fractions ( F ) were determined by qRT - PCR .

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Cells were cultured under normal or hypoxic condition. Relative levels of the HIF1A-AS transcripts in total vs. poly(A)+ RNA fractions (F) were determined by qRT-PCR.


Cells were cultured under normal or hypoxic condition . Relative levels of the HIF1A - AS transcripts in subcellular fractions ( G ) , were determined by qRT - PCR .

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Cells were cultured under normal or hypoxic condition. Relative levels of the HIF1A-AS transcripts in subcellular fractions (G), were determined by qRT-PCR.


FISH analysis of the spatial distribution of HIF - 1A mRNA ( red ) and HIF1A - AS2 transcripts ( white ) in HUVECs subjected to mock or CoCl2 treatment for 5 hrs . Nuclei were counterstained by DAPI . Scale bars , 10 μm .

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FISH analysis of the spatial distribution of HIF-1A mRNA (red) and HIF1A-AS2 transcripts (white) in HUVECs subjected to mock or CoCl2 treatment for 5 hrs. Nuclei were counterstained by DAPI. Scale bars, 10 μm.


Expression of HIF1A - AS2 was suppressed in HUVECs by oligo phosphodiester nucleotides ( ODN ) - based knockdown . Cells were then treated with CoCl2 for 5 hrs to mimic hypoxic condition , and subjected to gene expression analyses : The RNA abundance of HIF - 1A and HIF1A - AS2 was analyzed by real - time PCR and normalized by 18s rRNA ( A ) .

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Expression of HIF1A-AS2 was suppressed in HUVECs by oligo phosphodiester nucleotides (ODN)-based knockdown. Cells were then treated with CoCl2 for 5 hrs to mimic hypoxic condition, and subjected to gene expression analyses: The RNA abundance of HIF-1A and HIF1A-AS2 was analyzed by real-time PCR and normalized by 18s rRNA (A).


HIF - 1α protein level was monitored by western blot , with β - actin as the internal control ( B ) .

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HIF-1α protein level was monitored by western blot, with β-actin as the internal control (B).


Nuclear run - on assays were performed to assess the extent of nascent transcription of target genes under mock vs . CoCl2 treatment ( C )

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Nuclear run-on assays were performed to assess the extent of nascent transcription of target genes under mock vs. CoCl2 treatment (C)


Nuclear run - on assays were performed to assess the extent of nascent transcription of target genes in the HIF1A - AS2 knockdown cells ( D ) .

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Nuclear run-on assays were performed to assess the extent of nascent transcription of target genes in the HIF1A-AS2 knockdown cells (D).


HIF - 1A mRNA expression was detected in a region - specific manner ( 5 ' vs . 3 ' ) by primers corresponding to exons 1 - 2 ( E1 - 2 ) and exon 14 - 15 ( E14 - 15 ) . To further monitor transcription activity , ChIP was performed to characterize RNA polymerase II binding on chromatin isolated from MCF7 cells under CoCl2 treatment ( E ) , The precipitated DNA fragments were quantitatively analyzed by real - time PCR using primers for the HIF1A - AS2 and HIF - 1A promoters , and normalized to the values of IgG . HIF1A - AS2 corresponds to the transcribed region .

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HIF-1A mRNA expression was detected in a region-specific manner (5' vs. 3') by primers corresponding to exons 1-2 (E1-2) and exon 14-15 (E14-15). To further monitor transcription activity, ChIP was performed to characterize RNA polymerase II binding on chromatin isolated from MCF7 cells under CoCl2 treatment (E), The precipitated DNA fragments were quantitatively analyzed by real-time PCR using primers for the HIF1A-AS2 and HIF-1A promoters, and normalized to the values of IgG. HIF1A-AS2 corresponds to the transcribed region.


HIF - 1A mRNA expression was detected in a region - specific manner ( 5 ' vs . 3 ' ) by primers corresponding to exons 1 - 2 ( E1 - 2 ) and exon 14 - 15 ( E14 - 15 ) . To further monitor transcription activity , ChIP was performed to characterize RNA polymerase II binding on chromatin isolated from MCF7 cells in HIF1A - AS2 - knockdown vs . control cells ( F ) . The precipitated DNA fragments were quantitatively analyzed by real - time PCR using primers for the HIF1A - AS2 and HIF - 1A promoters , and normalized to the values of IgG . HIF1A - AS2 corresponds to the transcribed region .

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HIF-1A mRNA expression was detected in a region-specific manner (5' vs. 3') by primers corresponding to exons 1-2 (E1-2) and exon 14-15 (E14-15). To further monitor transcription activity, ChIP was performed to characterize RNA polymerase II binding on chromatin isolated from MCF7 cells in HIF1A-AS2-knockdown vs. control cells (F). The precipitated DNA fragments were quantitatively analyzed by real-time PCR using primers for the HIF1A-AS2 and HIF-1A promoters, and normalized to the values of IgG. HIF1A-AS2 corresponds to the transcribed region.


HUVECs were transfected with ADAR1 - targeting siRNAs and subjected to hypoxia treatment for 5 hrs . The expression levels of ADAR1 , HIF - 1A and HIF1A - AS2 transcripts were analyzed by qRT - PCR .

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HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. The expression levels of ADAR1, HIF-1A and HIF1A-AS2 transcripts were analyzed by qRT-PCR.


HUVECs were transfected with ADAR1 - targeting siRNAs and subjected to hypoxia treatment for 5 hrs . Nuclear run - on assay was done to measure the newly synthesized HIF - 1A and HIF1A - AS2 RNAs in ADAR1 - knockdown cells , followed by real - time PCR analysis of the indicated transcripts .

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HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. Nuclear run-on assay was done to measure the newly synthesized HIF-1A and HIF1A-AS2 RNAs in ADAR1-knockdown cells, followed by real-time PCR analysis of the indicated transcripts.


HUVECs were transfected with ADAR1 - targeting siRNAs and subjected to hypoxia treatment for 5 hrs . ChIP analysis of RNA polII binding on chromatin region of the HIF1A - AS2 and HIF - 1A promoters . The extent of occupancy was compared between control and ADAR1 - knockdown cells . The precipitated DNA fragments were quantitatively analyzed by real - time PCR and normalized by the values of IgG .

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HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. ChIP analysis of RNA polII binding on chromatin region of the HIF1A-AS2 and HIF-1A promoters. The extent of occupancy was compared between control and ADAR1-knockdown cells. The precipitated DNA fragments were quantitatively analyzed by real-time PCR and normalized by the values of IgG.


Chromatin binding of ADAR1 to HIF1A - AS2 and HIF - 1A promoters under nomoxia ( 20 % O2 ) vs . hypoxia ( 1 % O2 , 5 hrs ) conditions , as assessed by ChIP .

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Chromatin binding of ADAR1 to HIF1A-AS2 and HIF-1A promoters under nomoxia (20% O2) vs. hypoxia (1% O2, 5 hrs) conditions, as assessed by ChIP.


HUVECs were transfected with ADAR1 - targeting siRNAs and subjected to hypoxia treatment for 5 hrs . The 3 ' - end extension of HIF1A - AS2 RNA transcription in ADAR1 knockdown cells ( GFP , control ) was examined by PCR with primers corresponding to the depicted regions of HIF1A - AS2 locus . GAPDH serves as an internal control . Sequence locations are shown in kbp and relative to the transcription start site .

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HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. The 3'-end extension of HIF1A-AS2 RNA transcription in ADAR1 knockdown cells (GFP, control) was examined by PCR with primers corresponding to the depicted regions of HIF1A-AS2 locus. GAPDH serves as an internal control. Sequence locations are shown in kbp and relative to the transcription start site.


HUVECs were transfected with ADAR1 - targeting siRNAs and subjected to hypoxia treatment for 5 hrs . Expression levels of HIF - 1α protein ( F ) by western

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HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. Expression levels of HIF-1α protein (F) by western blot


HUVECs were transfected with ADAR1 - targeting siRNAs and subjected to hypoxia treatment for 5 hrs . Expression levels of HIF - 1α downstream genes ( G ) by real - time PCR , as indicated . qRT - PCR results were normalized by the expression of 18s rRNA .

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HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. Expression levels of HIF-1α downstream genes (G) by real-time PCR, as indicated. qRT-PCR results were normalized by the expression of 18s rRNA.


Transcriptome - wide changes in the cells subjected to CoCl2 treatment , HIF1A - AS2 or ADAR1 knockdown were profiled by RNA - Sequencing . For genes that undergo up - regulation upon CoCl2 treatment ( n = 505 ) , overall expression profiles of the different cell groups are represented by the heatmap . The expression values ( represented by normalized read counts ) were displayed in shades of red or blue ( linear scale ) relative to the means of all corresponding values within individual experimental groups . Clusters of genes ( based on unsupervised hierarchical clustering ) are indicated and denoted by their experimental types / conditions .

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Transcriptome-wide changes in the cells subjected to CoCl2 treatment, HIF1A-AS2 or ADAR1 knockdown were profiled by RNA-Sequencing. For genes that undergo up-regulation upon CoCl2 treatment (n = 505), overall expression profiles of the different cell groups are represented by the heatmap. The expression values (represented by normalized read counts) were displayed in shades of red or blue (linear scale) relative to the means of all corresponding values within individual experimental groups. Clusters of genes (based on unsupervised hierarchical clustering) are indicated and denoted by their experimental types/conditions.


GSEA comparing the gene expression profile by gene up - regulated upon CoCl2 treatment and those down - regulated in ADAR1 knockdown . The curves correspond to the observed enrichment score profiles in the CoCl2 gene set to the ADAR1 knockdown gene sets . The bar ‐ code plot below enrichment score plot denote the rank of each member in the CoCl2 up - regulated gene list .

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GSEA comparing the gene expression profile by gene up-regulated upon CoCl2 treatment and those down-regulated in ADAR1 knockdown. The curves correspond to the observed enrichment score profiles in the CoCl2 gene set to the ADAR1 knockdown gene sets. The bar‐code plot below enrichment score plot denote the rank of each member in the CoCl2 up-regulated gene list.


HUVECs were transfected with the indicated combinations of siRNA ( GFP as control versus ADAR1 ) and ODN ( GFP as control versus HIF1A - AS2 ) , followed by CoCl2 treatment for 5 hrs . The expression levels of ADAR1 and HIF - 1α protein ( E ) were analyzed respectively by immunoblotting .

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HUVECs were transfected with the indicated combinations of siRNA (GFP as control versus ADAR1) and ODN (GFP as control versus HIF1A-AS2), followed by CoCl2 treatment for 5 hrs. The expression levels of ADAR1 and HIF-1α protein (E) were analyzed respectively by immunoblotting.


HUVECs were transfected with the indicated combinations of siRNA ( GFP as control versus ADAR1 ) and ODN ( GFP as control versus HIF1A - AS2 ) , followed by CoCl2 treatment for 5 hrs . For cells in ( E ) , RNA expression of heat shock protein - encoding genes ( HSPA1A , HSPA1B , and HSPA6 ) with reported roles in angiogenesis were profiled by qRT - PCR .

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HUVECs were transfected with the indicated combinations of siRNA (GFP as control versus ADAR1) and ODN (GFP as control versus HIF1A-AS2), followed by CoCl2 treatment for 5 hrs. For cells in (E), RNA expression of heat shock protein-encoding genes (HSPA1A, HSPA1B, and HSPA6) with reported roles in angiogenesis were profiled by qRT-PCR.


Montage of representative images and quantification of 2D capillary network formation assays on HUVECs subjected to the above experimental manipulation . Scale bars , 2000 μm .

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Montage of representative images and quantification of 2D capillary network formation assays on HUVECs subjected to the above experimental manipulation. Scale bars, 2000 μm.


Sanger sequencing validation of RNA editing of the LIMD1 ( A ) and VHL ( B ) transcripts .

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Sanger sequencing validation of RNA editing of the LIMD1 (A) and VHL (B) transcripts.


Downregulation of ADAR1 levels in MCF7 cells was done using specific siRNAs . Cells were then subjected to hypoxia treatment for 5 hrs . The extent of knockdown and gene alterations was monitored by western blotting using antibodies targeting LIMD1 , VHL , PHD1 , PHD2 , ADAR1 and HIF - 1α , as indicated β - actin was used as loading control .

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Downregulation of ADAR1 levels in MCF7 cells was done using specific siRNAs. Cells were then subjected to hypoxia treatment for 5 hrs. The extent of knockdown and gene alterations was monitored by western blotting using antibodies targeting LIMD1, VHL, PHD1, PHD2, ADAR1 and HIF-1α, as indicated β-actin was used as loading control.


To characterize subcellular distribution of target transcripts , real - time PCR analysis was done on the fractionated MCF7 cells with the indicated experimental treatments .

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To characterize subcellular distribution of target transcripts, real-time PCR analysis was done on the fractionated MCF7 cells with the indicated experimental treatments.


Translation efficiency was determined by quantifying transcript association with the polyribosome fraction , using ADAR1 - depleted MCF7 cells with hypoxia treatment . The abundance of LIMD1 in polysomes was analyzed by real - time PCR and normalized by the eEF1α levels .

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Translation efficiency was determined by quantifying transcript association with the polyribosome fraction, using ADAR1-depleted MCF7 cells with hypoxia treatment. The abundance of LIMD1 in polysomes was analyzed by real-time PCR and normalized by the eEF1α levels.


To inspect HIF - 1α protein abundance and modification with ubiquitin , anti - HIF - 1α , anti - ubiquitylation ( Ub ) , and anti - VHL immunoprecipitation experiments were performed , coupled with immunoblotting detection of ubiquitylation and HIF - 1α , as indicated . MCF7 cells transfected with ADAR1 siRNA and treated with hypoxia were used . Protein signals were quantified using the ImageJ software . The fold changes of protein levels were determined on the basis of immunoreactive signals normalized by the input ( In ) , with the control ( siGFP ) group being represented as 1 .

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To inspect HIF-1α protein abundance and modification with ubiquitin, anti- HIF-1α, anti-ubiquitylation (Ub), and anti-VHL immunoprecipitation experiments were performed, coupled with immunoblotting detection of ubiquitylation and HIF-1α, as indicated. MCF7 cells transfected with ADAR1 siRNA and treated with hypoxia were used. Protein signals were quantified using the ImageJ software. The fold changes of protein levels were determined on the basis of immunoreactive signals normalized by the input (In), with the control (siGFP) group being represented as 1.


B , Western blot showing no detectable Wbp2 protein in 4 week old mutant brain compared to wt littermate controls . 5μg of the protein lysate were subjected to 10 % SDS - PAGE . β - tubulin was used as loading control . Wbp2 l and Wbp2 s refer to the long and short isoforms respectively .

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B, Western blot showing no detectable Wbp2 protein in 4 week old mutant brain compared to wt littermate controls. 5μg of the protein lysate were subjected to 10% SDS-PAGE. β-tubulin was used as loading control. Wbp2 l and Wbp2 s refer to the long and short isoforms respectively.


C , Quantitative real - time PCR showing severe knockdown of Wbp2 transcription in 4 week old mutants ( n = 3 ) inner ears and eyes , compared to wt littermate controls ( n = 3 ) . Heterozygotes show intermediate levels . Hprt was used as a control and levels are normalised to wt levels .

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C, Quantitative real-time PCR showing severe knockdown of Wbp2 transcription in 4 week old mutants (n=3) inner ears and eyes, compared to wt littermate controls (n=3). Heterozygotes show intermediate levels. Hprt was used as a control and levels are normalised to wt levels.


D , F , X - gal staining of Wbp2 hets at P14 showing Wbp2 expression ( blue ) in all the main cochlear structures : the stria vascularis ( black arrowhead in D ) , spiral prominence ( empty arrowhead in D ) , Reissner ' s membrane ( arrow in D ) , strong expression in the spiral ganglion cells ( red arrowheads in E ) , IHCs and OHCs in the organ of Corti ( arrowheads in F ) , pillar cells and spiral limbus ( empty arrowhead in F ) . Scale bars : D : 50μm , E , F : 20μm . ihc : inner hair cells ; ohc : outer hair cells . No X - gal staining is observed in wt controls ( not shown ) . The X - gal reaction is always cytoplasmic .

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D, F, X-gal staining of Wbp2 hets at P14 showing Wbp2 expression (blue) in all the main cochlear structures: the stria vascularis (black arrowhead in D), spiral prominence (empty arrowhead in D), Reissner's membrane (arrow in D), strong expression in the spiral ganglion cells (red arrowheads in E), IHCs and OHCs in the organ of Corti (arrowheads in F), pillar cells and spiral limbus (empty arrowhead in F). Scale bars: D: 50μm, E,F: 20μm. ihc: inner hair cells; ohc: outer hair cells. No X-gal staining is observed in wt controls (not shown). The X-gal reaction is always cytoplasmic.


A , E Mean ABR thresholds ( ± SD ) for clicks and tone pips are plotted for wt ( green ) , het ( blue ) and hom ( red ) mice at ages A , P14 ( wt , n = 3 ; het , n = 8 ; hom , n = 6 ) ; B , 4 weeks ( wt , n = 38 ; het , n = 26 ; hom , n = 37 ) ; C , 14 weeks ( wt , n = 10 ; hom , n = 14 ) ; D , 28 weeks ( wt , n = 15 ; het , n = 5 ; hom , n = 25 ) ; E , 44 weeks ( wt , n = 9 ; het , n = 2 ; hom , n = 11 ) . Grey symbols and lines indicate thresholds of individual mutants . In B , open symbols represent thresholds under urethane anaesthesia ( see methods ) , showing no difference compared with ketamine / xylazine used for all other thresholds . F , Mean thresholds for mutants aged 2 weeks ( yellow ) , 4 weeks ( purple ) , 14 weeks ( cyan ) , 28 weeks ( grey ) and 44 weeks ( black ) .

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A,E Mean ABR thresholds (± SD) for clicks and tone pips are plotted for wt (green), het (blue) and hom (red) mice at ages A, P14 (wt, n=3; het, n=8; hom, n=6); B, 4 weeks (wt, n=38; het, n=26; hom, n=37); C, 14 weeks (wt, n=10; hom, n=14); D, 28 weeks (wt, n=15; het, n=5; hom, n=25); E, 44 weeks (wt, n=9; het, n=2; hom, n=11). Grey symbols and lines indicate thresholds of individual mutants. In B, open symbols represent thresholds under urethane anaesthesia (see methods), showing no difference compared with ketamine/xylazine used for all other thresholds.F, Mean thresholds for mutants aged 2 weeks (yellow), 4 weeks (purple), 14 weeks (cyan), 28 weeks (grey) and 44 weeks (black).


G , Averaged click - evoked ABR waveforms from 4 weeks old wt ( n = 23 , green ) and mutants ( n = 34 , red ) , at 50dB sensation level ( SL ) ( left panel ) . SP and ABR Wave 1 ( W1 ) are indicated by grey areas . Expanded averaged SP and ABR W1 waveform patterns for 10 - 60dB SL in 10dB increments are plotted in green ( wt ; middle panel ) and red ( mutants ; right panel ) . , to illustrate the growth of SP and W1 with stimulus level .

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G, Averaged click-evoked ABR waveforms from 4 weeks old wt (n=23, green) and mutants (n=34, red), at 50dB sensation level (SL) (left panel). SP and ABR Wave 1 (W1) are indicated by grey areas. Expanded averaged SP and ABR W1 waveform patterns for 10-60dB SL in 10dB increments are plotted in green (wt; middle panel) and red (mutants; right panel)., to illustrate the growth of SP and W1 with stimulus level.


H , I . Mean 2f1 - f2 DPOAE thresholds ( ± SD ) are plotted for wt ( green ) , heterozygote ( blue ) and homozygous ( red ) mice aged 4 weeks ( H ) or 21 weeks ( I ) , as a function of f2 frequency .

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H, I. Mean 2f1-f2 DPOAE thresholds (± SD) are plotted for wt (green), heterozygote (blue) and homozygous (red) mice aged 4 weeks (H) or 21 weeks (I), as a function of f2 frequency.


A . Pure tone audiogram recorded from one of the probands , showing severe to profound bilateral asymmetric hearing loss .

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A. Pure tone audiogram recorded from one of the probands, showing severe to profound bilateral asymmetric hearing loss.


B . Agarose gel trace of cDNA obtained from mouse wt P28 inner ear ( IE ) , P28 brain , adult and P4 organ of Corti ( OC ) . Results show expression of two Wbp2 isoforms in the brain at P28 and very faint band for the short isoform together with a strong band for the long isoform in the inner ear at P28 . If we look at just the organ of Corti ( adult and P4 ) , we observe a strong band for the long isoform in the P4 OC and an almost undetectable band for the short isoform , which was not even picked up by sequencing ( see C ) . Wbp2 l : long isoform ( 550 bp ) ; Wbp2 s : short isoform ( 480 bp ) .

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B. Agarose gel trace of cDNA obtained from mouse wt P28 inner ear (IE), P28 brain, adult and P4 organ of Corti (OC). Results show expression of two Wbp2 isoforms in the brain at P28 and very faint band for the short isoform together with a strong band for the long isoform in the inner ear at P28. If we look at just the organ of Corti (adult and P4), we observe a strong band for the long isoform in the P4 OC and an almost undetectable band for the short isoform, which was not even picked up by sequencing (see C). Wbp2 l: long isoform (550 bp); Wbp2 s: short isoform (480 bp).


D . Western blot showing the predominant presence of the Wbp2 long isoform in the cochlea at P28 , with a short trace of the short isoform showing up only when a higher concentration of protein lysate ( 20μg ) is loaded . Both isoform are absent in the Wbp2 - deficient mouse . Gapdh was used as a loading control . Wbp2 l : long isoform ; Wbp2 s : short isoform .

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D. Western blot showing the predominant presence of the Wbp2 long isoform in the cochlea at P28, with a short trace of the short isoform showing up only when a higher concentration of protein lysate (20μg) is loaded. Both isoform are absent in the Wbp2-deficient mouse. Gapdh was used as a loading control. Wbp2 l: long isoform; Wbp2 s: short isoform.


A . At P14 , afferent terminals below IHCs are slightly swollen in the mutants ( neurofilament labelling in green , arrowheads to compare ; CtBP2 labels ribbons and IHCnuclei in red ) . Scale bars : 10 μm . At P28 , Neurofilament / CtBP2 labelling in the organ of Corti of 4 week old Wbp2 - deficient mice and littermate controls shows severe swelling of IHCafferent terminals in the mutants , especially in the 24kHz region ( yellow arrowheads ) . The pre - synaptic ribbons do not look as well aligned to the terminals in the mutants ( white arrows ) . At this stage , we also observe swelling of OHCafferent terminals in the 24kHz region ( empty arrowheads ) . Scale bars : 5 μm . ihc : IHCnucleus ; p : pillar side ; m : modiolar side . B . Counts of pre - synaptic ribbons per IHC in the 8 , 18 and 24kHz regions , showing no difference between mutants and controls at P28 .

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A. At P14, afferent terminals below IHCs are slightly swollen in the mutants (neurofilament labelling in green, arrowheads to compare; CtBP2 labels ribbons and IHCnuclei in red). Scale bars: 10 μm. At P28,Neurofilament/CtBP2 labelling in the organ of Corti of 4 week old Wbp2-deficient mice and littermate controls shows severe swelling of IHCafferent terminals in the mutants, especially in the 24kHz region (yellow arrowheads). The pre-synaptic ribbons do not look as well aligned to the terminals in the mutants (white arrows). At this stage, we also observe swelling of OHCafferent terminals in the 24kHz region (empty arrowheads). Scale bars: 5 μm. ihc: IHCnucleus; p: pillar side; m: modiolar side.B. Counts of pre-synaptic ribbons per IHC in the 8, 18 and 24kHz regions, showing no difference between mutants and controls at P28.


C . TEM of the organ of Corti performed at P28 showing swollen afferent terminals below inner and outer hair cells ( arrowheads for comparisons between mutants and controls ) . Scale bars : 5 μm .

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C. TEM of the organ of Corti performed at P28 showing swollen afferent terminals below inner and outer hair cells (arrowheads for comparisons between mutants and controls). Scale bars: 5 μm.


D . Neurofilament / CtBP2 labelling shows swollen and retracting terminals ( white arrows ) , especially in the 24kHz regions in the mutants at 8 weeks ( arrows ) . Scale bars : 10μm . E . Counts of pre - synaptic ribbons in the 8 , 18 and 24kHz regions , showing no difference in their number per IHC in the mutants compared to littermate controls at 8 weeks .

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D. Neurofilament/CtBP2 labelling shows swollen and retracting terminals (white arrows), especially in the 24kHz regions in the mutants at 8 weeks (arrows). Scale bars: 10μm.E. Counts of pre-synaptic ribbons in the 8, 18 and 24kHz regions, showing no difference in their number per IHC in the mutants compared to littermate controls at 8 weeks.


A . We arrayed synapse images aligned by ascending size of the post - synaptic site after GluR2 / 3 and CtBP2 labelling . This is a composite image made of several synapses taken from a single IHC from a single wt and a single hom , representing double labelling experiments performed on 3 mutants and 3 controls . Synapses in the mutants show abnormal morphology and smaller green patches , suggesting reduced expression of the GluR2 / 3 AMPA receptor subunits . Scale bar at bottom right : 1μm .

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A. We arrayed synapse images aligned by ascending size of the post-synaptic site after GluR2/3 and CtBP2 labelling. This is a composite image made of several synapses taken from a single IHC from a single wt and a single hom, representing double labelling experiments performed on 3 mutants and 3 controls. Synapses in the mutants show abnormal morphology and smaller green patches, suggesting reduced expression of the GluR2/3 AMPA receptor subunits. Scale bar at bottom right: 1μm.


B - G . TEM images of IHC ribbon synapses of wt ( B , arrowheads for synaptic vesicles ) and homs ( C - G ) at 4 weeks of age , showing a representative array of synaptic phenotypes in the 24 kHz cochlear region of mutants . While in C and D the ribbons look slightly abnormal in size with misplaced synaptic vesicles ( arrowheads in C ) , we also observe orphan post - synaptic densities surrounded by floating synaptic vesicles ( arrowheads in E ) , ribbons with synaptic membranes ( arrow in F ) that have detached from the IHC membrane ( the asterisk labels the original position of the synapse before detachment ) and are floating in the swollen nerve terminal ( F ) , ribbons ( arrow in G ) that are detached from their densities ( asterisk in G ) . Scale bars : B - E : 200 nm ; F , G : 500 nm . nt : nerve terminal ; psd : post - synaptic density ; snt : swollen nerve terminal .

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B-G. TEM images of IHC ribbon synapses of wt (B, arrowheads for synaptic vesicles) and homs (C-G) at 4 weeks of age, showing a representative array of synaptic phenotypes in the 24 kHz cochlear region of mutants. While in C and D the ribbons look slightly abnormal in size with misplaced synaptic vesicles (arrowheads in C), we also observe orphan post-synaptic densities surrounded by floating synaptic vesicles (arrowheads in E), ribbons with synaptic membranes (arrow in F) that have detached from the IHC membrane (the asterisk labels the original position of the synapse before detachment) and are floating in the swollen nerve terminal (F), ribbons (arrow in G) that are detached from their densities (asterisk in G). Scale bars: B-E: 200 nm; F,G: 500 nm. nt: nerve terminal; psd: post-synaptic density; snt: swollen nerve terminal.


B , Quantitative real - time PCR showing reduced mRNA levels for Esr1 , Esr2 and Pgr and upregulation of Shank3 and Psd - 95 in cochleae of 4 week old Wbp2 - deficient mice compared to littermate controls ( n = 3 for each genotype ) . Hprt is used as relative control . Error bars : ± SD . Two - tailed student t test : p = 0 . 03 for Psd - 95 ; p = 0 . 007 for Shank3 ; p = 0 . 03 for Esr2 ; p = 0 . 0016 for Esr1 ; p = 0 . 037 for Pgr .

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B, Quantitative real-time PCR showing reduced mRNA levels for Esr1, Esr2 and Pgr and upregulation of Shank3 and Psd-95 in cochleae of 4 week old Wbp2-deficient mice compared to littermate controls (n=3 for each genotype). Hprt is used as relative control. Error bars: ±SD. Two-tailed student t test: p=0.03 for Psd-95; p=0.007 for Shank3; p=0.03 for Esr2; p=0.0016 for Esr1; p=0.037 for Pgr.


C , Synapses from one mutant and one control IHC at 4 weeks of age after Psd - 95 and CtBP2 labelling , showing stronger Psd - 95 expression in the mutants compared to controls , representing double labelling experiments performed on 3 mutants and 3 controls . P indicates direction of pillar cells . Scale bar : 10μm . D , Quantification of Psd95 fluorescence in IHC synapses , representing expression in the apical ( 9kHz best frequency region of the cochlea ) and basal ( 24kHz best frequency region of the cochlea ) regions at 4 weeks old . Data from 2 wt and 2 homs were analysed ( 16 synapses per cochlear region per mouse ) . AU , Arbitrary Units . Wt : 24kHz 22 . 41 ± 10 . 70 , 9kHz 11 . 045 ± 2 . 128 ; Mutants : 24kHz 66 . 73 ± 13 . 70 , p = 0 . 069 ; 9kHz : 26 . 02 ± 5 . 79 p = 0 . 075 .

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C, Synapses from one mutant and one control IHC at 4 weeks of age after Psd-95 and CtBP2 labelling, showing stronger Psd-95 expression in the mutants compared to controls, representing double labelling experiments performed on 3 mutants and 3 controls. P indicates direction of pillar cells. Scale bar: 10μm.D, Quantification of Psd95 fluorescence in IHC synapses, representing expression in the apical (9kHz best frequency region of the cochlea) and basal (24kHz best frequency region of the cochlea) regions at 4 weeks old . Data from 2 wt and 2 homs were analysed (16 synapses per cochlear region per mouse). AU, Arbitrary Units.Wt: 24kHz 22.41±10.70, 9kHz 11.045±2.128; Mutants: 24kHz 66.73±13.70, p=0.069; 9kHz: 26.02±5.79 p=0.075.


( A ) Representative micrographs of optic nerve sections collected from mice 8 days after ONC , showing the accumulation of IB - 4 + activated myeloid cells in the injury site , which is demarcated by GFAP staining . Scale bar , 50 µm .

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(A) Representative micrographs of optic nerve sections collected from mice 8 days after ONC, showing the accumulation of IB-4+ activated myeloid cells in the injury site, which is demarcated by GFAP staining. Scale bar, 50 µm.


( B ) Representative micrograph of the lesion site in optic nerve sections 7 days after crush injury in [ Cx3cr1gfp / + WT ] BM chimeric mice , immunostained for GFP ( red ) , to depict monocyte - derived macrophages , and IB - 4 ( green ) to label activated myeloid cells . Scale bar , 100 µm . Inset scale , 20 µm .

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(B) Representative micrograph of the lesion site in optic nerve sections 7 days after crush injury in [Cx3cr1gfp/+ WT] BM chimeric mice, immunostained for GFP (red), to depict monocyte-derived macrophages, and IB-4 (green) to label activated myeloid cells. Scale bar, 100 µm. Inset scale, 20 µm.


( C ) Representative micrograph of optic nerve section 7 days after crush injury , immunostained for CD3 ( red , arrows ) . Scale bar , 100 µm .

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(C) Representative micrograph of optic nerve section 7 days after crush injury, immunostained for CD3 (red, arrows). Scale bar, 100 µm.


( D ) Representative dotplots showing the general gating strategy used in flow cytometry experiments for the analysis of immune cells in the retina . Leukocytes were identified by gating on CD45 + cells , and were further gated on CD11b + or TCRβ + to detect monocytes / macrophages and T cells , respectively . Of the CD11b + cells , infiltrating monocytes were regarded as CD45hi .

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(D) Representative dotplots showing the general gating strategy used in flow cytometry experiments for the analysis of immune cells in the retina. Leukocytes were identified by gating on CD45+ cells, and were further gated on CD11b+ or TCRβ+ to detect monocytes/macrophages and T cells, respectively. Of the CD11b+ cells, infiltrating monocytes were regarded as CD45hi.


( E ) Representative contour plot from [ Cx3cr1gfp / + WT ] BM chimeric mouse , verifying that infiltrating blood - derived macrophages ( CX3CR1 - GFP + ) are indeed CD45hi , and showing that they are detectable only in retinas from eyes that underwent ONC , while absent in noninjured controls ( red vs . blue line ) . Cells were pre - gated on CD11b . n = 3 - 4 per group .

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(E) Representative contour plot from [Cx3cr1gfp/+ WT] BM chimeric mouse, verifying that infiltrating blood-derived macrophages (CX3CR1-GFP+) are indeed CD45hi, and showing that they are detectable only in retinas from eyes that underwent ONC, while absent in noninjured controls (red vs. blue line). Cells were pre-gated on CD11b. n = 3-4 per group.


( F ) Quantitative analysis of flow cytometry data , showing the induction of an immune response in the retina after ONC , evident by a general increase in leukocytes ( CD45 + ) , including CD11b + CD45hi cells ( representing infiltrating macrophages ) and T cells ( TCRβ + ) . n = 4 per group . Data shown are representative of six independent experiments . Graphs show mean ± SE of each group . * * * P < 0 . 001 . FSC - forward scatter ; noninj - noninjured control ; ONC - optic nerve crush .

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(F) Quantitative analysis of flow cytometry data, showing the induction of an immune response in the retina after ONC, evident by a general increase in leukocytes (CD45+), including CD11b+CD45hi cells (representing infiltrating macrophages) and T cells (TCRβ+). n = 4 per group. Data shown are representative of six independent experiments. Graphs show mean ± SE of each group. *** P < 0.001. FSC - forward scatter; noninj - noninjured control; ONC - optic nerve crush.


( A , C - E ) Changes in mRNA transcript levels of an array of adhesion molecules ( A ) in retinas from ONC eyes compared with contralateral noninjured retinas , 8 hours , and 1 , 3 and 7 days after ONC . n = 6 - 13 per group . Data shown are representative of three independent experiments .

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(A, C-E) Changes in mRNA transcript levels of an array of adhesion molecules (A) in retinas from ONC eyes compared with contralateral noninjured retinas, 8 hours, and 1, 3 and 7 days after ONC. n = 6-13 per group. Data shown are representative of three independent experiments.


( B ) Representative micrographs showing the expression of ICAM - 1 and VCAM - 1 ( red ) by the RPE ( green ) 12 hours after ONC . Scale bar , 20 µm . Bar graphs throughout the figure show mean ± SE of each group . Asterisks above bars indicate significant differences compared with noninjured tissue ; significant differences between different time points are indicated by asterisks between bars . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * P < 0 . 001 . noninj - noninjured control ; ONC - optic nerve crush .

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(B) Representative micrographs showing the expression of ICAM-1 and VCAM-1 (red) by the RPE (green) 12 hours after ONC. Scale bar, 20 µm. Bar graphs throughout the figure show mean ± SE of each group. Asterisks above bars indicate significant differences compared with noninjured tissue; significant differences between different time points are indicated by asterisks between bars. *, P < 0.05; **, P < 0.01; *** P < 0.001. noninj - noninjured control; ONC - optic nerve crush.


( A , C - E ) Changes in mRNA transcript levels of an array of chemokines ( C , D ) and cytokine receptors ( E ) in retinas from ONC eyes compared with contralateral noninjured retinas , 8 hours , and 1 , 3 and 7 days after ONC ( optic nerve crush ) . n = 6 - 13 per group . Data shown are representative of three independent experiments .

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(A, C-E) Changes in mRNA transcript levels of an array of chemokines (C, D) and cytokine receptors (E) in retinas from ONC eyes compared with contralateral noninjured retinas, 8 hours, and 1, 3 and 7 days after ONC (optic nerve crush). n = 6-13 per group. Data shown are representative of three independent experiments.


( A , B ) Quantitative flow cytometry analysis of myeloid cells in the RPE complex ( A ) and retinas ( B ) from injured ( filled bars ) and contralateral noninjured eyes ( empty bars ) , on days 1 , 3 and 7 after ONC . Cells were pre - gated on total leukocytes ( CD45 + ) , and analyzed for the expression of CD11b . CD11b + cells were further discriminated based on their expression levels of Ly6C . n = 6 - 7 per group . Bar graphs throughout the figure show mean ± SE of each group . Asterisks above bars indicate significant differences compared with noninjured tissue ; significant differences between different time points are indicated by asterisks between bars . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * P < 0 . 001 . noninj - noninjured control ; ONC - optic nerve crush .

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(A, B) Quantitative flow cytometry analysis of myeloid cells in the RPE complex (A) and retinas (B) from injured (filled bars) and contralateral noninjured eyes (empty bars), on days 1, 3 and 7 after ONC. Cells were pre-gated on total leukocytes (CD45+), and analyzed for the expression of CD11b. CD11b+ cells were further discriminated based on their expression levels of Ly6C. n = 6-7 per group. Bar graphs throughout the figure show mean ± SE of each group. Asterisks above bars indicate significant differences compared with noninjured tissue; significant differences between different time points are indicated by asterisks between bars. *, P < 0.05; **, P < 0.01; *** P < 0.001. noninj - noninjured control; ONC - optic nerve crush.


Mice were subjected to ONC and i . v . - injected with either an anti - VCAM - 1 blocking antibody or an isotype control antibody . Eyes were collected after 2 or 3 days for flow cytometry . ( A ) Flow cytometry quantification of myeloid cells in retinas from mice that were treated with anti - VCAM - 1 antibody , as compared to isotype - treated controls . n = 3 per group . Asterisks above bars indicate significant differences compared with noninjured tissue ; significant differences between different antibody treatments are indicated by asterisks between bars .

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Mice were subjected to ONC and i.v.-injected with either an anti-VCAM-1 blocking antibody or an isotype control antibody. Eyes were collected after 2 or 3 days for flow cytometry. (A) Flow cytometry quantification of myeloid cells in retinas from mice that were treated with anti-VCAM-1 antibody, as compared to isotype-treated controls. n = 3 per group. Asterisks above bars indicate significant differences compared with noninjured tissue; significant differences between different antibody treatments are indicated by asterisks between bars.


Mice were subjected to ONC and i . v . - injected with either an anti - VCAM - 1 blocking antibody or an isotype control antibody . Eyes were collected after 2 or 3 days for Real - Time PCR analysis , respectively . ( B ) Expression ratios of Tnf , Il12a and Il1b , compared to Tgfb2 , showing that blocking VCAM - 1 results in skewing the retina towards a pro - inflammatory milieu after ONC . n = 11 - 13 per group . Data shown are representative of two independent experiments . Bar graphs throughout the figure show mean ± SE of each group . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * P < 0 . 001 . noninj - noninjured control ; ONC - optic nerve crush .

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Mice were subjected to ONC and i.v.-injected with either an anti-VCAM-1 blocking antibody or an isotype control antibody. Eyes were collected after 2 or 3 days for Real-Time PCR analysis, respectively. (B) Expression ratios of Tnf, Il12a and Il1b, compared to Tgfb2, showing that blocking VCAM-1 results in skewing the retina towards a pro-inflammatory milieu after ONC. n = 11-13 per group. Data shown are representative of two independent experiments. Bar graphs throughout the figure show mean ± SE of each group. *, P < 0.05; **, P < 0.01; *** P < 0.001. noninj - noninjured control; ONC - optic nerve crush.


( A ) Live cell microscopy imaging of noninjured and ONC eyes , showing the presence of DiR + monocyte clusters in injured eyes only . Some of the cell clusters are co - localized with GFP , indicating monocytes that have crossed the RPE and infiltrated the retina . n = 2 - 3 per group . Data shown are representative of two independent experiments .

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(A) Live cell microscopy imaging of noninjured and ONC eyes, showing the presence of DiR+monocyte clusters in injured eyes only. Some of the cell clusters are co-localized with GFP, indicating monocytes that have crossed the RPE and infiltrated the retina. n = 2-3 per group. Data shown are representative of two independent experiments.


( B ) Representative micrographs of eye sections collected from mice 1 day after ONC , or 29 days after the induction of EAU , stained for β - dystroglycan ( red , arrows ) and laminin ( green ) , showing a comparable structure of blood vessels in the inner retina of ONC eyes and noninjured controls , which is different from the appearance of blood vessels in EAU retina . Scale bar , 50 µm .

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(B) Representative micrographs of eye sections collected from mice 1 day after ONC, or 29 days after the induction of EAU, stained for β-dystroglycan (red, arrows) and laminin (green), showing a comparable structure of blood vessels in the inner retina of ONC eyes and noninjured controls, which is different from the appearance of blood vessels in EAU retina. Scale bar, 50 µm.


( C - E ) [ Actbegfp / + WT ] BM chimeric mice were generated and subjected to ONC ( optic nerve crush ) . ( C ) Representative micrographs showing the abundance of GFP + cells ( green ) in the RPE ( RPE65 , red ) and retina of the ONC eye compared to noninjured contralateral eye . Retinal layers can be distinguished by Hoechst staining ( blue ; scale bar , 100 µm ) . ( D ) Higher magnification micrographs of ONC eyes , showing the association of blood - derived GFP + cells ( green , arrows ) with the RPE ( red ) ; Note processes extending from the cells to the RPE ( scale bar , 20 µm ) . ( E ) Representative micrograph of ONC eye showing GFP + cells ( green ) in proximity to the GCL , the site of dying retinal ganglion cells ( scale bar , 20 µm ) . n = 4 per group .

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(C-E) [Actbegfp/+ WT] BM chimeric mice were generated and subjected to ONC (optic nerve crush). (C) Representative micrographs showing the abundance of GFP+ cells (green) in the RPE (RPE65, red) and retina of the ONC eye compared to noninjured contralateral eye. Retinal layers can be distinguished by Hoechst staining (blue; scale bar, 100 µm). (D) Higher magnification micrographs of ONC eyes, showing the association of blood-derived GFP+ cells (green, arrows) with the RPE (red); Note processes extending from the cells to the RPE (scale bar, 20 µm). (E) Representative micrograph of ONC eye showing GFP+ cells (green) in proximity to the GCL, the site of dying retinal ganglion cells (scale bar, 20 µm). n = 4 per group.


( A - D ) Changes in mRNA transcript levels of a series of adhesion molecules ( A ) , chemokines ( B , C ) and cytokine receptors ( D ) in injured retinas compared with contralateral noninjured retinas , assessed 1 day after GT intoxication . n = 6 per group .

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(A-D) Changes in mRNA transcript levels of a series of adhesion molecules (A), chemokines (B, C) and cytokine receptors (D) in injured retinas compared with contralateral noninjured retinas, assessed 1 day after GT intoxication. n = 6 per group.


( E ) Representative micrographs showing the expression of ICAM - 1 and VCAM - 1 ( red ) by the RPE ( green ) 1 day after GT intoxication . Bar graphs throughout the figure show mean ± SE of each group . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * P < 0 . 001 . GT - glutamate ; noninj - noninjured control .

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(E) Representative micrographs showing the expression of ICAM-1 and VCAM-1 (red) by the RPE (green) 1 day after GT intoxication. Bar graphs throughout the figure show mean ± SE of each group. *, P < 0.05; **, P < 0.01; *** P < 0.001. GT - glutamate; noninj - noninjured control.


( A ) Representative micrograph of an eye from day 7 after glutamate intoxication , from a mouse that was i . v . - injected with monocytes , showing monocyte localization to the SRS after this mode of application , and indicating a potential homing route for the cells ( IL - 10 , red ; CX3CR1 - GFP , green ) .

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(A) Representative micrograph of an eye from day 7 after glutamate intoxication, from a mouse that was i.v.-injected with monocytes, showing monocyte localization to the SRS after this mode of application, and indicating a potential homing route for the cells (IL-10, red; CX3CR1-GFP, green).


( B - E ) Mice were injected i . v . t . with either monocytes or PBS 1 day after GT intoxication , and ocular sections from 7 days after the injury were analyzed . ( B , C ) Representative micrographs of noninjured and injured retinas stained for the RGC marker , Brn3a ( red ; scale bar , 100 µm ) , and quantification of RGC survival . Arrows indicate Brn3a + cells . Bar graph shows mean ± SE of each group . * , P < 0 . 05 .

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(B-E) Mice were injected i.v.t. with either monocytes or PBS 1 day after GT intoxication, and ocular sections from 7 days after the injury were analyzed. (B, C) Representative micrographs of noninjured and injured retinas stained for the RGC marker, Brn3a (red; scale bar, 100 µm), and quantification of RGC survival. Arrows indicate Brn3a+ cells. Bar graph shows mean ± SE of each group. *, P < 0.05.


( B - E ) Mice were injected i . v . t . with either monocytes or PBS 1 day after GT intoxication , and ocular sections from 7 days after the injury were analyzed . ( D , E ) Representative micrographs of GT - intoxicated eyes that were i . v . t . - injected with monocytes , showing the expression of an array of cytokines and neurotrophic factors ( red ) by the injected monocytes ( CX3CR1 - GFP , green ) . The injected cells were found in the vitreous and in proximity to injured RGCs ( left panels ) , as well as in the SRS , adjacent to the RPE ( right panels ) . Insets show higher magnification of representative cells ( scale bar , 100 µm ; inset scale , 20 µm ) . n = 4 - 5 per group . GCL - ganglion cell layer ; GT - glutamate ; mon - monocytes ; RGC - retinal ganglion cell ; RPE - retinal pigment epithelium ; SRS - subretinal space ; vit - vitreous .

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(B-E) Mice were injected i.v.t. with either monocytes or PBS 1 day after GT intoxication, and ocular sections from 7 days after the injury were analyzed. (D, E) Representative micrographs of GT-intoxicated eyes that were i.v.t.-injected with monocytes, showing the expression of an array of cytokines and neurotrophic factors (red) by the injected monocytes (CX3CR1-GFP, green). The injected cells were found in the vitreous and in proximity to injured RGCs (left panels), as well as in the SRS, adjacent to the RPE (right panels). Insets show higher magnification of representative cells (scale bar, 100 µm; inset scale, 20 µm). n = 4-5 per group. GCL - ganglion cell layer; GT - glutamate; mon - monocytes; RGC - retinal ganglion cell; RPE - retinal pigment epithelium; SRS - subretinal space; vit - vitreous.


In panels ( A ) violin plots show the variability in continuous leaf 16 , ear phenotypes among the 60 individual plants used in downstream analyses . Data information : In all violin plots , the median is indicated by the white circle . The black box extends from the 25th to the 75th percentile , and black whiskers extend from each end of the box to the most extreme values within 1 . 5 times the interquartile range from the respective end . Data points beyond this range are shown as black dots . The red open circle indicates the mean of the distribution , with red whiskers extending to 1 standard deviation above and below the mean .

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In panels (A) violin plots show the variability in continuous leaf 16, ear phenotypes among the 60 individual plants used in downstream analyses. Data information: In all violin plots, the median is indicated by the white circle. The black box extends from the 25th to the 75th percentile, and black whiskers extend from each end of the box to the most extreme values within 1.5 times the interquartile range from the respective end. Data points beyond this range are shown as black dots. The red open circle indicates the mean of the distribution, with red whiskers extending to 1 standard deviation above and below the mean.


In panels ( E ) , violin plots show the variability in plant height phenotypes among the 60 individual plants used in downstream analyses . Data information : In all violin plots , the median is indicated by the white circle . The black box extends from the 25th to the 75th percentile , and black whiskers extend from each end of the box to the most extreme values within 1 . 5 times the interquartile range from the respective end . Data points beyond this range are shown as black dots . The red open circle indicates the mean of the distribution , with red whiskers extending to 1 standard deviation above and below the mean .

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In panels (E), violin plots show the variability in plant height phenotypes among the 60 individual plants used in downstream analyses. Data information: In all violin plots, the median is indicated by the white circle. The black box extends from the 25th to the 75th percentile, and black whiskers extend from each end of the box to the most extreme values within 1.5 times the interquartile range from the respective end. Data points beyond this range are shown as black dots. The red open circle indicates the mean of the distribution, with red whiskers extending to 1 standard deviation above and below the mean.


B . Genotype distribution in litters from heterozygous matings ( Fbxl4 + / - x Fbxl4 + / - ) at embryonic stage 13 . 5 ( E13 . 5 ) and of live animals at weaning . Data is presented as a percentage . Embryos n = 79 , weaned mice n = 339 . Dashed lines indicate the expected Mendelian ratios . Chi2 test vs expected Mendelian ratios ; * * * , p < 0 . 001 .

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B. Genotype distribution in litters from heterozygous matings (Fbxl4+/- x Fbxl4+/-) at embryonic stage 13.5 (E13.5) and of live animals at weaning. Data is presented as a percentage. Embryos n=79, weaned mice n=339. Dashed lines indicate the expected Mendelian ratios. Chi2 test vs expected Mendelian ratios; ***, p<0.001.


C . Representative images of wild - type ( + / + ) , heterozygous knockout ( + / - ) and homozygous Fbxl4 knockout embryos ( - / - ) at E13 . 5 .

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C. Representative images of wild-type (+/+), heterozygous knockout (+/-) and homozygous Fbxl4 knockout embryos (-/-) at E13.5.


D . Relative levels of mtDNA of Fbxl4 knockout ( - / - ) embryos and the corresponding wild type ( + / + ) embryos at E13 . 5 . Data represent mean ± SEM , n = 5 . Student ' s t - test ; * , p < 0 . 05 .

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D. Relative levels of mtDNA of Fbxl4 knockout (-/-) embryos and the corresponding wild type (+/+) embryos at E13.5. Data represent mean ± SEM, n=5. Student's t-test; *, p<0.05.


E . Representative images of male Fbxl4 knockout ( - / - ) and matched wild - type ( + / + ) animals at one year of age ( left ) . A hunch - back phenotype was observed both in male and female knockout animals ( right , indicated by arrow ) .

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E. Representative images of male Fbxl4 knockout (-/-) and matched wild-type (+/+) animals at one year of age (left). A hunch-back phenotype was observed both in male and female knockout animals (right, indicated by arrow).


F - I . Whole body ( F ) , heart ( G ) , liver ( H ) and kidney ( I ) weights of 1 - year old animals . Data represent mean ± SEM , n ≥ 5 animals . Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . , not significant ( p ≥ 0 . 05 ) .

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F-I. Whole body (F), heart (G), liver (H) and kidney (I) weights of 1-year old animals. Data represent mean ± SEM, n ≥ 5 animals. Student's t-test; *, p<0.05; **, p<0.01; ***, p<0.001; n.s., not significant (p ≥ 0.05).


A . Relative levels of mtDNA in tissues of 1 - year - old Fbxl4 knockout mice determined by quantitative real - time PCR ( normalized to controls ) . Data are presented as mean ± SEM , n = 6 for controls and n = 8 for knockout animals . Student ' s t - test ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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A. Relative levels of mtDNA in tissues of 1-year-old Fbxl4 knockout mice determined by quantitative real-time PCR (normalized to controls). Data are presented as mean ± SEM, n=6 for controls and n=8 for knockout animals. Student's t-test; **, p<0.01; ***, p<0.001.


B . Transcript levels in indicated tissues of 1 - year - old Fbxl4 knockout mice determined by quantitative real - time PCR . Relative mRNA levels in control animals were set as 1 ( dashed line ) . Data are presented as mean ± SEM , n = 6 for controls and n = 8 for knockout animals . Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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B. Transcript levels in indicated tissues of 1-year-old Fbxl4 knockout mice determined by quantitative real-time PCR. Relative mRNA levels in control animals were set as 1 (dashed line). Data are presented as mean ± SEM, n=6 for controls and n=8 for knockout animals. Student's t-test; *, p<0.05; **, p<0.01; ***, p<0.001.


C . Western blot analysis of protein steady - state levels in liver protein extracts from wild type ( Fbxl4 + / + ) and Fbxl4 knock - out ( Fbxl4 - / - ) animals . D . Quantification of the western blot in ( C ) . Signal normalized to the average of controls and presented as mean ± SEM , n = 4 for both controls and knockout animals . Student ' s t - test ; * * , p < 0 . 01 ; * * * , p < 0 , 001 .

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C. Western blot analysis of protein steady-state levels in liver protein extracts from wild type (Fbxl4+/+) and Fbxl4 knock-out (Fbxl4-/-) animals. D. Quantification of the western blot in (C). Signal normalized to the average of controls and presented as mean ± SEM, n=4 for both controls and knockout animals. Student's t-test; **, p<0.01; ***, p<0,001.


E . Quantitative TMT - based proteomic analysis of wild - type and Fbxl4 knock - out liver tissue protein extracts . Proteins from three control and three Fbxl4 knock - out liver protein extracts were TMT - labelled , quantified and analyzed separately as described in Materials and Methods ; the data from control and knock - out animals were averaged after the analysis and presented as - log10 of the adjusted p - value vs log2 - fold change ( logFC ) . Mitochondrial proteins were selected according to mouse MitoCarta 2 . 0 ( Calvo et al , 2016 ) and highlighted in red ; lysosomal proteins were selected according to hLGDB database ( Brozzi et al , 2013 ) and highlighted in yellow .

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E. Quantitative TMT-based proteomic analysis of wild-type and Fbxl4 knock-out liver tissue protein extracts. Proteins from three control and three Fbxl4 knock-out liver protein extracts were TMT-labelled, quantified and analyzed separately as described in Materials and Methods; the data from control and knock-out animals were averaged after the analysis and presented as -log10 of the adjusted p-value vs log2-fold change (logFC). Mitochondrial proteins were selected according to mouse MitoCarta 2.0(Calvo et al, 2016) and highlighted in red; lysosomal proteins were selected according to hLGDB database(Brozzi et al, 2013) and highlighted in yellow.


F . Enrichment analysis of lysosomal and mitochondrial proteins according to the logFC median of each group of proteins . Data is presented as 25 to 75 percentile box with the indicated median and whiskers representing the ± 1 . 5x inter - quartile range . Proteins included in each group : total = 1523 , mitochondrial = 324 , lysosomal = 50 .

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F. Enrichment analysis of lysosomal and mitochondrial proteins according to the logFC median of each group of proteins. Data is presented as 25 to 75 percentile box with the indicated median and whiskers representing the ±1.5x inter-quartile range. Proteins included in each group: total=1523, mitochondrial=324, lysosomal=50.


B . Relative mtDNA content in fibroblasts from patients and controls as determined by qRT - PCR using an ND6 probe and normalized to nuclear genomic DNA content using an 18S probe . Individual values are presented for each sample , as well as their mean ± SEM values .

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B. Relative mtDNA content in fibroblasts from patients and controls as determined by qRT-PCR using an ND6 probe and normalized to nuclear genomic DNA content using an 18S probe. Individual values are presented for each sample, as well as their mean ± SEM values.


C . Steady - state protein levels in three control and three FBXL4 patient - derived fibroblasts analyzed by western blotting .

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C. Steady-state protein levels in three control and three FBXL4 patient-derived fibroblasts analyzed by western blotting.


D . Quantification of the western blot in ( C ) . Signals from the control and FBXL4 - mutated cells were considered as biological replicates , normalized to the average of controls and displayed as mean ± SEM ( n = 3 for each ) . Student ' s t - test ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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D. Quantification of the western blot in (C). Signals from the control and FBXL4-mutated cells were considered as biological replicates, normalized to the average of controls and displayed as mean ± SEM (n=3 for each). Student's t-test; *, p<0.05; **, p<0.01; ***, p<0.001.


E . Quantification of nuclear and mitochondrial transcript levels in control and patient fibroblasts . The results from control and patient cells were averaged , normalized to controls and displayed as mean ± SEM ( n = 3 biological replicates for each condition ) .

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E. Quantification of nuclear and mitochondrial transcript levels in control and patient fibroblasts. The results from control and patient cells were averaged, normalized to controls and displayed as mean ± SEM (n=3 biological replicates for each condition).


F . Quantitative TMT - based proteomic analysis of control and patient fibroblasts . Proteins from three control and three patient fibroblast cell lines were TMT - labelled , quantified and analyzed separately as described in Materials and Methods ; the data from control and patients were averaged after the analysis and presented as - log10 of the adjusted p - value vs log2 - fold change ( logFC ) . Mitochondrial proteins were selected according to human MitoCarta 2 . 0 ( Calvo et al , 2016 ) and highlighted in blue ; lysosomal proteins were selected according to hLGDB database ( Brozzi et al , 2013 ) and highlighted in yellow .

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F. Quantitative TMT-based proteomic analysis of control and patient fibroblasts. Proteins from three control and three patient fibroblast cell lines were TMT-labelled, quantified and analyzed separately as described in Materials and Methods; the data from control and patients were averaged after the analysis and presented as -log10 of the adjusted p-value vs log2-fold change (logFC). Mitochondrial proteins were selected according to human MitoCarta 2.0 (Calvo et al, 2016) and highlighted in blue; lysosomal proteins were selected according to hLGDB database (Brozzi et al, 2013) and highlighted in yellow.


G . Log2FC correlation analysis of Mouse Liver proteomic dataset and Fibroblast Proteomic dataset . Proteins with a log2FC > 0 . 5 are highlighted in black and lysosomal proteins are highlighted in yellow .

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G. Log2FC correlation analysis of Mouse Liver proteomic dataset and Fibroblast Proteomic dataset. Proteins with a log2FC > 0.5 are highlighted in black and lysosomal proteins are highlighted in yellow.


B . Steady - state protein levels in wild - type ( WT ) and FBXL4 knockout ( KO ) RKO cells .

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B. Steady-state protein levels in wild-type (WT) and FBXL4 knockout (KO) RKO cells.


C . ( Upper panel ) Pulse - chase labeling of mitochondrial translation products in wild type ( WT ) and FBXL4 knockout ( KO ) cells . The cells were incubated for 1 hour with a 35S methionine - cysteine labeling mix in the presence of anisomycin to inhibit cytosolic translation ( pulse ) . After removal of the medium , the cells were incubated in complete growth medium without inhibitors and labeled amino acids for 16 or 24 hours ( chase ) . A representative experiment of 4 biological replicates is shown . A Coomassie - stained part of the gel is shown as loading control . ( Lower panel ) Quantification of the MTCO1 - ND4 signal presented as mean ± SEM ; n = 4 biological replicates ; Student ' s t - test ; * , p < 0 . 05 .

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C. (Upper panel) Pulse-chase labeling of mitochondrial translation products in wild type (WT) and FBXL4 knockout (KO) cells. The cells were incubated for 1 hour with a 35S methionine-cysteine labeling mix in the presence of anisomycin to inhibit cytosolic translation (pulse). After removal of the medium, the cells were incubated in complete growth medium without inhibitors and labeled amino acids for 16 or 24 hours (chase). A representative experiment of 4 biological replicates is shown. A Coomassie-stained part of the gel is shown as loading control. (Lower panel) Quantification of the MTCO1-ND4 signal presented as mean ± SEM; n=4 biological replicates; Student's t-test; *, p<0.05.


D . ( Upper panel ) Mitochondrial translation products were labeled in wild type ( WT ) and FBXL4 knock - out ( KO ) RKO cells for 60 minutes as in C , followed by 24 h chase in the presence of 5 μM epoxomicin ( epoxo ) or 20 mM ammonium chloride ( NH4Cl ) . A representative experiment of n ≥ 3 biological replicates is shown . A fragment of the Coomassie - stained gel is shown as loading control . ( Lower panel ) Quantification of the ND4 - MTCO1 signal . n ≥ 3 biological replicates , data are presented as mean ± SEM . Student ' s t - test ; * , p < 0 . 05 .

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D. (Upper panel) Mitochondrial translation products were labeled in wild type (WT) and FBXL4 knock-out (KO) RKO cells for 60 minutes as in C, followed by 24 h chase in the presence of 5 μM epoxomicin (epoxo) or 20 mM ammonium chloride (NH4Cl). A representative experiment of n≥3 biological replicates is shown. A fragment of the Coomassie-stained gel is shown as loading control. (Lower panel) Quantification of the ND4-MTCO1 signal. n≥3 biological replicates, data are presented as mean ± SEM. Student's t-test; *, p<0.05.


E - F . Steady - state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with

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E-F. Steady-state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with NH4Cl


E - F . Steady - state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with epoxomicin ( Epoxo , F ) .

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E-F. Steady-state levels of indicated proteins in wild type and FBXL4 knockout RKO cells treated with epoxomicin (Epoxo, F).


A . Confocal images from the mito - QC - expressing fibroblast lines . Scale bar 25 μm . B . Quantification of the mitolysosomes / cell area ( μm2 ) using the mito - QC counter plugin for ImageJ ( Montava - Garriga et al , 2020 ) . At least 3 technical replicates for each line and n > 15 cells analyzed for each technical replicate . Data is presented as 25 to 75 percentile box with the indicated median and whiskers representing 5 - 95 percentile interval . Non - parametric Mann - Whitney test ; * * * , p < 0 . 001 .

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A. Confocal images from the mito-QC-expressing fibroblast lines. Scale bar 25 μm. B. Quantification of the mitolysosomes/cell area (μm2) using the mito-QC counter plugin for ImageJ (Montava-Garriga et al, 2020). At least 3 technical replicates for each line and n>15 cells analyzed for each technical replicate. Data is presented as 25 to 75 percentile box with the indicated median and whiskers representing 5-95 percentile interval. Non-parametric Mann-Whitney test; ***, p<0.001.


( B ) Representative confocal microscopy sections of m6A - Tracer signal in HAP - 1 cells following pA - DamID with indicated antibodies . pA - Dam only : primary antibody was omitted , the dotted yellow line indicates DAPI segmentation . Free Dam : permeabilized cells were treated with freely diffusing pure Dam protein and SAM for 30 min . Scale bar corresponds to 2 µm . ( C ) Quantification of peripheral enrichment of antibody staining and m6A - Tracer signals after pA - DamID with indicated antibodies . The nuclear rim and interior were segmented using DAPI signal and the mean m6A or antibody signal was determined and tranformed to a log2 - ratio . The nuclear rim mask extends slightly beyond the NL , resulting in underestimation of the real enrichment .

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(B) Representative confocal microscopy sections of m6A-Tracer signal in HAP-1 cells following pA-DamID with indicated antibodies. pA-Dam only: primary antibody was omitted, the dotted yellow line indicates DAPI segmentation. Free Dam: permeabilized cells were treated with freely diffusing pure Dam protein and SAM for 30 min. Scale bar corresponds to 2 µm. (C) Quantification of peripheral enrichment of antibody staining and m6A-Tracer signals after pA-DamID with indicated antibodies. The nuclear rim and interior were segmented using DAPI signal and the mean m6A or antibody signal was determined and tranformed to a log2-ratio. The nuclear rim mask extends slightly beyond the NL, resulting in underestimation of the real enrichment.


( D ) HT1080 cells expressing inducible Dam - Lamin B1 were treated with Shield1 to induce m6A methylation . Cells were either fixed immediately or processed according to the pA - DamID protocol ( as negative controls ) and then fixed on poly - L - lysine coated cover slips . Cells were imaged by confocal microscopy for the NL ( Lamin B2 antibody ) and methylation ( m6A - Tracer ) for both conditions . Laser settings were changed between images to optimize image quality . The mask for defining the nuclear rim was obtained by segmentation of the DAPI image . The scale bar corresponds to 2 µm .

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(D) HT1080 cells expressing inducible Dam-Lamin B1 were treated with Shield1 to induce m6A methylation. Cells were either fixed immediately or processed according to the pA-DamID protocol (as negative controls) and then fixed on poly-L-lysine coated cover slips. Cells were imaged by confocal microscopy for the NL (Lamin B2 antibody) and methylation (m6A-Tracer) for both conditions. Laser settings were changed between images to optimize image quality. The mask for defining the nuclear rim was obtained by segmentation of the DAPI image.The scale bar corresponds to 2 µm.


( E ) For every cell , the 50 % decay distance from the nuclear periphery was determined for Lamin B2 and m6A - Tracer by fitting exponential decay functions from the nuclear rim as defined by segmentation of the DAPI image The difference in 50 % decay distance between Lamin B2 and m6A - Tracer was used as a measure of the thickness of the m6A - Tracer layer .

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(E) For every cell, the 50% decay distance from the nuclear periphery was determined for Lamin B2 and m6A-Tracer by fitting exponential decay functions from the nuclear rim as defined by segmentation of the DAPI image The difference in 50% decay distance between Lamin B2 and m6A-Tracer was used as a measure of the thickness of the m6A-Tracer layer.


( F ) Distribution of the m6A - Tracer layer thickness for HT1080 cells expressing Dam - Lamin B1 , visualized before and after the pA - DamID protocol . For comparison , a similar analysis was performed with HT1080 cells not expressing Dam - LaminB1 , subjected to Lamin B2 pA - DamID .

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(F) Distribution of the m6A-Tracer layer thickness for HT1080 cells expressing Dam-Lamin B1, visualized before and after the pA-DamID protocol. For comparison, a similar analysis was performed with HT1080 cells not expressing Dam-LaminB1, subjected to Lamin B2 pA-DamID.


( A ) Example of raw pA - DamID data tracks from 2 million HAP - 1 cells for Lamin B2 , H3K27me3 and the Dam control from a single experiment . Sequenced reads are counted in 20kb bins and normalized for library size . ( B ) Same pA - DamID tracks of Lamin B2 and H3K27me3 as in ( A ) but after normalization to the Dam - only control ( to correct for accessibility and amplification biases ) and log2 - transformation .

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(A) Example of raw pA-DamID data tracks from 2 million HAP-1 cells for Lamin B2, H3K27me3 and the Dam control from a single experiment. Sequenced reads are counted in 20kb bins and normalized for library size. (B) Same pA-DamID tracks of Lamin B2 and H3K27me3 as in (A) but after normalization to the Dam-only control (to correct for accessibility and amplification biases) and log2-transformation.


( C ) Correlation between the median peripheral enrichment of m6A - Tracer determined by confocal microscopy and enrichment of sequencing reads within LADs in HAP - 1 cells . The LAD definition is based on conventional Lamin B1 DamID data . Every point represents a single pA - DamID experiment for which both microscopy and sequencing data were generated , colored by the antibody used . The blue line represents a linear model with a standard error confidence interval in grey . The red dashed line represents the diagonal . The Pearson correlation coefficient was converted to a t - statistic and a one - sided t - test with n - 2 degrees of freedom was used to determine statistical significance .

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(C) Correlation between the median peripheral enrichment of m6A-Tracer determined by confocal microscopy and enrichment of sequencing reads within LADs in HAP-1 cells. The LAD definition is based on conventional Lamin B1 DamID data. Every point represents a single pA-DamID experiment for which both microscopy and sequencing data were generated, colored by the antibody used. The blue line represents a linear model with a standard error confidence interval in grey. The red dashed line represents the diagonal. The Pearson correlation coefficient was converted to a t-statistic and a one-sided t-test with n-2 degrees of freedom was used to determine statistical significance.


( D ) Comparisons of pA - DamID genome - wide data ( bin size 20 kb ) for different lamin antibodies in hTERT - RPE cells : Lamin B1 vs . Lamin B2 ( left panel ) and Lamin B2 vs . Lamin A / C ( right panel ) . n denotes the number of independent biological experiments that were averaged for each data track . The blue line represents a linear model . The red dashed line represents the diagonal .

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(D) Comparisons of pA-DamID genome-wide data (bin size 20 kb) for different lamin antibodies in hTERT-RPE cells: Lamin B1 vs. Lamin B2 (left panel) and Lamin B2 vs. Lamin A/C (right panel). n denotes the number of independent biological experiments that were averaged for each data track. The blue line represents a linear model. The red dashed line represents the diagonal.


( A ) Comparison of pA - DamID and DamID data for for various lamins across a representative genomic locus in HAP - 1 and hTERT - RPE cells . Arrow indicates a region with dissimilar pA - DamID and DamID scores .

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(A) Comparison of pA-DamID and DamID data for for various lamins across a representative genomic locus in HAP-1 and hTERT-RPE cells. Arrow indicates a region with dissimilar pA-DamID and DamID scores.


( B ) Genome - wide correlation of Lamin B1 pA - DamID and Lamin B1 DamID data . The blue line represents a linear model . The red dashed line represents the diagonal .

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(B) Genome-wide correlation of Lamin B1 pA-DamID and Lamin B1 DamID data. The blue line represents a linear model. The red dashed line represents the diagonal.


( D ) Multidimensional scaling ( MDS ) plot showing all individual replicates of DamID and pA - DamID experiments for various lamins and cell types .

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(D) Multidimensional scaling (MDS) plot showing all individual replicates of DamID and pA-DamID experiments for various lamins and cell types.


( B ) NL interactions in hTERT - RPE cells at indicated times after mitosis for chromosome 2 , as determined by Lamin B2 pA - DamID . Log2 - ratios were convered to z - scores to account for differences in dynamic range between replicates Average of two independent biological replicates . LADs are colored by their differential class ( down : red , stable : grey , up : green , The centromere is shown as grey box .

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(B) NL interactions in hTERT-RPE cells at indicated times after mitosis for chromosome 2, as determined by Lamin B2 pA-DamID. Log2-ratios were convered to z-scores to account for differences in dynamic range between replicates Average of two independent biological replicates. LADs are colored by their differential class (down:red, stable:grey, up:green, The centromere is shown as grey box.


( C ) pA - DamID signal as a function of time after mitosis , for stable ( grey , n = 717 ) and dynamic LADs ( red and green : decreasing ( n = 234 ) and increasing ( n = 225 ) NL interactions , respectively ) . Lines represent mean signals of the three LAD classes and the shaded area one standard deviation on both sides . ( D ) Rate of changes in NL interactions ( average pA - DamID z - score per hour ) as function of time after mitosis for stable and dynamics LADs Boxplots : horizontal lines represent 25th , 50th and 75th percentiles ; whiskers extend to the smallest values no further than 1 . 5 times distance between 25th and 75th percentiles .

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(C) pA-DamID signal as a function of time after mitosis, for stable (grey, n=717) and dynamic LADs (red and green: decreasing (n=234) and increasing (n=225) NL interactions, respectively). Lines represent mean signals of the three LAD classes and the shaded area one standard deviation on both sides. (D) Rate of changes in NL interactions (average pA-DamID z-score per hour) as function of time after mitosis for stable and dynamics LADs Boxplots: horizontal lines represent 25th, 50th and 75th percentiles; whiskers extend to the smallest values no further than 1.5 times distance between 25th and 75th percentiles.


( E - G ) LAD size distribution in basepairs ( E ) , density of active genes ( F ) and Repli - seq score distributions ( G ) for stable and dynamic LADs Statistical significance was determined using a two ‐ way Wilcoxin test with Benjamini - Hochberg p - value correction to account for multiple testing .

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(E-G) LAD size distribution in basepairs (E), density of active genes (F) and Repli-seq score distributions (G) for stable and dynamic LADs Statistical significance was determined using a two‐way Wilcoxin test with Benjamini-Hochberg p-value correction to account for multiple testing.


( H ) Difference in pA - DamID z - scores between 1h and 21h time points , plotted against the distance to telomeres ( leftpanel ) or centromeres ( rightpanel ) . Each dot represents a single LAD . Chromosomes are colored and ordered by size . Red line is a fitted loess curve . The Spearman correlation coefficient ( ρ ) was used to test for a significant monotonic , non - linear association .

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(H) Difference in pA-DamID z-scores between 1h and 21h time points, plotted against the distance to telomeres (leftpanel) or centromeres (rightpanel). Each dot represents a single LAD. Chromosomes are colored and ordered by size. Red line is a fitted loess curve. The Spearman correlation coefficient (ρ) was used to test for a significant monotonic, non-linear association.


Difference in pA - DamID z - scores plotted against the distance to telomeres ( leftpanel ) or centromeres ( rightpanel ) . Each dot represents a single LAD . Chromosomes are colored and ordered by size . Red line is a fitted loess curve . The Spearman correlation coefficient ( ρ ) was used to test for a significant monotonic , non - linear association . pA - DamID z - scores for individual time points .

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Difference in pA-DamID z-scores plotted against the distance to telomeres (leftpanel) or centromeres (rightpanel). Each dot represents a single LAD. Chromosomes are colored and ordered by size. Red line is a fitted loess curve. The Spearman correlation coefficient (ρ) was used to test for a significant monotonic, non-linear association. pA-DamID z-scores for individual time points.


( A ) Lamin B2 pA - DamID z - scores of a representative locus in HAP - 1 cells sorted for G1 , mid - S and G2 phases . Arrows point to regions that lose NL interaction from G1 to G2 . The red star indicates the end of the chromosome . Average of three biological replicates .

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(A) Lamin B2 pA-DamID z-scores of a representative locus in HAP-1 cells sorted for G1, mid-S and G2 phases. Arrows point to regions that lose NL interaction from G1 to G2. The red star indicates the end of the chromosome. Average of three biological replicates.


difference in pA - DamID z - scores between G2 and G1 sorted pools as function of distance to either telomere or centromere for HAP - 1 ( n = 3 ) , HCT116 ( n = 2 ) and K562 cells ( n = 2 ) .

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difference in pA-DamID z-scores between G2 and G1 sorted pools as function of distance to either telomere or centromere for HAP-1 (n=3), HCT116 (n=2) and K562 cells (n=2).


( C - E ) LAD size distribution in basepairs ( C ) , density of active genes ( D ) and Repli - seq scores ( E ) for stable and dynamic LADs . Repli - seq data are not available for HAP - 1 cells .

(C-E) LAD size distribution in basepairs (C), density of active genes (D) and Repli-seq scores (E) for stable and dynamic LADs. Repli-seq data are not available for HAP-1 cells.


( F ) Overlapping fractions of stable and dynamic LADs and iLADs with Hi - C subcompartments in HAP - 1 and K562 cells , as imputed by SNIPER A permutation test with 1000 permutations ( R - package regioneR was used to test for a significant enrichment of B1 subcompartments in decreasing LADs , resulting in P - values of 1e - 3 and 1e - 3 for HAP - 1 and K562 , respectively .

(F) Overlapping fractions of stable and dynamic LADs and iLADs with Hi-C subcompartments in HAP-1 and K562 cells, as imputed by SNIPER A permutation test with 1000 permutations (R-package regioneR was used to test for a significant enrichment of B1 subcompartments in decreasing LADs, resulting in P-values of 1e-3 and 1e-3 for HAP-1 and K562, respectively.


( A ) Representative K562 locus comparing Lamin B2 pA - DamID z - scores in cell cycle sorted cells with replication timing . Arrows point to regions with increased mid - S signal compared to G1 and G2 , which tend to overlap with regions that exhibit intermediate replication timing .

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(A) Representative K562 locus comparing Lamin B2 pA-DamID z-scores in cell cycle sorted cells with replication timing. Arrows point to regions with increased mid-S signal compared to G1 and G2, which tend to overlap with regions that exhibit intermediate replication timing.


( B ) Difference tracks in pA - DamID z - scores between the cell cycle stages Data were smoothed by a running mean of 5

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(B) Difference tracks in pA-DamID z-scores between the cell cycle stages Data were smoothed by a running mean of 5 bins