( C ) The early - flowering phenotype of the mbd9 - 1 , mbd9 - 3 , mbd9 - 4 and hta9 - 1 / 11 - 1 mutants compared to the WT . 24 - day - old plants in soil are shown .

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(C) The early-flowering phenotype of the mbd9-1, mbd9-3, mbd9-4 and hta9-1/11-1 mutants compared to the WT. 24-day-old plants in soil are shown.


( D ) Numbers of rosette leaves of the WT and mutant plants shortly after bolting . Rosette leaves from at least 20 plants were counted for each genotype . Values are mean ± SD . * * P < 0 . 01 , student ' s t test .

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(D) Numbers of rosette leaves of the WT and mutant plants shortly after bolting. Rosette leaves from at least 20 plants were counted for each genotype. Values are mean ± SD. ** P<0.01, student's t test.


( E ) Determination of the effect of the arp4 - 1 , arp6 and swc6 - 1 mutations on flowering time . The top panel shows the number of rosette leaves in the WT , arp4 - 1 , arp6 , and swc6 - 1 mutant plants ; the bottom panel shows a photograph of the plants . Rosette leaves from at least 20 plants were counted for each genotype . Values are mean ± SD . * * P < 0 . 01 , student ' s t test .

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(E) Determination of the effect of the arp4-1, arp6 and swc6-1 mutations on flowering time. The top panel shows the number of rosette leaves in the WT, arp4-1, arp6, and swc6-1 mutant plants; the bottom panel shows a photograph of the plants. Rosette leaves from at least 20 plants were counted for each genotype. Values are mean ± SD. ** P<0.01, student's t test.


( F ) Determination of the effect of the tra1a and tra1b mutations on flowering time . The top panel shows numbers of rosette leaves of the WT , tra1a - 1 , tra1a - 2 , and tra1b mutant plants ; the bottom panel shows a photograph of the plants . Rosette leaves from at least 20 null were counted for each genotype . Values are mean ± SD . * * P < 0 . 01 , student ' s t test .

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(F) Determination of the effect of the tra1a and tra1b mutations on flowering time. The top panel shows numbers of rosette leaves of the WT, tra1a-1, tra1a-2, and tra1b mutant plants; the bottom panel shows a photograph of the plants. Rosette leaves from at least 20 null were counted for each genotype. Values are mean ± SD. ** P<0.01, student's t test.


( B ) Heat maps showing the effect of indicated mutations on the expression of differentially expressed genes identified in the mbd9 - 1 mutant . The color bar shows log2 ( fold change ) . Up - and down - regulation are shown by red and blue , respectively .

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(B) Heat maps showing the effect of indicated mutations on the expression of differentially expressed genes identified in the mbd9-1 mutant. The color bar shows log2(fold change). Up- and down-regulation are shown by red and blue, respectively.


( C ) Scatter plots showing that the expression pattern in the hta9 - 1 / 11 - 1 mutant is correlated with that in the chr11 / 17 , mbd9 - 1 , and pie1 mutants . The differentially expressed genes identified in the hta9 - 1 / 11 - 1 mutant were subjected to the analysis .

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(C) Scatter plots showing that the expression pattern in the hta9-1/11-1 mutant is correlated with that in the chr11/17, mbd9-1, and pie1 mutants. The differentially expressed genes identified in the hta9-1/11-1 mutant were subjected to the analysis.


( A ) The effect of indicated mutations on H2A . Z deposition as determined by immunoblotting . Chromatin - associated proteins were extracted and subjected to immunoblotting by the anti - H2A . Z antibody . The histone H4 level was detected by the anti - H4 antibody and indicated as a loading control .

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(A) The effect of indicated mutations on H2A.Z deposition as determined by immunoblotting. Chromatin-associated proteins were extracted and subjected to immunoblotting by the anti-H2A.Z antibody. The histone H4 level was detected by the anti-H4 antibody and indicated as a loading control.


( B ) Meta plots showing H2A . Z occupancy on H2A . Z target genes . TSS , transcription start site ; TTS , transcription termination site . RPKM , reads per kilobase per million . The H2A . Z ChIP - seq results were generated from two independent biological replicates .

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(B) Meta plots showing H2A.Z occupancy on H2A.Z target genes. TSS, transcription start site; TTS, transcription termination site. RPKM, reads per kilobase per million. The H2A.Z ChIP-seq results were generated from two independent biological replicates.


( D ) Heat maps showing H2A . Z enrichment in the WT , pie1 , chr11 / 17 , and mbd9 - 1 mutants over the PIE1

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(D) Heat maps showing H2A.Z enrichment in the WT, pie1, chr11/17, and mbd9-1 mutants over the PIE1


( D ) Heat maps showing H2A . Z enrichment in the WT , pie1 , chr11 / 17 , and mbd9 - 1 mutants over the

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( E ) Shown are H2A . Z changes in the pie1 , chr11 / 17 , and mbd9 - 1 mutants relative to the WT . PIE1 - dependent H2A . Z target genes were subjected to the analysis and sorted by H2A . Z density in the WT . The color bar shows log2 ( fold change ) .

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(E) Shown are H2A.Z changes in the pie1, chr11/17, and mbd9-1 mutants relative to the WT. PIE1-dependent H2A.Z target genes were subjected to the analysis and sorted by H2A.Z density in the WT. The color bar shows log2(fold change).


( F ) Meta plots showing CHR11 and PIE1 occupancy on PIE1 - dependent H2A . Z target genes . TSS , transcription start site ; TTS , transcription termination site . The CHR11 - Flag and PIE1 - Flag ChIP - seq data are from two independent biological replicates .

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(F) Meta plots showing CHR11 and PIE1 occupancy on PIE1-dependent H2A.Z target genes. TSS, transcription start site; TTS, transcription termination site. The CHR11-Flag and PIE1-Flag ChIP-seq data are from two independent biological replicates.


( G ) Heat maps showing the enrichment of PIE1 - Flag and CHR11 - Flag over the PIE1 - dependent H2A . Z target genes . The peaks were sorted by H2A . Z density in the WT .

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(G) Heat maps showing the enrichment of PIE1-Flag and CHR11-Flag over the PIE1-dependent H2A.Z target genes. The peaks were sorted by H2A.Z density in the WT.


( H ) Box plot showing the enrichment of CHR11 and PIE1 over H2A . Z target genes and non - H2A . Z target genes . The log2 ( fold change ) values of RPKM between the CHR11 - Flag or PIE1 - Flag signals and the WT signals are shown . Center lines and box edges represent medians and the interquartile range ( IQR ) , respectively . Whiskers extend to minimum and maximum values within 1 . 5 times of the IQR . The data shown are from two independent biological replicates . The difference between the CHR11 - Flag and PIE1 - Flag ChIP - seq levels was determined by student ' s t test . * * P < 0 . 01 .

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

(H) Box plot showing the enrichment of CHR11 and PIE1 over H2A.Z target genes and non-H2A.Z target genes. The log2(fold change) values of RPKM between the CHR11-Flag or PIE1-Flag signals and the WT signals are shown. Center lines and box edges represent medians and the interquartile range (IQR), respectively. Whiskers extend to minimum and maximum values within 1.5 times of the IQR. The data shown are from two independent biological replicates. The difference between the CHR11-Flag and PIE1-Flag ChIP-seq levels was determined by student's t test. ** P<0.01.


( A ) Increasing amounts of CHR11 ( left panel ) or PIE1 ( right panel ) were incubated with end - positioned ( lane 1 - 4 and 9 - 12 ) or center - positioned ( lane 5 - 8 and 13 - 16 ) mono - nucleosomes in the presence of ATP . The sliding results were then visualized on native PAGE gels by EB staining .

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(A) Increasing amounts of CHR11 (left panel) or PIE1 (right panel) were incubated with end-positioned (lane 1-4 and 9-12) or center-positioned (lane 5-8 and 13-16) mono-nucleosomes in the presence of ATP. The sliding results were then visualized on native PAGE gels by EB staining.


( B ) Distribution of nucleosomes over TSS of all protein - coding genes in the WT , chr11 / 17 , pie1 , and mbd9 - 1 mutants . The MNase - seq results were generated from two independent biological replicates .

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(B) Distribution of nucleosomes over TSS of all protein-coding genes in the WT, chr11/17, pie1, and mbd9-1 mutants. The MNase-seq results were generated from two independent biological replicates.


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 ) Silver staining of proteins extracted from N2A mouse neuronal cells expressing Flag - HA - tagged C9ORF72 and captured through consecutive anti - Flag and anti - HA affinity purification steps .

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(A) Silver staining of proteins extracted from N2A mouse neuronal cells expressing Flag-HA-tagged C9ORF72 and captured through consecutive anti-Flag and anti-HA affinity purification steps.


( B ) Immunoblot analysis of HA - immunoprecipitated proteins and lysate of HEK293 cells co - expressing HA - tagged C9ORF72 and / or Flag - tagged SMCR8 and / or Flag - tagged WDR41 .

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(B) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and/ or Flag-tagged SMCR8 and/or Flag-tagged WDR41.


( C ) Coomassie blue staining of Nickel - NTA affinity purification of HIS - C9ORF72 , SMCR8 and WDR41 co - expressed in baculovirus infected insect cells .

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(C) Coomassie blue staining of Nickel-NTA affinity purification of HIS-C9ORF72, SMCR8 and WDR41 co-expressed in baculovirus infected insect cells.


( D ) Immunoblot analysis of HA - immunoprecipitated proteins and lysate of HEK293 cells co - expressing HA - tagged C9ORF72 and HA - tagged SMCR8 with various Flag - tagged Rab GTPases .

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(D) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and HA-tagged SMCR8 with various Flag-tagged Rab GTPases.


( E ) Immunoblot against endogenous C9orf72 , Wdr41 , Rab8a , Rab39b , Rab5 and Rab7 of control ( IgG alone ) or endogenous Smcr8 immunoprecipitated from adult mouse brain .

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(E) Immunoblot against endogenous C9orf72, Wdr41, Rab8a, Rab39b, Rab5 and Rab7 of control (IgG alone) or endogenous Smcr8 immunoprecipitated from adult mouse brain.


( F ) αP32 - radiolabelled GDP release from GST - tagged purified RAB8a as a function of increased concentration of either recombinant purified C9ORF72 alone or in complex with SMCR8 and WDR41 .

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(F)αP32-radiolabelled GDP release from GST-tagged purified RAB8a as a function of increased concentration of either recombinant purified C9ORF72 alone or in complex with SMCR8 and WDR41.


( G ) Identical GDP release assay as in ( F ) but using recombinant purified GST - tagged RAB39b instead of RAB8a . Error bars indicate s . e . m . Experiments were repeated 3 times ( n = 3 ) .

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(G) Identical GDP release assay as in (F) but using recombinant purified GST-tagged RAB39b instead of RAB8a. Error bars indicate s.e.m. Experiments were repeated 3 times (n=3).


( A ) Left panel , representative images of organotypic cultures of E18 mouse cortical neurons transfected with GFP - RFP - LC3B and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin . Right panel , quantification of LC3B puncta .

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(A) Left panel, representative images of organotypic cultures of E18 mouse cortical neurons transfected with GFP-RFP-LC3B and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin. Right panel, quantification of LC3B puncta.


( B ) Upper panel , immunoblot analysis of endogenous LC3B ( Map1lc3b ) , C9orf72 and control Actin of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin . Lower panel , real time RT - qPCR quantification of endogenous C9orf72 mRNA expression relative to Rplp0 mRNA .

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(B) Upper panel, immunoblot analysis of endogenous LC3B (Map1lc3b), C9orf72 and control Actin of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin. Lower panel, real time RT-qPCR quantification of endogenous C9orf72 mRNA expression relative to Rplp0 mRNA.


( C ) Left panel , representative images of immunofluorescence labeling of endogenous P62 ( Sqstm1 ) on organotypic cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72 . Right panel , quantification of P62 aggregates .

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(C) Left panel, representative images of immunofluorescence labeling of endogenous P62 (Sqstm1) on organotypic cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72. Right panel, quantification of P62 aggregates.


( D ) Left panel , representative images of immunofluorescence labeling of transfected constructs ( Green ) and endogenous P62 ( Sqstm1 , red ) on GT1 - 7 neuronal cells transfected with siRNA targeting C9orf72 and plasmids expressing control GFP , HA tagged wild type or mutant RAB39b ( CA , Q68L or CN , S22N ) , RAB8a or RAB7 . Right panel , quantification of P62 aggregates . Scale bars , 10 µm . Nuclei were counterstained with DAPI . Error bars indicate s . e . m . Student T - test , * indicates p < 0 . 05 , * * * indicates p < 0 . 001 . n = 3 .

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(D) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting C9orf72 and plasmids expressing control GFP, HA tagged wild type or mutant RAB39b (CA, Q68L or CN, S22N), RAB8a or RAB7. Right panel, quantification of P62 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, * indicates p<0.05, *** indicates p<0.001. n=3.


( A ) Immunoblot analysis of HA - immunoprecipitated proteins and lysate of HEK293 cells co - expressing HA - tagged C9ORF72 and HA - tagged SMCR8 with Flag - tagged TBK1 , NAP1 , TANK or SINTBAD .

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(A) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and HA-tagged SMCR8 with Flag-tagged TBK1, NAP1, TANK or SINTBAD.


( B ) Immunoprecipitated HA - tagged C9ORF72 , HA - tagged SMCR8 and HA - tagged WDR41 expressed in HEK293 were subjected to in vitro TBK1 kinase assay in the presence of γP32 - radiolabelled ATP . Proteins were separated by SDS page migration and phosphorylation was detected by autoradiography ( upper panel ) , while expression was detected by western blotting ( lower panel ) .

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(B) Immunoprecipitated HA-tagged C9ORF72, HA-tagged SMCR8 and HA-tagged WDR41 expressed in HEK293 were subjected to in vitro TBK1 kinase assay in the presence of γP32-radiolabelled ATP. Proteins were separated by SDS page migration and phosphorylation was detected by autoradiography (upper panel), while expression was detected by western blotting (lower panel).


( C ) Mass spectrometry identification of SMCR8 phosphorylation sites by in vitro TBK1 kinase assay of HIS - tagged C9ORF72 : SMCR8 : WDR41 complex purified from baculovirus infected insect cells .

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(C) Mass spectrometry identification of SMCR8 phosphorylation sites by in vitro TBK1 kinase assay of HIS-tagged C9ORF72:SMCR8:WDR41 complex purified from baculovirus infected insect cells.


( D ) Left panel , representative images of immunofluorescence labeling of transfected constructs ( Green ) and endogenous P62 ( Sqstm1 , red ) on GT1 - 7 neuronal cells transfected with siRNA targeting the 3 ' UTR of Smcr8 mRNA and plasmids expressing control GFP , HA tagged wild type or mutant SMCR8 ( TA , S402A and T796A ; TD , S402D and T796D ; UD , S400D , S492D , S562D and T666D ) . Right panel , quantification of P62 aggregates .

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(D) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting the 3'UTR of Smcr8 mRNA and plasmids expressing control GFP, HA tagged wild type or mutant SMCR8 (TA, S402A and T796A; TD, S402D and T796D; UD, S400D, S492D, S562D and T666D). Right panel, quantification of P62 aggregates.


( E ) Left panel , representative images of immunofluorescence labeling of transfected constructs ( Green ) and endogenous P62 ( Sqstm1 , red ) on GT1 - 7 neuronal cells transfected with siRNA targeting Tbk1 and plasmids expressing control GFP , HA tagged wild type or mutant SMCR8 or RAB39b . Right panel , quantification of P62 aggregates . Scale bars , 10 µm . Nuclei were counterstained with DAPI . Error bars indicate s . e . m . Student T - test , * indicates p < 0 . 05 , * * * indicates p < 0 . 001 . n = 3 .

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(E) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting Tbk1 and plasmids expressing control GFP, HA tagged wild type or mutant SMCR8 or RAB39b. Right panel, quantification of P62 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, * indicates p<0.05, *** indicates p<0.001. n=3.


( A ) Immunoblot analysis of endogenous Tdp - 43 , C9orf72 and control Gapdh of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA .

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(A) Immunoblot analysis of endogenous Tdp-43, C9orf72 and control Gapdh of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA.


( B ) Left panel , representative images of immunofluorescence labeling of endogenous phosphorylated Ser409 / 410 - Tdp - 43 on primary cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72 . Right panel , quantification of cytoplasmic Tdp - 43 aggregates .

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(B) Left panel, representative images of immunofluorescence labeling of endogenous phosphorylated Ser409/410-Tdp-43 on primary cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72. Right panel, quantification of cytoplasmic Tdp-43 aggregates.


( C ) Left panel , representative images of immunofluorescence labeling of D169G mutant GFP - tagged TDP - 43 on primary cultures of E18 mouse cortical neurons transfected with either HA - tagged control or HA - C9ORF72 plasmid . Right panel , quantification of cells with cytoplasmic aggregates of TDP - 43 . Scale bars , 10 µm . Nuclei were counterstained with DAPI . Error bars indicate s . e . m . Student T - test , * * * indicates p < 0 . 001 . n = 3 .

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(C) Left panel, representative images of immunofluorescence labeling of D169G mutant GFP-tagged TDP-43 on primary cultures of E18 mouse cortical neurons transfected with either HA-tagged control or HA-C9ORF72 plasmid. Right panel, quantification of cells with cytoplasmic aggregates of TDP-43. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, *** indicates p<0.001. n=3.


( A ) Left panel , representative images of organotypic cultures of E18 mouse cortical neurons co - transfected with HA - tagged ATXN2 with control ( Q22x ) or intermediate ( Q30x ) polyQ size and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA . Right panel , quantification of ATXN2 aggregates . Scale bars , 10 µm . Nuclei were counterstained with DAPI .

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(A) Left panel, representative images of organotypic cultures of E18 mouse cortical neurons co-transfected with HA-tagged ATXN2 with control (Q22x) or intermediate (Q30x) polyQ size and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA. Right panel, quantification of ATXN2 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI.


( B ) Cell viability ( tetrazolium assay ) of GT1 - 7 neuronal cells co - transfected with HA - tagged ATXN2 with control ( Q22x ) or intermediate ( Q30x ) polyQ size and control siRNA or siRNA targeting C9orf72 mRNA . Error bars indicate s . e . m . Student T - test , * * * indicates p < 0 . 001 . n = 3 .

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(B) Cell viability (tetrazolium assay) of GT1-7 neuronal cells co-transfected with HA-tagged ATXN2 with control (Q22x) or intermediate (Q30x) polyQ size and control siRNA or siRNA targeting C9orf72 mRNA. Error bars indicate s.e.m. Student T-test, *** indicates p<0.001. n=3.


( C ) Tracing of the swimming trajectories of 48 hours post - fertilization zebrafish larvae following light touch . ( D - F ) Quantification of the touch - evoked swimming distance ( D ) , average velocity ( E ) , and maximum velocity attained ( F ) shows significant functional impairment of the zebrafish injected with HA - tagged ATXN2 with intermediate length of polyQ ( Q30x ) and antisense morpholino oligonucleotides ( AMOs ) against C9orf72 compared to control HA - tagged ATXN2 ( Q22x ) or to the sole injection of AMO against C9orf72 .

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(C) Tracing of the swimming trajectories of 48 hours post-fertilization zebrafish larvae following light touch. (D-F) Quantification of the touch-evoked swimming distance (D), average velocity (E), and maximum velocity attained (F) shows significant functional impairment of the zebrafish injected with HA-tagged ATXN2 with intermediate length of polyQ (Q30x) and antisense morpholino oligonucleotides (AMOs) against C9orf72 compared to control HA-tagged ATXN2 (Q22x) or to the sole injection of AMO against C9orf72.


( G ) Representative images of motor neurons visualized with anti - Sv2 immunohistochemistry show severe axonopathy in fish injected with both ATXN2 Q30x and AMO against C9orf72 compared to zebrafish injected with control ATXN2 Q22x or with AMO against C9orf72 alone . ( H ) Quantification of the motor neuron axonal length demonstrates a significant decrease of axonal length in 48 hours post - fertilization zebrafish larvae injected with both ATXN2 Q30x and AMO against C9orf72 compared to controls . Error bars indicate s . e . m . One - way ANOVA , * * indicates p < 0 . 01 . n = 3 .

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(G) Representative images of motor neurons visualized with anti-Sv2 immunohistochemistry show severe axonopathy in fish injected with both ATXN2 Q30x and AMO against C9orf72 compared to zebrafish injected with control ATXN2 Q22x or with AMO against C9orf72 alone. (H) Quantification of the motor neuron axonal length demonstrates a significant decrease of axonal length in 48 hours post-fertilization zebrafish larvae injected with both ATXN2 Q30x and AMO against C9orf72 compared to controls. Error bars indicate s.e.m. One-way ANOVA, ** indicates p<0.01. n=3.


Cell surface H2 - Kb levels of wild type and TAP1 - / - BMDC transduced with control vector or a vector expressing hβ2m were analyzed .

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Cell surface H2-Kb levels of wild type and TAP1-/- BMDC transduced with control vector or a vector expressing hβ2m were analyzed.


Cross - presentation of OVA by wild type and TAP1 - / - BMDC expressing hβ2m were compared to control BMDC , by measuring IL - 2 production by a Kb - SIINFEKL specific T - cell hybridoma ( B3Z ) cultured with paraformaldehyde fixed BMDC that were incubated with varying numbers of OVA - coated latex beads for 6hr , prior to fixation .

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Cross-presentation of OVA by wild type and TAP1-/- BMDC expressing hβ2m were compared to control BMDC, by measuring IL-2 production by a Kb-SIINFEKL specific T-cell hybridoma (B3Z) cultured with paraformaldehyde fixed BMDC that were incubated with varying numbers of OVA-coated latex beads for 6hr, prior to fixation.


The effect of hβ2m on the presentation of peptides derived from endogenous antigen was assessed by measuring IL - 2 levels in the culture supernatant of the B3Z hybridoma incubated with fixed VV - OVA infected WT and TAP1 - / - BMDC transduced with control vector or hβ2m .

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The effect of hβ2m on the presentation of peptides derived from endogenous antigen was assessed by measuring IL-2 levels in the culture supernatant of the B3Z hybridoma incubated with fixed VV-OVA infected WT and TAP1-/- BMDC transduced with control vector or hβ2m.


Surface H2 - Kb levels of wild - type and TAP1 - / - BMDC co - expressing Rab mutants and hβ2m were analyzed by flow cytometry .

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Surface H2-Kb levels of wild-type and TAP1-/- BMDC co-expressing Rab mutants and hβ2m were analyzed by flow cytometry.


Cross - presentation of OVA by wild - type and TAP1 - / - BMDC co - expressing Rab mutants and hβ2m were analyzed and IL - 2 release is

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Cross-presentation of OVA by wild-type and TAP1-/- BMDC co-expressing Rab mutants and hβ2m were analyzed and IL-2 release is shown


TAP1 - / - BMDC transduced with vector control ( A ) or hβ2m ( C ) were incubated with varying concentrations of Epoxomicin for 6hr in absence or presence of exogenously added Kb - binding SIINFEKL peptide and analyzed for surface expression of Kb by flow cytometry . Representative plots are shown ( A , C ) . The impact of Epoxomicin on cell surface Kb was analyzed by plotting Kb surface expression on cells incubated with Epoxomicin , normalized to that on cell incubated without Epoxomicin ( n = 3 ) ( B , D ) .

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TAP1-/- BMDC transduced with vector control (A) or hβ2m (C) were incubated with varying concentrations of Epoxomicin for 6hr in absence or presence of exogenously added Kb-binding SIINFEKL peptide and analyzed for surface expression of Kb by flow cytometry. Representative plots are shown (A, C). The impact of Epoxomicin on cell surface Kb was analyzed by plotting Kb surface expression on cells incubated with Epoxomicin, normalized to that on cell incubated without Epoxomicin (n=3) (B, D).


The effect of Epoxomicin on cross - presentation of OVA - coated beads by TAP1 - / - BMDC expressing hβ2m was determined by incubating the BMDC with OVA coated latex beads in the presence of varying doses of Epoxomicin and 10μM gB peptide . After 6hrs the cells were fixed , incubated with B3Z cells and IL - 2 production was

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The effect of Epoxomicin on cross-presentation of OVA-coated beads by TAP1-/- BMDC expressing hβ2m was determined by incubating the BMDC with OVA coated latex beads in the presence of varying doses of Epoxomicin and 10μM gB peptide. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured


293T - FcR - Kb cells co - expressing empty vector or Rab mutants ( Rab5ACA , Rab22ACA , Rab7ADN ) individually , with either LacZ as a control or US6 were analyzed for cross - presentation of OVA using B3Z cells . The effects of US6 on cross - presentation were analyzed by plotting the mean percentage of IL - 2 release by cells co - expressing US6 ( A ) with control vector or Rab mutants compared to cells co - expressing LacZ with control vector or Rab mutants , respectively .

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293T-FcR-Kb cells co-expressing empty vector or Rab mutants (Rab5ACA, Rab22ACA, Rab7ADN) individually, with either LacZ as a control or US6 were analyzed for cross-presentation of OVA using B3Z cells. The effects of US6 on cross-presentation were analyzed by plotting the mean percentage of IL-2 release by cells co-expressing US6 (A) with control vector or Rab mutants compared to cells co-expressing LacZ with control vector or Rab mutants, respectively.


293T - FcR - Kb cells co - expressing empty vector or Rab mutants ( Rab5ACA , Rab22ACA , Rab7ADN ) individually , with either LacZ as a control or ICP47 ( B ) , were analyzed for cross - presentation of OVA using B3Z cells . The effects of ICP47 on cross - presentation were analyzed by plotting the mean percentage of IL - 2 release by cells co - expressing ICP47 ( B ) with control vector or Rab mutants compared to cells co - expressing LacZ with control vector or Rab mutants , respectively .

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293T-FcR-Kb cells co-expressing empty vector or Rab mutants (Rab5ACA, Rab22ACA, Rab7ADN) individually, with either LacZ as a control or ICP47 (B), were analyzed for cross-presentation of OVA using B3Z cells. The effects of ICP47 on cross-presentation were analyzed by plotting the mean percentage of IL-2 release by cells co-expressing ICP47 (B) with control vector or Rab mutants compared to cells co-expressing LacZ with control vector or Rab mutants, respectively.


293T - FcR - Kb cells expressing Rab5ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin . After 6hrs the cells were fixed , incubated with B3Z cells and IL - 2 production was measured .

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293T-FcR-Kb cells expressing Rab5ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured.


293T - FcR - Kb cells expressing Rab22ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin . After 6hrs the cells were fixed , incubated with B3Z cells and IL - 2 production was measured .

293T-FcR-Kb cells expressing Rab22ACA were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured.


293T - FcR - Kb cells expressing Rab7ADN ( E ) were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin . After 6hrs the cells were fixed , incubated with B3Z cells and IL - 2 production was measured .

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293T-FcR-Kb cells expressing Rab7ADN (E) were incubated with opsonized OVA coated latex beads in the presence of varying doses of Epoxomicin. After 6hrs the cells were fixed, incubated with B3Z cells and IL-2 production was measured.


EM micrographs of double immuno - gold labeling using antibodies against immunoproteasome subunit LMP2 and the endolysosomal membrane marker LAMP1 in BMDC , WT MEF and LMP2 KO MEF . Large gold particles ( 15 nm , black arrow head ) labels LMP2 and small gold particles labels LAMP1 ( 5 nm , white arrow head ) ( Scale bars = 500nm ) . The insets are the magnification of region of interest containing LAMP1 positive vacuole ( Scale bars = 100nm ) , marked by the black rectangle .

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EM micrographs of double immuno-gold labeling using antibodies against immunoproteasome subunit LMP2 and the endolysosomal membrane marker LAMP1 in BMDC, WT MEF and LMP2 KO MEF. Large gold particles (15 nm, black arrow head) labels LMP2 and small gold particles labels LAMP1 (5 nm, white arrow head) (Scale bars = 500nm). The insets are the magnification of region of interest containing LAMP1 positive vacuole (Scale bars = 100nm), marked by the black rectangle.


The distribution of LMP2 positive signals ( n = 22 images ) assessed by plotting the number of gold particles labeling LMP2 within the LAMP1 positive membrane compartment versus those outside the LAMP1 positive organelles .

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The distribution of LMP2 positive signals (n = 22 images) assessed by plotting the number of gold particles labeling LMP2 within the LAMP1 positive membrane compartment versus those outside the LAMP1 positive organelles.


Immuno - gold labeling with antibodies against constitutive proteasome subunits , α5 , β5 and 19 S2 ( 15 nm , black arrow head ) , co - labeled with an anti - LAMP1 antibody ( 5 nm , white arrow head ) in BMDC ( Scale bars = 500nm ) . Regions of interest containing LAMP1 positive vacuoles are marked by the black rectangles and magnified in the insets ( Scale bars = 100nm ) .

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Immuno-gold labeling with antibodies against constitutive proteasome subunits, α5, β5 and 19 S2 (15 nm, black arrow head), co-labeled with an anti-LAMP1 antibody (5 nm, white arrow head) in BMDC (Scale bars = 500nm). Regions of interest containing LAMP1 positive vacuoles are marked by the black rectangles and magnified in the insets (Scale bars = 100nm).


Immuno - electron microscopy images ( n = 23 ) were analyzed by plotting the total number of gold particles labeling proteasome subunits inside and outside the LAMP1 positive membrane compartments ( D ) . These data are also presented as the percentage of the total gold particles that are within the LAMP1 positive organelle ( E ) .

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Immuno-electron microscopy images (n = 23) were analyzed by plotting the total number of gold particles labeling proteasome subunits inside and outside the LAMP1 positive membrane compartments (D). These data are also presented as the percentage of the total gold particles that are within the LAMP1 positive organelle (E).


BMDC transduced with genes encoding GFP , GFP - Rab5ACA and GFP - Rab22ACA under the control of an inducible promoter were fixed and stained for LMP2 and LAMP1 and analyzed by confocal microscopy 24hr after doxycycline induction and 4hr after Alexa 647 - OVA coated bead uptake . A single optical section of 10 optical sections of a representative cell are shown ( Scale Bar 10μm ) . Region of interest containing LAMP1 positive vacuole containing beads coated with Alexa - 647 conjugated OVA , marked by white rectangles , are magnified as inset ( Scale bars = 1μm )

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BMDC transduced with genes encoding GFP, GFP-Rab5ACA and GFP-Rab22ACA under the control of an inducible promoter were fixed and stained for LMP2 and LAMP1 and analyzed by confocal microscopy 24hr after doxycycline induction and 4hr after Alexa 647-OVA coated bead uptake. A single optical section of 10 optical sections of a representative cell are shown (Scale Bar 10μm). Region of interest containing LAMP1 positive vacuole containing beads coated with Alexa-647 conjugated OVA, marked by white rectangles, are magnified as inset (Scale bars = 1μm)


BMDC transduced with genes encoding GFP , GFP - Rab5ACA and GFP - Rab22ACA under the control of an inducible promoter were fixed and stained for LMP2 and LAMP1 and analyzed by confocal microscopy 3D - rendering of 10 optical sections of a representative cell are shown ( Scale Bar 10μm ) . Region of interest containing LAMP1 positive vacuole containing beads coated with Alexa - 647 conjugated OVA , marked by white rectangles , are magnified as inset ( Scale bars = 1μm )

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BMDC transduced with genes encoding GFP, GFP-Rab5ACA and GFP-Rab22ACA under the control of an inducible promoter were fixed and stained for LMP2 and LAMP1 and analyzed by confocal microscopy 3D-rendering of 10 optical sections of a representative cell are shown (Scale Bar 10μm). Region of interest containing LAMP1 positive vacuole containing beads coated with Alexa-647 conjugated OVA, marked by white rectangles, are magnified as inset (Scale bars = 1μm)


The same cells were analyzed for LMP2 fluorescence intensity within the phagosomal lumen as defined by a region of interest positive for Alexa 647 - OVA enclosed within a limiting membrane positive for LAMP1 . The fluorescence intensity of LMP2 within the phagosomal lumen normalized to Alexa647 - OVA was calculated and data compiled from independent experiments ( n = 3 ) are plotted ( H ) . For each transduced cell type , images of at the least 27 cells acquired across three independent experiments were analyzed and each cell is represented as a data point .

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The same cells were analyzed for LMP2 fluorescence intensity within the phagosomal lumen as defined by a region of interest positive for Alexa 647-OVA enclosed within a limiting membrane positive for LAMP1. The fluorescence intensity of LMP2 within the phagosomal lumen normalized to Alexa647-OVA was calculated and data compiled from independent experiments (n=3) are plotted (H).For each transduced cell type, images of at the least 27 cells acquired across three independent experiments were analyzed and each cell is represented as a data point.


BMDC co - expressing LMP2 - RFP and GFP - Rab5ACA were live imaged 16hr after doxycycline induction and 30min post uptake of Alexa647 coated latex beads ( Bar 10μm ) . Images were acquired every 3min and images taken at 12min interval are presented ( I - K ) . The arrowheads in panel ( I and J ) mark the phagosome or vacuoles containing LMP2 , respectively .

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BMDC co-expressing LMP2-RFP and GFP-Rab5ACA were live imaged 16hr after doxycycline induction and 30min post uptake of Alexa647 coated latex beads (Bar 10μm). Images were acquired every 3min and images taken at 12min interval are presented (I-K). The arrowheads in panel (I and J) mark the phagosome or vacuoles containing LMP2, respectively.


BMDC co - expressing LMP2 - RFP and GFP - Rab22ACA ( J , K ) were live imaged 16hr after doxycycline induction and 30min post uptake of Alexa647 coated latex beads ( Bar 10μm ) . Images were acquired every 3min and images taken at 12min interval are presented ( I - K ) . The arrowheads in panel ( I and J ) mark the phagosome or vacuoles containing LMP2 , respectively . The boxed region in panel ( J ) marks a phagosome that has been enlarged in panel ( K ) ( Bar 1μm ) .

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BMDC co-expressing LMP2-RFP and GFP-Rab22ACA (J, K) were live imaged 16hr after doxycycline induction and 30min post uptake of Alexa647 coated latex beads (Bar 10μm). Images were acquired every 3min and images taken at 12min interval are presented (I-K). The arrowheads in panel (I and J) mark the phagosome or vacuoles containing LMP2, respectively. The boxed region in panel (J) marks a phagosome that has been enlarged in panel (K) (Bar 1μm).


Activity of proteasomes within the phagosomes was determined by measuring the fluorescence intensity of fluorogenic proteasome substrate incubated with latex beads from detergent - treated phagosomes treated or untreated with Epoxomicin . The fluorescence intensities were measured at 1hr intervals and values plotted represent the ratio of the signals in the absence versus the presence of

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Activity of proteasomes within the phagosomes was determined by measuring the fluorescence intensity of fluorogenic proteasome substrate incubated with latex beads from detergent-treated phagosomes treated or untreated with Epoxomicin. The fluorescence intensities were measured at 1hr intervals and values plotted represent the ratio of the signals in the absence versus the presence of Epoxomicin


Intraluminal proteasome dependent degradation of phagocytosed antigen was measured by flow cytometric analysis of phagocytosed latex beads covalently conjugated with Alexa647 - OVA . Latex beads were extracted by mechanical disruption of BMDC and incubated with ATP , lysosomal protease inhibitor cocktail and varying dose of Epoxomicin at 37oC for 16hrs . Post incubation latex beads were treated with detergent and analyzed by flow cytometry ( B ) . The mean of Alexa647 fluorescence associated with the latex beads incubated with Epoxomicin were normalized to fluorescence of untreated beads from independent experiments were plotted ( n = 4 ) ( C ) .

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Intraluminal proteasome dependent degradation of phagocytosed antigen was measured by flow cytometric analysis of phagocytosed latex beads covalently conjugated with Alexa647-OVA. Latex beads were extracted by mechanical disruption of BMDC and incubated with ATP, lysosomal protease inhibitor cocktail and varying dose of Epoxomicin at 37oC for 16hrs. Post incubation latex beads were treated with detergent and analyzed by flow cytometry (B). The mean of Alexa647 fluorescence associated with the latex beads incubated with Epoxomicin were normalized to fluorescence of untreated beads from independent experiments were plotted (n=4) (C).


Ubiquitination of phagosomal cargo within the phagosomes was assessed by measuring the acquisition of GFP fluorescence by phagocytosed latex beads conjugated with OVA post - detergent extraction from BMDC expressing GFP or GFP - Ub ( D ) . The mean of fluorescence intensities of GFP on the beads from independent experiments was plotted ( E ) .

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Ubiquitination of phagosomal cargo within the phagosomes was assessed by measuring the acquisition of GFP fluorescence by phagocytosed latex beads conjugated with OVA post-detergent extraction from BMDC expressing GFP or GFP-Ub (D). The mean of fluorescence intensities of GFP on the beads from independent experiments was plotted (E).


Whole cell extracts of High Five insect cells individually expressing FLAG - SHLD3 , Strep - RIF1 , and 6xHis - REV7 through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for REV7 or the Strep or FLAG epitopes . Results are representative of three biologically independent experiments . IB : immunoblot , FL : full - length . For SHLD3 , N and C correspond to residues 1 - 125 and 126 - 250 , respectively .

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Whole cell extracts of High Five insect cells individually expressing FLAG-SHLD3, Strep-RIF1, and 6xHis-REV7 through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for REV7 or the Strep or FLAG epitopes. Results are representative of three biologically independent experiments. IB: immunoblot, FL: full-length. For SHLD3, N and C correspond to residues 1-125 and 126-250, respectively.


Representative micrographs of the LacR / LacO assay using mCherry - LacR - RIF1N as bait to evaluate chromatin recruitment of eGFP - tagged SHLD3 variants . SHLD3 : residues 2 - 250 . SHLD3N : residues 2 - 125 . SHLD3C : residues 126 - 250 . RIF1N : residues 1 - 967 .

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Representative micrographs of the LacR/LacO assay using mCherry-LacR-RIF1N as bait to evaluate chromatin recruitment of eGFP-tagged SHLD3 variants. SHLD3: residues 2-250. SHLD3N: residues 2-125. SHLD3C: residues 126-250. RIF1N: residues 1-967.


Quantification of LacR / LacO assay measuring chromatin recruitment of eGFP - tagged SHLD3 variants to LacO arrays by mCherry - LacR - RIF1N . GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry - labeled LacR focus and the average nuclear intensity . Bars represent means ( n = 138 , 126 , 129 , 137 for eGFP , eGFP - SHLD3 , eGFP - SHLD3N , eGFP - SHLD3C from 3 biologically independent experiments ) . Analysis was performed by Kruskal - Wallis test followed by Dunn ' s multiple comparisons against empty vector control or full - length eGFP - SHLD3 . * * * * : P < 0 . 0001 . ns : P > 0 . 05 .

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Quantification of LacR/LacO assay measuring chromatin recruitment of eGFP-tagged SHLD3 variants to LacO arrays by mCherry-LacR-RIF1N. GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry-labeled LacR focus and the average nuclear intensity. Bars represent means (n = 138, 126, 129, 137 for eGFP, eGFP-SHLD3, eGFP-SHLD3N, eGFP-SHLD3C from 3 biologically independent experiments). Analysis was performed by Kruskal-Wallis test followed by Dunn's multiple comparisons against empty vector control or full-length eGFP-SHLD3. ****: P < 0.0001. ns: P > 0.05.


Representative micrographs of the LacR - FokI assay to evaluate DNA damage recruitment of eGFP - tagged SHLD3 variants after induction of LacR - FokI expression .

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Representative micrographs of the LacR-FokI assay to evaluate DNA damage recruitment of eGFP-tagged SHLD3 variants after induction of LacR-FokI expression.


Quantification of LacR - FokI assay evaluating recruitment of eGFP - tagged SHLD3 variants to FokI - induced DSBs at LacO arrays . GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry - labeled LacR - FokI focus and the average nuclear intensity . Bars represent mean ( n = 143 , 147 , 147 , 141 for eGFP , eGFP - SHLD3 , eGFP - SHLD3N , eGFP - SHLD3C from 3 biologically independent experiments ) . Analysis was performed by Kruskal - Wallis test followed by Dunn ' s multiple comparisons against empty vector control or full - length eGFP - SHLD3 . * * * * : P < 0 . 0001 . ns : P > 0 . 05 .

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Quantification of LacR-FokI assay evaluating recruitment of eGFP-tagged SHLD3 variants to FokI-induced DSBs at LacO arrays. GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry-labeled LacR-FokI focus and the average nuclear intensity. Bars represent mean (n = 143, 147, 147, 141 for eGFP, eGFP-SHLD3, eGFP-SHLD3N, eGFP-SHLD3C from 3 biologically independent experiments). Analysis was performed by Kruskal-Wallis test followed by Dunn's multiple comparisons against empty vector control or full-length eGFP-SHLD3. ****: P < 0.0001. ns: P > 0.05.


Whole cell extracts of High Five insect cells individually expressing the wild - type ( WT ) or indicated alanine substitution FLAG - SHLD3C ( residues 126 - 250 ) variants and Strep - RIF1N ( residues 1 - 980 ) through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for the Strep or FLAG epitopes . The results are representative of two biologically independent experiments . IB : immunoblot . IP : immunoprecipitation .

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

Whole cell extracts of High Five insect cells individually expressing the wild-type (WT) or indicated alanine substitution FLAG-SHLD3C (residues 126-250) variants and Strep-RIF1N (residues 1-980) through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for the Strep or FLAG epitopes. The results are representative of two biologically independent experiments. IB: immunoblot. IP: immunoprecipitation.


Quantification of LacR / LacO assay measuring recruitment of eGFP - SHLD3C wild - type ( WT ) or alanine substitution variants to LacO arrays in U2OS 2 - 6 - 3 cells by mCherry - LacR - RIF1N . GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry - labeled LacR - RIF1N focus and the average nuclear intensity . Bars represent means ( n = 134 , 125 , 120 , 130 , 104 , 129 , 117 for EV , WT , S131A , W132A , R166A , N201A , D216A from 3 biologically independent experiments ) . EV : empty vector . Analysis was performed by Kruskal - Wallis test followed by Dunn ' s multiple comparisons against empty vector and WT controls . * * * * : P < 0 . 0001 . * * * : P = 0 . 0004 . * * : P = 0 . 002 . * : P = 0 . 02 . ns : P > 0 . 05 .

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

Quantification of LacR/LacO assay measuring recruitment of eGFP-SHLD3C wild-type (WT) or alanine substitution variants to LacO arrays in U2OS 2-6-3 cells by mCherry-LacR-RIF1N. GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry-labeled LacR-RIF1N focus and the average nuclear intensity. Bars represent means (n = 134, 125, 120, 130, 104, 129, 117 for EV, WT, S131A, W132A, R166A, N201A, D216A from 3 biologically independent experiments). EV: empty vector. Analysis was performed by Kruskal-Wallis test followed by Dunn's multiple comparisons against empty vector and WT controls. ****: P < 0.0001. ***: P = 0.0004. **: P = 0.002. *: P = 0.02. ns: P > 0.05.


Quantification of LacR - FokI assay measuring recruitment of eGFP - SHLD3C wild - type ( WT ) or alanine substitution variants to sites of DNA double - strand breaks induced by mCherry - LacR - FokI in U2OS 2 - 6 - 3 cells . GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry - labeled LacR - FokI focus and the average nuclear intensity . Bars represent means ( n = 103 , 136 , 159 , 151 , 139 , 145 , 140 for EV , WT , S131A , W132A , R166A , N201A , D216A from 3 biologically independent experiments ) . Analysis was performed by Kruskal - Wallis test followed by Dunn ' s multiple comparisons against empty vector and WT controls . * * * * : P < 0 . 0001 . ns : P > 0 . 05 .

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

Quantification of LacR-FokI assay measuring recruitment of eGFP-SHLD3C wild-type (WT) or alanine substitution variants to sites of DNA double-strand breaks induced by mCherry-LacR-FokI in U2OS 2-6-3 cells. GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry-labeled LacR-FokI focus and the average nuclear intensity. Bars represent means (n = 103, 136, 159, 151, 139, 145, 140 for EV, WT, S131A, W132A, R166A, N201A, D216A from 3 biologically independent experiments). Analysis was performed by Kruskal-Wallis test followed by Dunn's multiple comparisons against empty vector and WT controls. ****: P < 0.0001. ns: P > 0.05.


Representative micrographs of immunofluorescence experiments ( from 3 biologically independent experiments ) analyzing localization of SHLD3 to 53BP1 bodies in RPE SHLD3 - KO cells . 3xFLAG - SHLD3 - complemented RPE SHLD3 - KO cells were treated with 200 nM aphidicolin ( Aphi ) for 24 h and processed for immunofluorescence microscopy using antibodies against 53BP1 , FLAG , and cyclin A . 53BP1 bodies are defined as distinct foci visible in cyclin A - negative cells . Quantification of FLAG and 53BP1 colocalization from A performed in CellProfiler . Pearson ' s correlation coefficients ( PCC ) were calculated for pixels within each cyclin A - negative , 53BP1 body - positive nucleus between the 53BP1 and FLAG channels . Each point represents the PCC value of an individual nucleus . Bars represent means . ( n = 120 , 130 for EV , SHLD3 from three biologically independent experiments ) . Analysis was performed using two - tailed Mann - Whitney test . * * * * : P < 0 . 0001 .

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

Representative micrographs of immunofluorescence experiments (from 3 biologically independent experiments) analyzing localization of SHLD3 to 53BP1 bodies in RPE SHLD3-KO cells. 3xFLAG-SHLD3-complemented RPE SHLD3-KO cells were treated with 200 nM aphidicolin (Aphi) for 24 h and processed for immunofluorescence microscopy using antibodies against 53BP1, FLAG, and cyclin A. 53BP1 bodies are defined as distinct foci visible in cyclin A-negative cells. Quantification of FLAG and 53BP1 colocalization from A performed in CellProfiler. Pearson's correlation coefficients (PCC) were calculated for pixels within each cyclin A-negative, 53BP1 body-positive nucleus between the 53BP1 and FLAG channels. Each point represents the PCC value of an individual nucleus. Bars represent means. (n = 120, 130 for EV, SHLD3 from three biologically independent experiments). Analysis was performed using two-tailed Mann-Whitney test. ****: P < 0.0001.


Representative micrographs of immunofluorescence experiments analyzing localization of FLAG - SHLD3 alanine substitution variants stably expressed in RPE SHLD3 - KO cells by lentivirus transduction . Cells were treated with 200 nM Aphi for 24 hours and processed for immunofluorescence microscopy using antibodies raised against 53BP1 , FLAG , and cyclin A . 53BP1 bodies are defined as distinct foci visible in cyclin A - negative cells .

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

Representative micrographs of immunofluorescence experiments analyzing localization of FLAG-SHLD3 alanine substitution variants stably expressed in RPE SHLD3-KO cells by lentivirus transduction. Cells were treated with 200 nM Aphi for 24 hours and processed for immunofluorescence microscopy using antibodies raised against 53BP1, FLAG, and cyclin A. 53BP1 bodies are defined as distinct foci visible in cyclin A-negative cells.


Quantification of the percentage of cells containing the indicated number of 53BP1 bodies in RPE WT and SHLD3 - KO cells with or without 24 - hour 200 nM Aphi treatment . Bars represent means ± s . d . ( n = 3 biologically independent experiments with ≥ 30 nuclei imaged each ) .

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

Quantification of the percentage of cells containing the indicated number of 53BP1 bodies in RPE WT and SHLD3-KO cells with or without 24-hour 200 nM Aphi treatment. Bars represent means ± s.d. (n = 3 biologically independent experiments with ≥ 30 nuclei imaged each).


Left : Representation of the region of the RIF1 N - terminal HEAT repeats that is predicted to bind the SHLD3 eIF4E - like domain . Residues 1 - 173 are highlighted . Right : Quantification of LacR / LacO assay measuring eGFP - SHLD3 recruitment to LacO arrays in U2OS 2 - 6 - 3 cells by the indicated truncated mCherry - LacR - RIF1N variants . GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry - labeled LacR - RIF1N focus and the average nuclear intensity . Bars represent means ( n = 148 , 123 , 122 for EV , 1 - 567 , 174 - 567 from 3 biologically independent experiments ) . Analysis was performed using Mann - Whitney two - tailed test . * * * * : P < 0 . 001 .

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

Left: Representation of the region of the RIF1 N-terminal HEAT repeats that is predicted to bind the SHLD3 eIF4E-like domain. Residues 1-173 are highlighted. Right: Quantification of LacR/LacO assay measuring eGFP-SHLD3 recruitment to LacO arrays in U2OS 2-6-3 cells by the indicated truncated mCherry-LacR-RIF1N variants. GFP intensities are presented as a ratio between the average fluorescence intensity within the mCherry-labeled LacR-RIF1N focus and the average nuclear intensity. Bars represent means (n = 148, 123, 122 for EV, 1-567, 174-567 from 3 biologically independent experiments). Analysis was performed using Mann-Whitney two-tailed test. ****: P < 0.001.


Whole cell extracts of High Five insect cells individually expressing the FLAG - SHLD3C ( residues 126 - 250 ) and the indicated alanine substitution Strep - RIF1 ( residues 1 - 980 ) variants through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for the Strep or FLAG epitopes . The results are representative of two biologically independent experiments . IB : immunoblot . IP : immunoprecipitation .

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

Whole cell extracts of High Five insect cells individually expressing the FLAG-SHLD3C (residues 126-250) and the indicated alanine substitution Strep-RIF1 (residues 1-980) variants through baculovirus infection were combined and subjected to FLAG immunoprecipitation or streptactin pulldown and immunoblotted for the Strep or FLAG epitopes. The results are representative of two biologically independent experiments. IB: immunoblot. IP: immunoprecipitation.


Representative micrographs of UV laser microirradiation experiments measuring DNA damage recruitment of REV7 in U2OS 2 - 6 - 3 cells with endogenously mutated RIF1 . DNA damage was induced in U2OS 2 - 6 - 3 cells through irradiation in the form of linear stripes and analyzed by immunofluorescence microscopy with RIF1 and REV7 antibodies . Quantification of UV laser microirradiation immunofluorescence experiment measuring DNA damage recruitment of REV7 in U2OS 2 - 6 - 3 cells with endogenously mutated RIF1 . REV7 immunofluorescence intensities are presented as a ratio between the average fluorescence intensity within the RIF1 - labeled irradiation stripe and the average nuclear intensity . Only nuclei containing RIF1 stripes are quantified . Bars represent means ( n = 206 , 215 for RIF1WT , RIF1D28N from 2 biologically independent experiments ) . Analysis was performed using Welch ' s t - test . * * * * : P < 0 . 0001 .

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

Representative micrographs of UV laser microirradiation experiments measuring DNA damage recruitment of REV7 in U2OS 2-6-3 cells with endogenously mutated RIF1. DNA damage was induced in U2OS 2-6-3 cells through irradiation in the form of linear stripes and analyzed by immunofluorescence microscopy with RIF1 and REV7 antibodies. Quantification of UV laser microirradiation immunofluorescence experiment measuring DNA damage recruitment of REV7 in U2OS 2-6-3 cells with endogenously mutated RIF1. REV7 immunofluorescence intensities are presented as a ratio between the average fluorescence intensity within the RIF1-labeled irradiation stripe and the average nuclear intensity. Only nuclei containing RIF1 stripes are quantified. Bars represent means (n = 206, 215 for RIF1WT, RIF1D28N from 2 biologically independent experiments). Analysis was performed using Welch's t-test. ****: P < 0.0001.


Quantification of class switch recombination in stimulated CH12F3 Shld3 - / - cells stably expressing the indicated FLAG - SHLD3 variants through lentiviral transduction . Bars represent means ± s . d . , n = 3 biologically independent experiments . Analysis was performed using one - way ANOVA followed by Dunnett ' s multiple comparisons test against Shld3 - / - cells complemented with wild - type SHLD3 . * * : P < 0 . 01 , * : P < 0 . 05 , ns : P > 0 . 05 .

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

Quantification of class switch recombination in stimulated CH12F3 Shld3-/- cells stably expressing the indicated FLAG-SHLD3 variants through lentiviral transduction. Bars represent means ± s.d., n = 3 biologically independent experiments. Analysis was performed using one-way ANOVA followed by Dunnett's multiple comparisons test against Shld3-/- cells complemented with wild-type SHLD3. **: P < 0.01, *: P < 0.05, ns: P > 0.05.


Competitive growth assay of RPE BRCA1 - KO SHLD3 - KO cells complemented with 3xFLAG - SHLD3 variants . Growth of the indicated eGFP - expressing cells were compared to mCherry - expressing RPE BRCA1 - KO SHLD3 - KO cells transduced with empty vector after mixing 1 : 1 in 24 - well plates . Plates were imaged 1 , 5 , 9 , and 13 days after plating . Dotted black lines represent an eGFP : mCherry ratio of 1 . Points represent means ± s . d . ( n = 3 biologically independent experiments ) . Analysis was performed using a one sample t - test against a hypothetical eGFP : mCherry ratio value of 1 for the day 13 time point . * * : P < 0 . 01 , ns : P > 0 . 05 .

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

Competitive growth assay of RPE BRCA1-KO SHLD3-KO cells complemented with 3xFLAG-SHLD3 variants. Growth of the indicated eGFP-expressing cells were compared to mCherry-expressing RPE BRCA1-KO SHLD3-KO cells transduced with empty vector after mixing 1:1 in 24-well plates. Plates were imaged 1, 5, 9, and 13 days after plating. Dotted black lines represent an eGFP:mCherry ratio of 1. Points represent means ± s.d. (n = 3 biologically independent experiments). Analysis was performed using a one sample t-test against a hypothetical eGFP:mCherry ratio value of 1 for the day 13 time point. **: P < 0.01, ns: P > 0.05.


( B ) IHC for CDC50A and RIM1 on cultured neurons at DIV21 . The left panel shows CDC50A is expressed in both neuronal soma and processes . The right panel exhibits CDC50A colocalize to pre - synapses alongside neuronal processes . MAP2 , Microtubule - associated protein 2 , was used to identify neuronal cells and trace dendritic processes . RIM1 is an active zone protein , which marks pre - synapses . Left scale bar = 50 µm ; right scale bar = 2 µm . ( C ) Quantification of the percentage of RIM1 + pre - synapses colocalizing to CDC50A . N = 3 for biological replicates .

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

(B) IHC for CDC50A and RIM1 on cultured neurons at DIV21. The left panel shows CDC50A is expressed in both neuronal soma and processes. The right panel exhibits CDC50A colocalize to pre-synapses alongside neuronal processes. MAP2, Microtubule-associated protein 2, was used to identify neuronal cells and trace dendritic processes. RIM1 is an active zone protein, which marks pre-synapses. Left scale bar = 50 µm; right scale bar = 2 µm. (C) Quantification of the percentage of RIM1+ pre-synapses colocalizing to CDC50A. N = 3 for biological replicates.


( E ) IHC for CDC50A and vGluT2 in the developmental dLGN at P14 , P21 and P30 . The left panels show the overview of immunostaining in the dLGN , scale bar = 200 µm . The right panels are the zoomed - in images , scale bar = 5 µm . Arrows indicate the colocalized CDC50A and vGluT2 . ( F ) Quantification of the percentage of CDC50A + / vGlut2 + synapses . The result shows that a significantly higher percentage of CDC50A + / vGlut2 + synapses at P21 and P30 than P14 . N = 6 for biological replicates .

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

(E) IHC for CDC50A and vGluT2 in the developmental dLGN at P14, P21 and P30. The left panels show the overview of immunostaining in the dLGN, scale bar = 200 µm. The right panels are the zoomed-in images, scale bar = 5 µm. Arrows indicate the colocalized CDC50A and vGluT2. (F) Quantification of the percentage of CDC50A+/vGlut2+ synapses. The result shows that a significantly higher percentage of CDC50A+/vGlut2+ synapses at P21 and P30 than P14. N = 6 for biological replicates.


( C ) IHC for CDC50A . CDC50A protein intensities were reduced after neuronal inactivation ( day 7 ) , and was recovered after removing neuronal inhibitors ( day 13 ) . Scale bar , 10 µm . ( D ) Relative CDC50A intensities of neurons . N = 5 for biological replicates , mean ± SD , * p < 0 . 5 , * * * * p < 0 . 0001 by one - way ANOVA with Holm - Sidak ' s multiple comparisons test .

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(C) IHC for CDC50A. CDC50A protein intensities were reduced after neuronal inactivation (day 7), and was recovered after removing neuronal inhibitors (day 13). Scale bar, 10 µm. (D) Relative CDC50A intensities of neurons. N = 5 for biological replicates, mean ± SD, * p < 0.5, **** p < 0.0001 by one-way ANOVA with Holm-Sidak's multiple comparisons test.


( E ) CDC50A expression at RGC inputs after 5 - day consecutive TTX treatment . Scale bar , 10 µm . ( F ) Quantification of the percentage of CDC50A + / vGlut2 + synapses . N = 5 for biological replicates , mean ± SD , * * * * p < 0 . 0001 by Student ' s test .

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(E) CDC50A expression at RGC inputs after 5-day consecutive TTX treatment. Scale bar, 10 µm. (F) Quantification of the percentage of CDC50A+/vGlut2+ synapses. N = 5 for biological replicates, mean ± SD, **** p < 0.0001 by Student's test.


( A ) Immunostaining of CDC50A on Cdc50a knockdown neurons and controls . Accell Cdc50a siRNA pool was used to knock down Cdc50a in neurons for 7 days . CDC50A expression alongside neuronal processed was reduced after Cdc50a siRNA treatment . Scale bar , 10 µm .

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(A) Immunostaining of CDC50A on Cdc50a knockdown neurons and controls. Accell Cdc50a siRNA pool was used to knock down Cdc50a in neurons for 7 days. CDC50A expression alongside neuronal processed was reduced after Cdc50a siRNA treatment. Scale bar, 10 µm.


( D ) IHC of Cdc50a - RNAi neurons exhibited pSIVA colocalized with synapsin I , a general synapse marker . The dotted square was zoomed - in shown on the right side . Arrows indicated where pSIVA colocalized with synapsin I . Scale bar , 5 µm .

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

(D) IHC of Cdc50a-RNAi neurons exhibited pSIVA colocalized with synapsin I, a general synapse marker. The dotted square was zoomed-in shown on the right side. Arrows indicated where pSIVA colocalized with synapsin I. Scale bar, 5 µm.