B , C Aligned pair - end read data were stratified into two classes , ( B ) 122 - 137 bp ( C ) 162 - 188 bp for pl - iPSC NPC . Plots show average distribution of nucleosomes centred at surrounding RE1 site ( n = 871 ) . Peaks correspond to NRSF / , nucleosomes in B C respectively .

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B, C Aligned pair-end sequence read data were stratified into two size classes, (B) 122-137 bp and (C) 162-188 bp for pl-iPSC and NPC. Plots show the average distribution of nucleosomes centred at and surrounding the RE1 site (n=871). Peaks correspond to the NRSF/REST complex, and nucleosomes in B and C respectively.


D Cluster of nucleosome positioning centred on RE1 site ± 300 bp . RE1 sites were clustered on 122 - 137 bp mid - point read values ( NRSF / ) from pl - iPSC , shown as a frequency distribution in upper left panel . frequency distribution data for same sites was plotted using 122 - 137 bp data from NPC ( upper right panel ) 162 - 188 bp data ( nucleosomes ; bottom panels ) for strongest ( solid ) weakest ( dotted ) clusters .

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D Cluster analysis of nucleosome positioning centred on the RE1 site ± 300 bp. RE1 sites were clustered based on the 122-137 bp mid-point sequence read values (NRSF/REST protein complex) from pl-iPSC, shown as a frequency distribution in upper left panel. The frequency distribution data for the same sites was plotted using 122-137 bp data from NPC (upper right panel) and 162-188 bp data (nucleosomes; bottom panels) for the strongest (solid line) and weakest (dotted line) clusters.


A Average frequency distribution for read mid - point data in range of 122 - 137 bp centred on CTCF binding site ( n = 9516 ) . This shows a distinctive peak corresponding to CTCF , which is present in pl - iPSC , but reduced in NPC . B Average frequency distribution for read mid - point data centred on CTCF binding site in range of 162 - 188 bp , corresponding to larger nucleosome footprints , for pl - iPSC NPC . This shows that pattern of nucleosome positioning is independent of amount CTCF . Although positioned nucleosomes are retained flanking CTCF sites , their positions are shifted closer to CTCF site their spacing is altered . C Average frequency distribution for read mid - point data centred on CTCF site for pl - iPSC ( 162 - 188 bp ) K562 ( total MNase - seq data ) . Positioned nucleosomes are retained flanking CTCF sites in K562 , but with shifted positions altered spacing .

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

A Average frequency distribution for sequence read mid-point data in the size range of 122-137 bp centred on the CTCF binding site (n=9516). This shows a distinctive peak corresponding to the CTCF protein complex, which is present in pl-iPSC, but reduced in NPC. B Average frequency distribution for sequence read mid-point data centred on the CTCF binding site in the size range of 162-188 bp, corresponding to larger nucleosome footprints, for pl-iPSC and NPC cells. This shows that the pattern of nucleosome positioning is independent of the amount CTCF protein complex. Although positioned nucleosomes are retained flanking CTCF sites, their positions are shifted closer to the CTCF site and their spacing is altered. C Average frequency distribution for sequence read mid-point data centred on the CTCF site for pl-iPSC (162-188 bp) and K562 (total MNase-seq data) cells. Positioned nucleosomes are retained flanking CTCF sites in K562 cells, but with shifted positions and altered spacing.


D Average frequency distribution for read mid - point data in range of 122 - 137 bp for pl - iPSC NPC centred on 2 , 522 CTCF binding sites identified from NPC specific ChIP - seq data . A distinctive peak corresponding to CTCF is present in NPC state , but reduced to background in pl - iPSC . E Average frequency distribution for read mid - point data centred on CTCF binding sites in range of 162 - 188 bp , corresponding to larger nucleosome footprints , for pl - iPSC NPC centred on 2 , 522 CTCF binding sites identified from NPC specific ChIP - seq data . Nucleosome arrays are present in both states , independent of CTCF . Nucleosomes in CTCF sites in pl - iPSC tend to position closer to CTCF site than in NPC . data derived from unique NPC CTCF sites has greater noise due to smaller number of sites analysed .

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D Average frequency distribution for sequence read mid-point data in the size range of 122-137 bp for pl-iPSC and NPC cells centred on the 2,522 CTCF binding sites identified from NPC specific ChIP-seq data. A distinctive peak corresponding to the CTCF protein complex is present in the NPC state, but reduced to background levels in pl-iPSC. E Average frequency distribution for sequence read mid-point data centred on the CTCF binding sites in the size range of 162-188 bp, corresponding to larger nucleosome footprints, for pl-iPSC and NPC centred on the 2,522 CTCF binding sites identified from NPC specific ChIP-seq data. Nucleosome arrays are present in both cell states, independent of the CTCF protein complex. Nucleosomes in the CTCF sites in pl-iPSC tend to position closer to the CTCF site than in NPC. The data derived from the unique NPC CTCF sites has greater noise due to the smaller number of sites analysed.


( a ) HACE1 mRNA expression is markedly increased in ischaemic dilated cardiomyopathy ( ICM DCM ) heart failure ( HF ) heart tissue compared with non - failing hearts ( NF ) . Expression were normalized to HPRT GAPDH mRNAs . n = 15 per group . Control , non - failing heart .

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(a) HACE1 mRNA expression is markedly increased in ischaemic and dilated cardiomyopathy (ICM and DCM) heart failure (HF) patient heart tissue compared with non-failing hearts (NF). Expression levels were normalized to HPRT and GAPDH mRNAs. n=15 per group. Control, non-failing heart.


( b ) Elevated Hace1 mRNA expression in sTAC mousehearts compared with sham controls , expression were normalized to Hprt Gapdh mRNAs . n = 6 - 9 per group , P = 0 . 004 ( unpaired two - Student ' s t - ) .

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(b) Elevated Hace1 mRNA expression in sTAC mousehearts compared with sham controls, expression levels were normalized to Hprt and Gapdh mRNAs. n=6-9 per group, P=0.004 (unpaired two-tailed Student's t-tests).


( c - e ) Hace1 − / − WT mice were subjected to sTAC or sham surgery analysed 4 days after operation . Echocardiography measurement of ( c ) LV fractional shortening ( FS = ( LVEDD − LVESD ) / LVEDD × 100 % ) ; ( d ) LV end - diastolic dimension ( LVEDD ) ( mm ) ( e ) LV end - systolic dimension ( LVESD ) ( mm ) are shown . n = 6 - 8 for each group .

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(c-e) Hace1−/− and WT mice were subjected to sTAC or sham surgery and analysed 4 days after the operation. Echocardiography measurement of (c) LV fractional shortening (FS=(LVEDD−LVESD)/LVEDD × 100%); (d) LV end-diastolic dimension (LVEDD) (mm) and (e) LV end-systolic dimension (LVESD) (mm) are shown. n=6-8 for each group.


( f ) Mortality rate of WT Hace1 − / − mice after sTAC is shown by Kaplan - Meier plots as survival proportion of each cohort of mice . Survival incidence in mice over indicated follow - up period is shown as of number of mice survived / total mice analysed for each group . This represents 60 % ( 12 / 20 ) for Hace1 − / − sTAC mice , 84 % ( 16 / 19 ) for Hace1 + / + sTAC mice 100 % ( 12 / 12 ) for both Hace1 − / − Hace1 + / + sham mice on day 4 ; P value as indicated ( log - rank χ2 - ) .

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(f) Mortality rate of WT and Hace1−/− mice after sTAC is shown by Kaplan-Meier plots as the survival proportion of each cohort of mice. Survival incidence in mice over the indicated follow-up period is shown as the ratio of the number of mice survived/total mice analysed for each group. This represents 60% (12/20) for Hace1−/− sTAC mice, 84% (16/19) for Hace1+/+ sTAC mice and 100% (12/12) for both Hace1−/− and Hace1+/+ sham mice on day 4; P value as indicated (log-rank χ2-test).


( g ) Heart / ratios ( HW / BW ) ( h ) lung / ( LW / BW ) of WT Hace1 − / − mice 4 days after sTAC or sham surgery . n = 15 - 21 for each group .

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(g) Heart weight/body weight ratios (HW/BW) and (h) lung weight/body weight ratio (LW/BW) of WT and Hace1−/− mice 4 days after sTAC or sham surgery. n=15-21 for each group.


( i ) Representative haematoxylin eosin transverse heart cross - sections showing myocyte cross - sectional ( scale bars , 20 μm ) .

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(i) Representative haematoxylin and eosin staining transverse heart cross-sections showing myocyte cross-sectional area (scale bars, 20 μm).


( j ) Real - time RT - PCR analyses reveal a significant increase in expression of atrial natriuretic peptide gene ( Nppa ) , a clinical biomarker for heart hypertrophy , in Hace1 − / − sTAC heart ; expression were normalized to Hprt Gapdh mRNAs . n = 6 - 9 per group . In panels , error bars represent s . e . m . , one - way of variance were used to calculate P - values .

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(j) Real-time RT-PCR analyses reveal a significant increase in the expression of the atrial natriuretic peptide gene (Nppa), a clinical biomarker for heart hypertrophy, in Hace1−/− sTAC heart; expression levels were normalized to Hprt and Gapdh mRNAs. n=6-9 per group. In all panels, error bars represent s.e.m., one-way analysis of variance were used to calculate P-values.


Representative immunohistochemistry for ( a ) ubiquitinated ( brown ) ; ( b ) p62 ( brown ) ( c ) GFP - LC3 ( brown ) in paraffin sections of heart revealing increased accumulation of p62 , ubiquitinated LC3 in Hace1 − / − sTAChearts . Hearts from three mice in each group were analysed ( scale bars , 10 μm ) .

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Representative immunohistochemistry staining for (a) ubiquitinated protein (brown); (b) p62 (brown) and (c) GFP-LC3 (brown) in paraffin sections of heart revealing increased accumulation of p62, ubiquitinated protein and LC3 in Hace1−/− sTAChearts. Hearts from three mice in each group were analysed (scale bars, 10 μm).


Representative immunoblot for ( d , e ) Ub - conjugated , ( d , f ) p62 ( g , h ) LC3 in heart extracts ( n = 3 per group ) , Gapdh were used as a loading control .

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Representative immunoblot and quantification for (d,e) Ub-conjugated proteins, (d,f) p62 and (g,h) LC3 in heart extracts (n=3 per group), Gapdh were used as a loading control.


( i ) Increased Ub proteasome activity in Hace1 − / − sTAC hearts as measured by 20S proteasome activity ( n = 4 - 6 per group ) . In panels , error bars represent s . e . m . , P value between KO WT sTAC as indicated ( one - way of variance ) .

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(i) Increased Ub proteasome activity in Hace1−/− sTAC hearts as measured by 20S proteasome activity assay (n=4-6 per group). In all panels, error bars represent s.e.m., P value between KO and WT sTAC as indicated (one-way analysis of variance).


( a ) Representative confocal microscopy showing HACE1 ( green ) localizing to Ub + ( red ) induced by puromycin treatment in HA - HACE1 MEF . Arrows indicate co - localized . Scale bar , 10 μm .

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(a) Representative confocal microscopy images showing HACE1 (green) localizing to the Ub+ protein aggregates (red) induced by puromycin treatment in HA-HACE1 MEF. Arrows indicate co-localized puncta. Scale bar, 10 μm.


( b ) Representative confocal of Hace1 − / − Hace1 + / + NCM treated with puromycin for 4 h ( puromycin ) , puromycin followed by 8 h recover ( puromycin + recover ) . Vehicle ( PBS ) - treated were used as control . Hace1 deficiency in cardiomyocytes ( Hace1 − / − NCM ) blocks ubiquitinated aggregate degradation . Scale bars , 10 μm .

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(b) Representative confocal micrographs of Hace1−/− and Hace1+/+ NCM treated with puromycin for 4 h (puromycin), puromycin followed by 8 h recover (puromycin+recover). Vehicle (PBS)-treated cells were used as control. Hace1 deficiency in cardiomyocytes (Hace1−/− NCM) blocks ubiquitinated protein aggregate degradation. Scale bars, 10 μm.


( c , d ) WT Hace1 − / − MEF as well as Hace1 − / − MEF stably transfected with WT HACE1 C876S - mutant HACE1were subjected to clearance by pulsing with puromycin for 4 h to induce aggregate formation followed by an 8 - h chase to monitor for aggregate clearance . were fixed stained for ubiquitinated to visualize . Vehicle ( PBS ) - treated MEFs were used as control . ( c ) Representative confocal of used for of efficiency of clearance in d ( of that cleared by 8 h after puromycin removal ) . At least 10 from each group were used for calculation , error bars represent s . e . m . , P0 . 001 ( one - way of variance ) . Scale bars , 10 μm .

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(c,d) WT and Hace1−/− MEF as well as Hace1−/− MEF stably transfected with WT HACE1 and C876S-mutant HACE1were subjected to protein aggregates clearance assay by pulsing with puromycin for 4 h to induce protein aggregate formation followed by an 8-h chase to monitor for aggregate clearance. Cells were fixed and stained for ubiquitinated proteins to visualize aggregates. Vehicle (PBS)-treated MEFs were used as control. (c) Representative confocal micrographs of images used for quantification of the efficiency of aggregates clearance in d (percentage of cells that cleared all aggregates by 8 h after puromycin removal). At least 10 images from each group were used for the calculation, error bars represent s.e.m., P0.001 (one-way analysis of variance). Scale bars, 10 μm.


( a ) Diagrams representing GFP - tagged WT HACE1 mutant HACE1 ( HACE1Δ HECT HACE1Δ ANK ) constructs transiently transfected into Hace1 − / − MEF for aggregate clearance . ( b - d ) Representative confocal of ( b ) WT , ( c ) ΔHECT ( d ) ΔANK MEF subjected to aggregate clearance by pulsing with puromycin for 4 h to induce aggregate formation followed by an 8 - h chase to monitor for aggregate clearance . Vehicle - treated MEFs were used as control . Scale bars , 10 μm .

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(a) Diagrams representing GFP-tagged WT HACE1 and mutant HACE1 (HACE1Δ HECT and HACE1Δ ANK) constructs transiently transfected into Hace1−/− MEF for protein aggregate clearance assay. (b-d) Representative confocal micrographs of (b) WT, (c) ΔHECT and (d) ΔANK MEF subjected to protein aggregate clearance assay by pulsing with puromycin for 4 h to induce protein aggregate formation followed by an 8-h chase to monitor for aggregate clearance. Vehicle-treated MEFs were used as control. Scale bars, 10 μm.


( a ) microscopy showing HACE1 ( green ) co - aggregating with p62 ( red ) in perinuclear space on puromycin treatment ( 8 h ) in Hace1 − / − NCM transiently expressing HACE1 - GFP . p62 was visualized by immunofluorescent . Arrows indicate co - localized .

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(a) Fluorescence microscopy images showing HACE1 (green) co-aggregating with p62 (red) in the perinuclear space on puromycin treatment (8 h) in Hace1−/− NCM transiently expressing HACE1-GFP. p62 was visualized by immunofluorescent staining. Arrows indicate co-localized puncta.


( b ) Representative confocal showing HACE1 ( red ) localizes to LC3 + ( green ) induced by MG132 treatment ( 8 h ) in Hace1 − / − GFP - Lc3Tg NCM transiently expressing HACE1 - RFP . Arrows indicate co - localized .

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(b) Representative confocal images showing HACE1 (red) localizes to LC3+ puncta (green) induced by MG132 treatment (8 h) in Hace1−/−GFP-Lc3Tg NCM cells transiently expressing HACE1-RFP. Arrows indicate co-localized puncta.


( c ) NCMs from Hace1 + / + Lc3Tg Hace1 − / − Lc3Tg newborn pups were treated with or without MG132 for 8 h lysates were subjected to western blot with anti - GFP antibodies . Gapdh was used as a loading control .

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(c) Primary NCMs from Hace1+/+Lc3Tg and Hace1−/−Lc3Tg newborn pups were treated with or without MG132 for 8 h and the cell lysates were subjected to western blot with anti-GFP antibodies. Gapdh was used as a loading control.


( d ) microscopy showing co - localization of HACE1 ( green ) with Lamp1 ( red ) on MG132 treatment ( 8 h ) in Hace1 − / − NCM transiently expressing HACE1 - GFP . Lamp1 was visualized by immunofluorescent . Scale bar , 10 μm in .

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(d) Fluorescence microscopy images showing co-localization of HACE1 (green) with Lamp1 (red) on MG132 treatment (8 h) in Hace1−/−NCM cells transiently expressing HACE1-GFP. Lamp1 was visualized by immunofluorescent staining. Scale bar, 10 μm in all images.


( a , b ) NCMs from WT Hace1 − / − newborn pups stably transfected with WT HACE1 C876S - mutant HACE1 , were transiently transfected with a tandem tagged mRFP - GFP - LC3 reporter ( tfLC3 ) plasmid , followed by 8 h treatment with MG132 or MG132 plus Baf in last 4 h , were subjected to confocal microscopy . DMSO treatment only was used in control group . ( a , c ) Representative confocal of used for of of yellow dots in b d . At least 10 from each group were used for calculation ; error bars represent s . e . m . , P - value as indicated ( one - way ANAVO ) . Scale bars , 10 μm . Autophagosomes show both GFP mRFP - LC3 signals ( yellow ) while autolysosomes exhibit mRFP - L3 signals only ( red ) .

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(a,b) Primary NCMs from WT and Hace1−/− newborn pups stably transfected with WT HACE1 and C876S-mutant HACE1, were transiently transfected with a tandem tagged mRFP-GFP-LC3 reporter (tfLC3) plasmid, followed by 8 h treatment with MG132 or MG132 plus Baf in the last 4 h, and were subjected to confocal microscopy. DMSO treatment only was used in the control group. (a,c) Representative confocal micrographs of images used for quantification of the percentage of yellow dots in b and d. At least 10 images from each group were used for the calculation; error bars represent s.e.m., P-value as indicated (one-way ANAVO). Scale bars, 10 μm. Autophagosomes show both GFP and mRFP-LC3 signals (yellow) while autolysosomes exhibit mRFP-L3 signals only (red).


( c , d ) WT Hace1 − / − MEF , as well as Hace1 − / − MEF stably transfected with WT HACE1 C876S - mutant HACE1 , were transiently transfected with a tandem tagged mRFP - GFP - LC3 reporter ( tfLC3 ) plasmid , followed by 8 h treatment with MG132 or MG132 plus Baf in last 4 h , were subjected to confocal microscopy . DMSO treatment only was used in control group . ( a , c ) Representative confocal of used for of of yellow dots in b d . At least 10 from each group were used for calculation ; error bars represent s . e . m . , P - value as indicated ( one - way ANAVO ) . Scale bars , 10 μm . Autophagosomes show both GFP mRFP - LC3 signals ( yellow ) while autolysosomes exhibit mRFP - L3 signals only ( red ) .

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(c,d) WT and Hace1−/− MEF, as well as Hace1−/− MEF stably transfected with WT HACE1 and C876S-mutant HACE1, were transiently transfected with a tandem tagged mRFP-GFP-LC3 reporter (tfLC3) plasmid, followed by 8 h treatment with MG132 or MG132 plus Baf in the last 4 h, and were subjected to confocal microscopy. DMSO treatment only was used in the control group. (a,c) Representative confocal micrographs of images used for quantification of the percentage of yellow dots in b and d. At least 10 images from each group were used for the calculation; error bars represent s.e.m., P-value as indicated (one-way ANAVO). Scale bars, 10 μm. Autophagosomes show both GFP and mRFP-LC3 signals (yellow) while autolysosomes exhibit mRFP-L3 signals only (red).


( a , b ) Hace1 − / − MEF were stably transfected with HA - tagged WT HACE1 ( HA - HACE1 ) empty vector MSCV . ( a ) Immunoblot for HA shows stable expression of HA - tagged HACE1 in HA - HACE1MEF lysate . ( b ) HA - HACE1MEF control MSCVMEF lysates were subjected to Co - IP with anti - HA antibody coomassie blue shows a couple of distinct bands only detected in HA - HACE1MEFIP pull - down as indicated by red arrows .

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(a,b) Hace1−/− MEF cells were stably transfected with HA-tagged WT HACE1 (HA-HACE1) and empty vector MSCV. (a) Immunoblot for HA shows stable expression of HA-tagged HACE1 in HA-HACE1MEF cell lysate. (b) HA-HACE1MEF and control MSCVMEF cell lysates were subjected to Co-IP with anti-HA antibody and coomassie blue staining shows a couple of distinct bands only detected in HA-HACE1MEFIP pull-down as indicated by red arrows.


( c - f ) Identification of HACE1 interacting by MS of Co - IP products . ( c ) Hierarchical clustering ( heat ) map demonstrating clustering of on their expression intensities in HA - HACE1MEF control MSCVMEFHA - IP pull - down products . To left of heat map , selected annotations found to be enriched in clusters are shown . n = 5 for HA - HACE1 group n = 4 for control MSCV group . ( d ) Pie chart shows enriched representative HACE1 - interacting according to molecular function by GO term . ( e ) Selected HACE1 - interacting identified by HA - IP MS were evaluated by western blot with indicated antibodies ( IB ) . HA - HACE1 denotes HA - IP pull - down from HA - HACE1MEF ; MSCV denotes HA - IP pull - down from control MSCVMEF ; input indicates HA - HACE1MEF lysate before IP . ( f ) HA - HACE1MEF control MSCVMEF lysates were subjected to co - IP with an anti - Unc - 45b antibody pull - down were analysed by western blot with an anti - HA antibody .

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(c-f) Identification of HACE1 interacting proteins by MS analysis of Co-IP products. (c) Hierarchical clustering (heat) map demonstrating the clustering of proteins based on their expression intensities in HA-HACE1MEF and control MSCVMEFHA-IP pull-down products. To the left of the heat map, selected annotations found to be enriched in the protein clusters are shown. n=5 for HA-HACE1 group and n=4 for control MSCV group. (d) Pie chart shows enriched representative HACE1-interacting proteins according to molecular function by GO term. (e) Selected HACE1-interacting proteins identified by HA-IP MS were evaluated by western blot with the indicated antibodies (IB). HA-HACE1 denotes HA-IP pull-down from HA-HACE1MEF; MSCV denotes HA-IP pull-down from control MSCVMEF; and input indicates HA-HACE1MEF lysate before IP. (f) HA-HACE1MEF and control MSCVMEF lysates were subjected to co-IP with an anti-Unc-45b antibody and the pull-down were analysed by western blot with an anti-HA antibody.


( g , h ) HA - IP pull - down products from HA - HACE1MEF ( HA - HACE1 ) Hace1 KO MSCVMEF ( MSCV ) immortalized in HA beads were further subjected to Co - Co - IP with WT mouseheart lysates , pull - down products were analysed by western blot using indicated antibodies . HACE1 those with which it interacts were still detectable ( g ) . There were also increased ubiquitinated in Co - Co - IPheart tissues ( h ) .

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(g,h) HA-IP pull-down products from HA-HACE1MEF (HA-HACE1) and Hace1 KO MSCVMEF (MSCV) immortalized in HA beads were further subjected to Co-Co-IP with WT mouseheart lysates, and the pull-down products were analysed by western blot using the indicated antibodies. HACE1 and those proteins with which it interacts were still detectable (g). There were also increased ubiquitinated proteins in the Co-Co-IPheart tissues (h).


( a - c ) Representative western blots ( d ) show increased accumulation of some of HACE1 - interacting in Hace1 − / − sTAC myocardium . n = 3 mice per group , error bars represent s . e . m . , P - value between KO WT sTAC as indicated ( one - way of variance ) . Gapdh was used as a loading control .

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(a-c) Representative western blots and (d) quantification show increased accumulation of some of the HACE1-interacting proteins in the Hace1−/− sTAC myocardium. n=3 mice per group, error bars represent s.e.m., P-value between KO and WT sTAC as indicated (one-way analysis of variance). Gapdh was used as a loading control.


( A ) lysates prepared from HeLa were solubilized with Triton X - 100 subjected to immunoprecipitation using preimmune sera ( lane 1 ) or anti - Beclin antibodies ( lanes 2 3 ) . immunoprecipitates were incubated with phosphatidylinositol , [ γ - 32P ] ATP 60 μM cold ATP in presence of Mn2 + ( lanes 1 2 ) or Mg2 + ( lane 3 ) for 5 min at 30 ° C .

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(A) Cell lysates prepared from HeLa cells were solubilized with Triton X-100 and subjected to immunoprecipitation using preimmune sera (lane 1) or anti-Beclin antibodies (lanes 2 and 3). The immunoprecipitates were incubated with phosphatidylinositol, [γ-32P]ATP and 60 μM cold ATP in the presence of Mn2+ (lanes 1 and 2) or Mg2+ (lane 3) for 5 min at 30°C.


( B ) Immunoprecipitates of anti ‐ Beclin antibodies prepared as described in ( A ) were assayed for PI 3 ‐ kinase activity in presence of wortmannin at concentrations of 0 nM ( lane 1 ) , 1 nM ( lane 2 ) , 10 nM ( lane 3 ) , 100 nM ( lane 4 ) . labeled lipids were extracted separated by thin layer chromatography , followed by detection by autoradiography using a bioimaging analyzer BAS2000 ( Fuji Film ) .

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(B) Immunoprecipitates of anti‐Beclin antibodies prepared as described in (A) were assayed for PI 3‐kinase activity in the presence of wortmannin at concentrations of 0 nM (lane 1), 1 nM (lane 2), 10 nM (lane 3), 100 nM (lane 4). The labeled lipids were extracted and separated by thin layer chromatography, followed by detection by autoradiography using a bioimaging analyzer BAS2000 (Fuji Film).


( C ) lysates prepared from HeLa were solubilized with Triton X ‐ 100 incubated with A ‐ immobilized anti ‐ Beclin ( lane 2 ) or anti ‐ PtdIns 3 ‐ kinase antibodies ( lane 4 ) . As controls , preimmune sera of respective antibodies ( lanes 1 3 ) were used . Retained were separated by SDS - PAGE detected by immunoblotting with anti ‐ PtdIns 3 ‐ kinase ( upper panels ) anti ‐ Beclin ( lower panels ) antibodies .

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(C) Cell lysates prepared from HeLa cells were solubilized with Triton X‐100 and incubated with protein A‐immobilized anti‐Beclin (lane 2) or anti‐PtdIns 3‐kinase antibodies (lane 4). As controls, preimmune sera of the respective antibodies (lanes 1 and 3) were used. Retained proteins were separated by SDS-PAGE and detected by immunoblotting with anti‐PtdIns 3‐kinase (upper panels) and anti‐Beclin (lower panels) antibodies.


( A ) Total lysates were treated with DSP ( lanes 3 , 4 , 7 8 ) or its solvent , dimethylsulfoxide ( lanes 1 , 2 , 5 6 ) , at 4 ° C for 2 h . were solubilized with SDS subjected to immunoprecipitation using A ‐ immobilized anti ‐ PtdIns 3 ‐ kinase ( PtdIns 3 ‐ K ) ( lanes 1 - 4 ) or Beclin ( lanes 5 - 8 ) antibodies . Immunoprecipitates were eluted with 1 × SDS sample buffer ( 62 . 5 mM Tris - HCl pH 6 . 8 , 2 % SDS , 10 % glycerol , a trace amount of Bromophenol blue ) . After treatment with 5 % 2 ‐ mercaptoethanol , were separated by SDS - PAGE , followed by detection by immunoblotting with anti ‐ Beclin ( upper panels ) anti ‐ PtdIns 3 ‐ kinase ( lower panels ) antibodies .

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(A) Total cell lysates were treated with DSP (lanes 3, 4, 7 and 8) or its solvent, dimethylsulfoxide (lanes 1, 2, 5 and 6), at 4°C for 2 h. Proteins were solubilized with SDS and subjected to immunoprecipitation using protein A‐immobilized anti‐PtdIns 3‐kinase (PtdIns 3‐K) (lanes 1-4) or Beclin (lanes 5-8) antibodies. Immunoprecipitates were eluted with 1× SDS sample buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, a trace amount of Bromophenol blue). After treatment with 5% 2‐mercaptoethanol, proteins were separated by SDS-PAGE, followed by detection by immunoblotting with anti‐Beclin (upper panels) and anti‐PtdIns 3‐kinase (lower panels) antibodies.


( B ) lysates prepared from HeLa were subjected to subcellular fractionation by centrifugation at 100 000 g for 1 h . ( lanes 1 3 ) supernatant ( lanes 2 4 ) fractions were subjected to SDS - PAGE , followed by immunoblotting with anti ‐ PtdIns 3 ‐ kinase ( lanes 1 2 ) anti ‐ Beclin ( lanes 3 4 ) antibodies .

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(B) Cell lysates prepared from HeLa cells were subjected to subcellular fractionation by centrifugation at 100 000 g for 1 h. The membrane (lanes 1 and 3) and supernatant (lanes 2 and 4) fractions were subjected to SDS-PAGE, followed by immunoblotting with anti‐PtdIns 3‐kinase (lanes 1 and 2) and anti‐Beclin (lanes 3 and 4) antibodies.


( C ) ( lanes 1 - 4 ; 100 000 g , 30 min , pellet ) soluble fractions ( lanes 5 - 8 ; 100 000 g , 30 min , supernatant ) prepared from HeLa were treated with DSP ( lanes 3 , 4 , 7 8 ) or dimethylsulfoxide ( lanes 1 , 2 , 5 6 ) . were solubilized with SDS subjected to immunoprecipitation using A - immobilized anti - PtdIns 3 - kinase antibodies . Immunoprecipitates were separated by SDS - PAGE under a reducing condition , followed by detection by immunoblotting with Beclin antibodies . F E indicate flow - through elute fracions , respectively .

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(C) Membrane (lanes 1-4; 100 000 g, 30 min, pellet) and soluble fractions (lanes 5-8; 100 000 g, 30 min, supernatant) prepared from HeLa cells were treated with DSP (lanes 3, 4, 7 and 8) or dimethylsulfoxide (lanes 1, 2, 5 and 6). Proteins were solubilized with SDS and subjected to immunoprecipitation using protein A-immobilized anti-PtdIns 3-kinase antibodies. Immunoprecipitates were separated by SDS-PAGE under a reducing condition, followed by detection by immunoblotting with Beclin antibodies. F and E indicate flow-through and elute fracions, respectively.


Beclin localizes to TGN . HeLa ( A , D , E F ) , BNL CL . 2 ( B ) H ‐ 4 ‐ II ‐ E ( C ) were grown on cover slips . were permeabilized with digitonin before ( C , D E ) or after ( A , B F ) fixation with 3 % paraformaldehyde . were double ‐ labeled with anti ‐ Beclin ( A - F , left panels ) either anti ‐ syntaxin 6 ( A B , middle panels ) , anti ‐ TGN38 ( C , middle panel ) , anti ‐ EEA1 ( D , middle panel ) , anti ‐ LBPA ( E , middle panel ) or anti ‐ TfR ( F , middle panel ) antibodies . In merged ( right panels ) , yellow indicates co ‐ localization . Bar , 10 μm .

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Beclin localizes to the TGN. HeLa (A, D, E and F), BNL CL.2 (B) and H‐4‐II‐E cells (C) were grown on cover slips. Cells were permeabilized with digitonin before (C, D and E) or after (A, B and F) fixation with 3% paraformaldehyde. Cells were double‐labeled with anti‐Beclin (A-F, left panels) and either anti‐syntaxin 6 (A and B, middle panels), anti‐TGN38 (C, middle panel), anti‐EEA1 (D, middle panel), anti‐LBPA (E, middle panel) or anti‐TfR (F, middle panel) antibodies. In the merged images (right panels), yellow indicates co‐localization. Bar, 10 μm.


Immunofluorescence localization of PtdIns 3 ‐ kinase . HeLa ( A C ) H ‐ 4 ‐ II ‐ E ( B ) were permeabilized , fixed , then subjected to immunostaining . were double ‐ labeled with anti ‐ PtdIns 3 ‐ kinase ( A - C , left panels ) either anti ‐ syntaxin 6 ( A , middle panel ) , anti ‐ TGN38 ( B , middle panel ) or anti ‐ LBPA ( C , middle panel ) antibodies . Bar , 10 μm .

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Immunofluorescence localization of PtdIns 3‐kinase. HeLa (A and C) and H‐4‐II‐E cells (B) were permeabilized, fixed, and then subjected to immunostaining. Cells were double‐labeled with anti‐PtdIns 3‐kinase (A-C, left panels) and either anti‐syntaxin 6 (A, middle panel), anti‐TGN38 (B, middle panel) or anti‐LBPA (C, middle panel) antibodies. Bar, 10 μm.


( A ) MEFs were transfected with GFP - tagged wild - type ( WT ) or mutant Parkin expression plasmid followed by an 18 - h treatment of CCCP . are immunostained with a Tom20 antibody to visualize mitochondria ( red ) . GFP - parkin - transfected are marked by dotted lines . Arrows indicate parkin - positive mitochondria or mitochondrial . Bar , 10 µm . ( B ) average percentages of mitochondria - free from three independent experiments from A are presented with standard deviation as error bar . * * , P < 0 .

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(A) MEFs were transfected with GFP-tagged wild-type (WT) or mutant Parkin expression plasmid followed by an 18-h treatment of CCCP. Cells are immunostained with a Tom20 antibody to visualize mitochondria (red). GFP-parkin-transfected cells are marked by dotted lines. Arrows indicate parkin-positive mitochondria or mitochondrial aggregates. Bar, 10 µm. (B) The average percentages of mitochondria-free cells from three independent experiments from A are presented with standard deviation as error bar. **, P < 0.01


( C ) MEFs were transfected treated with CCCP as described in A , followed by an immunoblotting with antibodies for cytochrome c , actin , parkin . Note that of parkin R275W R42P mutant were lower , as previously reported ( Wang et al . , 2005 ) .

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(C) MEFs were transfected and treated with CCCP as described in A, followed by an immunoblotting analysis with antibodies for cytochrome c, actin, and parkin. Note that levels of parkin R275W and R42P mutant were lower, as previously reported (Wang et al., 2005).


CCCP - induced parkin - mitochondrial aggregate formation is an intermediate step for mitophagy . ( A ) MEFs expressing WT GFP - parkin were treated with CCCP or CCCP nocodazole ( NOC , 10 µM ) for 8 , 16 , 24 h as indicated . were immunostained with anti - Tom20 to visualize mitochondria . GFP - parkin - positive are marked by dotted lines . Arrows indicate parkin - positive mitochondria or mitochondrial . Bar , 25 µm . ( B ) average percentages of with mitochondrial or without mitochondria from three independent experiments from A are presented with standard deviation as error bar . * * , P < 0 . 01 .

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CCCP-induced parkin-mitochondrial aggregate formation is an intermediate step for mitophagy. (A) MEFs expressing WT GFP-parkin were treated with CCCP or CCCP and nocodazole (NOC, 10 µM) for 8, 16, and 24 h as indicated. Cells were immunostained with anti-Tom20 to visualize mitochondria. GFP-parkin-positive cells are marked by dotted lines. Arrows indicate parkin-positive mitochondria or mitochondrial aggregates. Bar, 25 µm. (B) The average percentages of cells with mitochondrial aggregates or without mitochondria from three independent experiments from A are presented with standard deviation as error bar. **, P < 0.01.


( A ) MEFs were transfected with GFP wild - type ( WT ) , A240R , T415N mutant parkin followed by CCCP treatment for 8 h . were immunostained with antibodies for cytochrome c ( red ) ubiquitin ( blue ) . Arrows indicate mitochondrial . Bar , 10 µm . ( B ) average percentages of ubiquitin - positive mitochondrial from three independent experiments from A are presented with standard deviation as error bar .

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(A) MEFs were transfected with GFP wild-type (WT), A240R, and T415N mutant parkin followed by CCCP treatment for 8 h. Cells were immunostained with antibodies for cytochrome c (red) and ubiquitin (blue). Arrows indicate mitochondrial aggregates. Bar, 10 µm. (B) The average percentages of ubiquitin-positive mitochondrial aggregates from three independent experiments from A are presented with standard deviation as error bar.


( C D ) MEFs were transfected treated with CCCP as described in A . were subjected to fractionation to isolate crude mitochondria . mitochondrial fractions were subjected to immunoblotting using antibodies for ubiquitin , parkin , cytochrome c , Hsp90 .

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(C and D) MEFs were transfected and treated with CCCP as described in A. Cells were subjected to the fractionation to isolate crude mitochondria. Cytoplasmic and mitochondrial fractions were subjected to immunoblotting analysis using antibodies for ubiquitin, parkin, cytochrome c, and cytosolic Hsp90.


Parkin - mediated ubiquitination recruits p62 HDAC6 . MEFs were transfected with WT , A240R , T415N mutant GFP - parkin followed by CCCP treatment for 8 h . were double immunostained with cytochrome c ( red ) p62 antibody ( blue ) in A , cytochrome c ( red ) HDAC6 antibody in C . Arrows indicate mitochondrial . ( B D ) average percentages of p62 - or HDAC6 - positive mitochondrial from three independent experiments are presented with standard deviation as error bar .

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Parkin-mediated ubiquitination recruits p62 and HDAC6. MEFs were transfected with WT, A240R, and T415N mutant GFP-parkin followed by CCCP treatment for 8 h. Cells were double immunostained with cytochrome c (red) and p62 antibody (blue) in A, and cytochrome c (red) and HDAC6 antibody in C. Arrows indicate mitochondrial aggregates. (B and D) The average percentages of p62- or HDAC6-positive mitochondrial aggregates from three independent experiments are presented with standard deviation as error bar.


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( E ) Wild - type HDAC6 knockout ( KO ) MEFs were transfected with parkin - GFP or cotransfected with a Flag - tagged HDAC6 followed by CCCP treatment for 16 h as indicated . are immunostained with Tom20 ( red ) Flag ( blue ) antibodies . Bar , 25 µm .

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(E) Wild-type and HDAC6 knockout (KO) MEFs were transfected with parkin-GFP or cotransfected with a Flag-tagged HDAC6 followed by CCCP treatment for 16 h as indicated. Cells are immunostained with Tom20 (red) and Flag (blue) antibodies. Bar, 25 µm.


( F ) Wild - type MEFs were transfected with control or HDAC6 siRNA parkin - GFP , treated with or without CCCP for 16 h . lysates were subjected to immunoblotting using antibodies for Tom20 , parkin , HDAC6 , actin .

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(F) Wild-type MEFs were transfected with control or HDAC6 siRNA and parkin-GFP, and treated with or without CCCP for 16 h. Cell lysates were subjected to immunoblotting analysis using antibodies for Tom20, parkin, HDAC6, and actin.


MEFs were transfected with control or cortactin RNAi parkin - GFP , incubated with or without CCCP for 16 h as indicated . ( A ) were subjected to immunostaining with antibodies for Tom20 parkin . Bar , 10 µm .

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MEFs were transfected with control or cortactin RNAi and parkin-GFP, and incubated with or without CCCP for 16 h as indicated. (A) Cells were subjected to immunostaining with antibodies for Tom20 and parkin. Bar, 10 µm.


MEFs were transfected with control or cortactin RNAi parkin - GFP , incubated with or without CCCP for 16 h as indicated . ( B ) lysates were subjected to immunoblotting with antibodies for Tom20 , parkin , cortactin , actin .

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MEFs were transfected with control or cortactin RNAi and parkin-GFP, and incubated with or without CCCP for 16 h as indicated.(B) Cell lysates were subjected to immunoblotting with antibodies for Tom20, parkin, cortactin, and actin.


( B ) SOFG of spinal entheseal tissue Immunohistochemical of CaSR + Runx2 + in human spinal tissue . bottom panels show higher magnification of boxed in top panels . Data information : SP : spinous process ; IL : interspinous ligament ; SL : supraspinous ligament ; UIL : uncalcified interspinous ligament ; TZ : transitional zone . Data shown as mean ± SD . . * * P < 0 . 01 compared between groups . Scale bar : 100μm

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(B) SOFG staining of spinal entheseal tissue and Immunohistochemical analysis of CaSR+ and Runx2+ cells in human spinal tissue. The bottom panels show higher magnification of the boxed area in the top panels. Data information: SP: spinous process; IL: interspinous ligament; SL: supraspinous ligament; UIL: uncalcified interspinous ligament; TZ: transitional zone. Data shown as mean ± SD.. ** P < 0.01 compared between groups. Scale bar: 100μm


( B ) SOFG of spinal entheseal tissue Immunohistochemical of CaSR + Runx2 + in human spinal tissue . bottom panels show higher magnification of boxed in top panels . Data information : SP : spinous process ; IL : interspinous ligament ; SL : supraspinous ligament ; UIL : uncalcified interspinous ligament ; TZ : transitional zone . Data shown as mean ± SD . . * * P < 0 . 01 compared between groups . Scale bar :

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( C ) Quantitative of number of CaSR + Runx2 + per ( mm2 ) . n = 5 , Student ' s t - .

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(C) Quantitative analysis of the number of CaSR+ and Runx2+ cells per area (mm2). n=5, Student's t-test.


D ) RT - qPCR of expression of CaSR in human spinal tissue . n = 3 , Student ' s t - .

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D) RT-qPCR analysis of expression of CaSR in human spinal tissue. n=3, Student's t-test.


E ) Western blot of expression of CaSR in human spinal tissue .

E) Western blot analysis of expression of CaSR in human spinal tissue.


F ) Immunofluorescence of CaSR + ( Green ) Runx2 + ( Red ) in human spinal tissue . ( G ) Quantitative of CaSR + , Runx2 + CaSR + Runx2 + number per ( mm2 ) CaSR + . n = 5 , Student ' s t - . ata information : SP : spinous process ; IL : interspinous ligament ; SL : supraspinous ligament ; UIL : uncalcified interspinous ligament ; TZ : transitional zone . Data shown as mean ± SD . . * * P < 0 . 01 compared between groups . Scale bar : 100μm .

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

F) Immunofluorescence analysis of CaSR+ (Green) and Runx2+ (Red) cells in human spinal tissue. (G) Quantitative analysis of CaSR+, Runx2+ and CaSR+ Runx2+ cells number per area (mm2) and CaSR+ cell percentage. n=5, Student's t-test. ata information: SP: spinous process; IL: interspinous ligament; SL: supraspinous ligament; UIL: uncalcified interspinous ligament; TZ: transitional zone. Data shown as mean ± SD.. ** P < 0.01 compared between groups. Scale bar: 100μm.


F ) Immunofluorescence of CaSR + ( Green ) Runx2 + ( Red ) in human spinal tissue .

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

F) Immunofluorescence analysis of CaSR+ (Green) and Runx2+ (Red) cells in human spinal tissue.


H ) Immunofluorescence of CaSR + ( Green ) OCN + ( Red ) in human spinal tissue . ( I ) Quantitative of CaSR + , OCN + CaSR + OCN + number per ( mm2 ) . n = 5 , Student ' s t - . ata information : SP : spinous process ; IL : interspinous ligament ; SL : supraspinous ligament ; UIL : uncalcified interspinous ligament ; TZ : transitional zone . Data shown as mean ± SD . . * * P < 0 . 01 compared between groups . Scale bar : 100μm .

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

H) Immunofluorescence analysis of CaSR+ (Green) and OCN+ (Red) cells in human spinal tissue. (I) Quantitative analysis of CaSR+, OCN+ and CaSR+ OCN+ cells number per area (mm2). n=5, Student's t-test. ata information: SP: spinous process; IL: interspinous ligament; SL: supraspinous ligament; UIL: uncalcified interspinous ligament; TZ: transitional zone. Data shown as mean ± SD.. ** P < 0.01 compared between groups. Scale bar: 100μm.


H ) Immunofluorescence of CaSR + ( Green ) OCN + ( Red ) in human spinal tissue .

H) Immunofluorescence analysis of CaSR+ (Green) and OCN+ (Red) cells in human spinal tissue.


( A ) μCT of spine in PGIS model ( 2D 3D reconstruction ) . Arrow head shows spinal ankylosis . n = 5 per group . μCT of new bone ( red ) in hind paws ( 2D 3D reconstruction ) in DBA / 1 model compared to baseline .

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

(A) μCT images of the spine in PGIS model (2D and 3D reconstruction). Arrow head shows spinal ankylosis. n=5 per group. μCT images of new bone (red area) in hind paws (2D and 3D reconstruction) in DBA/1 model compared to baseline.


( B ) Incidence of spinal ankylosis in PGIS model . n = 5 per cage of total 3 cages per group , Fisher ' s exact . ( C ) Quantitative of structural parameters of spinal ankylosis by μCT . n = 5 per group , one ‐ way ANOVA , Bonferroni post hoc .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Incidence of spinal ankylosis in PGIS model. n=5 per cage of total 3 cages per group, Fisher's exact test. (C) Quantitative analysis of structural parameters of spinal ankylosis by μCT analysis. n=5 per group, one‐way ANOVA, Bonferroni post hoc.


( B ) Incidence of spinal ankylosis in PGIS model . n = 5 per cage of total 3 cages per group , Fisher ' s exact .

O O O O 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) Incidence of spinal ankylosis in PGIS model. n=5 per cage of total 3 cages per group, Fisher's exact test.


( D ) RT - qPCR of expression of CaSR in spinal ligament tissuse . n = 3 , Student ' s t - .

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

(D) RT-qPCR analysis of expression of CaSR in spinal ligament tissuse. n=3, Student's t-test.


( E ) H & E , SOFG Immunofluorescence analyses of spine specimen of PGIS model compare to baseline . n = 5 per group . ( F ) Quantitative of CaSR + , Runx2 + CaSR + Runx2 + number CaSR + in spine specimen of PGIS model . n = 5 per group , one ‐ way ANOVA , Bonferroni post hoc . Data information : AF : AF : Annulus fibrosus ; GP : Growth plate ; NB : New bone . Data shown as mean ± SD . * * P < 0 . 01 compared between groups . Scale bar : 100μm .

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

(E) H&E, SOFG and Immunofluorescence analyses of the spine specimen of PGIS model compare to baseline. n=5 per group. (F) Quantitative analysis of CaSR+, Runx2+ and CaSR+ Runx2+ cells number and CaSR+ cell percentage in the spine specimen of PGIS model. n=5 per group, one‐way ANOVA, Bonferroni post hoc. Data information: AF: AF: Annulus fibrosus; GP: Growth plate; NB: New bone. Data shown as mean ± SD. ** P < 0.01 compared between groups. Scale bar: 100μm.


( E ) H & E , SOFG Immunofluorescence analyses of spine specimen of PGIS model compare to baseline . n = 5 per group .

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

(E) H&E, SOFG and Immunofluorescence analyses of the spine specimen of PGIS model compare to baseline. n=5 per group.


( G ) H & E , SOFG Immunofluorescence analyses of spine specimen of PGIS model at 30 compare to baseline . GP : Growth plate . n = 5 per group . ( H ) Quantitative of CaSR + , OCN + CaSR + OCN + number in spine specimen of PGIS model . n = 5 per group , one ‐ way ANOVA , Bonferroni post hoc . Data information : AF : AF : Annulus fibrosus ; GP : Growth plate ; NB : New bone . Data shown as mean ± SD . * * P < 0 . 01 compared between groups . Scale bar : 100μm .

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

(G) H&E, SOFG and Immunofluorescence analyses of the spine specimen of PGIS model at 30 Weeks compare to baseline. GP: Growth plate. n=5 per group. (H) Quantitative analysis of CaSR+, OCN+ and CaSR+ OCN+ cells number in the spine specimen of PGIS model. n=5 per group, one‐way ANOVA, Bonferroni post hoc. Data information: AF: AF: Annulus fibrosus; GP: Growth plate; NB: New bone. Data shown as mean ± SD. ** P < 0.01 compared between groups. Scale bar: 100μm.


( G ) H & E , SOFG Immunofluorescence analyses of spine specimen of PGIS model at 30 compare to baseline . GP : Growth plate . n = 5 per group .

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

(G) H&E, SOFG and Immunofluorescence analyses of the spine specimen of PGIS model at 30 Weeks compare to baseline. GP: Growth plate. n=5 per group.


( A ) μCT of pathological new bone formation in DBA / 1 model . n = 9 per group .

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

(A) μCT images of pathological new bone formation in DBA/1 model. n=9 per group.


( B ) Incidence of ankle pathological new bone formation in DBA / 1 model . n = 9 per cage of total 3 cages per group , Fisher ' s exact . ( C ) Quantitative of structural parameters of pathological new bone by μCT . n = 9 per group , one ‐ way ANOVA , Bonferroni post hoc .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Incidence of ankle pathological new bone formation in DBA/1 model. n=9 per cage of total 3 cages per group, Fisher's exact test.  (C) Quantitative analysis of structural parameters of pathological new bone by μCT analysis. n=9 per group, one‐way ANOVA, Bonferroni post hoc.


( B ) Incidence of ankle pathological new bone formation in DBA / 1 model . n = 9 per cage of total 3 cages per group , Fisher ' s exact .

(B) Incidence of ankle pathological new bone formation in DBA/1 model. n=9 per cage of total 3 cages per group, Fisher's exact test.


( D ) RT - qPCR of expression of CaSR in hind paw tissue . n = 3 , Student ' s t - .

(D) RT-qPCR analysis of expression of CaSR in hind paw tissue. n=3, Student's t-test.


( E ) H & E , SOFG Immunofluorescence analyses of pathological new bone formation in hind paws . n = 9 per group . ( F ) Quantitative of CaSR + , Runx2 + CaSR + Runx2 + number CaSR + in pathological new bone formation sites . n = 9 per group , one ‐ way ANOVA , Bonferroni post hoc . Data information : CB : Cortical bone ; NB : New bone . Data shown as mean ± SD . * * P < 0 . 01 compared between groups . Scale bar : 100μm .

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

(E) H&E, SOFG and Immunofluorescence analyses of pathological new bone formation in hind paws. n=9 per group. (F) Quantitative analysis of CaSR+, Runx2+ and CaSR+ Runx2+ cells number and CaSR+ cell percentage in pathological new bone formation sites. n=9 per group, one‐way ANOVA, Bonferroni post hoc. Data information: CB: Cortical bone; NB: New bone. Data shown as mean ± SD. ** P < 0.01 compared between groups. Scale bar: 100μm.


( E ) H & E , SOFG Immunofluorescence analyses of pathological new bone formation in hind paws . n = 9 per group .

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

(E) H&E, SOFG and Immunofluorescence analyses of pathological new bone formation in hind paws. n=9 per group.


G ) H & E , SOFG Immunofluorescence analyses of pathological new bone in hind paws . n = 9 per group . ( H ) Quantitative of CaSR + , OCN + CaSR + OCN + number in pathological new bone formation sites . n = 9 per group , one ‐ way ANOVA , Bonferroni post hoc . Data information : CB : Cortical bone ; NB : New bone . Data shown as mean ± SD . * * P < 0 . 01 compared between groups . Scale bar : 100μm .

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

G) H&E, SOFG and Immunofluorescence analyses of pathological new bone in hind paws. n=9 per group. (H) Quantitative analysis of CaSR+, OCN+ and CaSR+ OCN+ cells number in pathological new bone formation sites. n=9 per group, one‐way ANOVA, Bonferroni post hoc. Data information: CB: Cortical bone; NB: New bone. Data shown as mean ± SD. ** P < 0.01 compared between groups. Scale bar: 100μm.


G ) H & E , SOFG Immunofluorescence analyses of pathological new bone in hind paws . n = 9 per group .

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

G) H&E, SOFG and Immunofluorescence analyses of pathological new bone in hind paws. n=9 per group.


( A ) μCT of spine in PGIS model ( 2D 3D reconstruction ) . Arrow head shows spinal ankylosis .

(A) μCT images of the spine in PGIS model (2D and 3D reconstruction). Arrow head shows spinal ankylosis.


( B ) H & E SOFG of spine in PGIS model .

(B) H&E and SOFG staining of spine in PGIS model.


( C ) Incidence of spinal ankylosis in PGIS model . n = 5 per cage of total 6 cages per group , Fisher ' s exact . ( D ) Quantitative of structural parameters of new bone by μCT . n = 5 , one ‐ way ANOVA , Bonferroni post hoc .

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

(C) Incidence of spinal ankylosis in PGIS model. n=5 per cage of total 6 cages per group, Fisher's exact test.  (D) Quantitative analysis of structural parameters of new bone by μCT analysis. n=5, one‐way ANOVA, Bonferroni post hoc.


( C ) Incidence of spinal ankylosis in PGIS model . n = 5 per cage of total 6 cages per group , Fisher ' s exact .

(C) Incidence of spinal ankylosis in PGIS model. n=5 per cage of total 6 cages per group, Fisher's exact test.


( E ) μCT of new bone formation ( red ) in hind paws ( 2D 3D reconstruction ) . ( F ) Incidence of new bone formation in DBA / 1 model . n = 9 per cage of total 3 cages per group , Fisher ' s exact . ( G ) Quantitative of structural parameters of new bone by μCT . n = 9 , one ‐ way ANOVA , Bonferroni post hoc .

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

(E) μCT images of new bone formation (red area) in hind paws (2D and 3D reconstruction).  (F) Incidence of new bone formation in DBA/1 model. n=9 per cage of total 3 cages per group, Fisher's exact test. (G) Quantitative analysis of structural parameters of new bone by μCT analysis. n=9, one‐way ANOVA, Bonferroni post hoc.


( E ) μCT of new bone formation ( red ) in hind paws ( 2D 3D reconstruction ) .

(E) μCT images of new bone formation (red area) in hind paws (2D and 3D reconstruction).


( H ) H & E SOFG of new bone in hind paws . Data information : CB : Cortical bone ; NB : New bone

(H) H&E and SOFG staining of new bone in hind paws. Data information: CB: Cortical bone; NB: New bone


( H ) H & E SOFG of new bone in hind paws . Data information : CB : Cortical bone ; NB : New


( I ) μCT of PCT in SMTS model ( 2D ) . Arrow heads shows bony projection .

(I) μCT images of the PCT in SMTS model (2D). Arrow heads shows bony projection.


( J ) H & E SOFG of Achilles tendon enthesis compartment in SMTS model . Data information : UF : Uncalcified fibrocartilage ; CF : Calcified fibrocartilage ; PCT : posterior calcaneal tuberosity .

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

(J) H&E and SOFG staining of Achilles tendon enthesis compartment in SMTS model. Data information: UF: Uncalcified fibrocartilage; CF: Calcified fibrocartilage; PCT: posterior calcaneal tuberosity.


( K ) Quantitative of of UF . n = 5 , one ‐ way ANOVA , Bonferroni post hoc . ( L ) Quantitative of of CF . n = 5 , one ‐ way ANOVA , Bonferroni post hoc . Data information : UF : Uncalcified fibrocartilage ; CF : Calcified

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

(K) Quantitative analysis of area of UF. n=5, one‐way ANOVA, Bonferroni post hoc. (L) Quantitative analysis of area of CF. n=5, one‐way ANOVA, Bonferroni post hoc. Data information: UF: Uncalcified fibrocartilage; CF: Calcified fibrocartilage


( K ) Quantitative of of UF . n = 5 , one ‐ way ANOVA , Bonferroni post hoc .

(K) Quantitative analysis of area of UF. n=5, one‐way ANOVA, Bonferroni post hoc.


( A ) Alizarin red of hBMSC for 11 days .

(A) Alizarin red staining of hBMSC cells for 11 days.


( B ) of Alizarin Red at 570 nm ( A570 ) . n = 3 , one ‐ way ANOVA , Bonferroni post hoc .

(B) Quantification of Alizarin Red staining at 570 nm (A570). n=3, one‐way ANOVA, Bonferroni post hoc.


( C ) RT - qPCR of osteogenesis markers in hBMSC . n = 3 , one ‐ way ANOVA , Bonferroni post hoc .

(C) RT-qPCR analysis of osteogenesis markers in hBMSC cells. n=3, one‐way ANOVA, Bonferroni post hoc.


(D) Alizarin red staining of hBMSC cells.


( E ) of Alizarin Red at 570 nm ( A570 ) . n = 3 , one ‐ way ANOVA , Bonferroni post hoc .

(E) Quantification of Alizarin Red staining at 570 nm (A570). n=3, one‐way ANOVA, Bonferroni post hoc.


( F ) RT - qPCR of osteogenesis markers in hBMSC . n = 3 , one ‐ way ANOVA , Bonferroni post hoc .

(F) RT-qPCR analysis of osteogenesis markers in hBMSC cells. n=3, one‐way ANOVA, Bonferroni post hoc.


( G ) Western blot of phosphorylation of PLCγ in hBMSC .

(G) Western blot analysis of phosphorylation of PLCγ protein levels in hBMSC cells.


( H ) Western blot of phosphorylation of PLCγ in hBMSC treated with SR NPS - 2143 .

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

(H) Western blot analysis of phosphorylation of PLCγ protein levels in hBMSC cells treated with SR and NPS-2143.


( I ) Immunofluorescence of spine in PGIS model . Quantitative of CaSR + , p - PLCγ + CaSR + p - PLCγ + number in spine of PGIS model . n = 5 , one ‐ way ANOVA , Bonferroni post hoc . Data information : AF : Annulus fibrosus ; NB : New bone

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

(I) Immunofluorescence analysis of spine in PGIS model. Quantitative analysis of CaSR+, p-PLCγ+ and CaSR+ p-PLCγ+ cells number in spine of PGIS model. n=5, one‐way ANOVA, Bonferroni post hoc. Data information: AF: Annulus fibrosus; NB: New bone


( I ) Immunofluorescence of spine in PGIS model . Quantitative of CaSR + , p - PLCγ + CaSR + p - PLCγ + number in spine of PGIS model . n = 5 , one ‐ way ANOVA , Bonferroni post hoc . Data information : AF : Annulus fibrosus ; NB : New

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


( J ) Immunofluorescence of Achilles tendon enthesis compartment in SMTS model . Quantitative of CaSR + , p - PLCγ + CaSR + p - PLCγ + number in Achilles tendon enthesis compartment . n = 5 , one ‐ way ANOVA , Bonferroni post hoc . Data information : UF : Uncalcified fibrocartilage ; CF : Calcified fibrocartilage

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

(J) Immunofluorescence analysis of Achilles tendon enthesis compartment in SMTS model. Quantitative analysis of CaSR+, p-PLCγ+ and CaSR+ p-PLCγ+ cells number in Achilles tendon enthesis compartment. n=5, one‐way ANOVA, Bonferroni post hoc. Data information: UF: Uncalcified fibrocartilage; CF: Calcified fibrocartilage


( J ) Immunofluorescence of Achilles tendon enthesis compartment in SMTS model . Quantitative of CaSR + , p - PLCγ + CaSR + p - PLCγ + number in Achilles tendon enthesis compartment . n = 5 , one ‐ way ANOVA , Bonferroni post hoc . Data information : UF : Uncalcified fibrocartilage ; CF : Calcified

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


( K ) Immunofluorescence of pathological new bone on dorsal surface of hind paws . Quantitative of CaSR + , p - PLCγ + CaSR + p - PLCγ + number in pathological new bone formation sites . n = 9 , one ‐ way ANOVA , Bonferroni post hoc . Data information : CB : Cortical bone ; NB : New bone

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

(K) Immunofluorescence analysis of pathological new bone on dorsal surface of hind paws. Quantitative analysis of CaSR+, p-PLCγ+ and CaSR+ p-PLCγ+ cells number in pathological new bone formation sites. n=9, one‐way ANOVA, Bonferroni post hoc. Data information: CB: Cortical bone; NB: New bone


( K ) Immunofluorescence of pathological new bone on dorsal surface of hind paws . Quantitative of CaSR + , p - PLCγ + CaSR + p - PLCγ + number in pathological new bone formation sites . n = 9 , one ‐ way ANOVA , Bonferroni post hoc . Data information : CB : Cortical bone ; NB : New

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


( L ) μCT of PCT in SMTS model ( 2D ) . Arrow heads shows bony projection .

(L) μCT images of the PCT in SMTS model (2D). Arrow heads shows bony projection.


( M ) SOFG of Achilles tendon enthesis compartment in SMTS model . Data information : UF : Uncalcified fibrocartilage ; CF : Calcified fibrocartilage ; PCT : posterior calcaneal tuberosity .

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

(M) SOFG staining of Achilles tendon enthesis compartment in SMTS model. Data information: UF: Uncalcified fibrocartilage; CF: Calcified fibrocartilage; PCT: posterior calcaneal tuberosity.


( N ) Quantitative of of UF CF . n = 5 , Student ' s t -

(N) Quantitative analysis of area of UF and CF. n=5, Student's t-test


( A ) RT - qPCR Western Blot analyses of CaSR expressions in MC3T3 - E1 treated with IL - 1β . n = 3 , one ‐ way ANOVA , Bonferroni post hoc .

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

(A) RT-qPCR and Western Blot analyses of CaSR expressions in MC3T3-E1 cells treated with IL-1β. n=3, one‐way ANOVA, Bonferroni post hoc.


( B ) RT - qPCR Western Blot analyss of CaSR expressions in MC3T3 - E1 treated with TNFα . n = 3 , one ‐ way ANOVA , Bonferroni post hoc .

O O O O O O O O O O B-MEASURED_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

(B) RT-qPCR and Western Blot analyss of CaSR expressions in MC3T3-E1 cells treated with TNFα. n=3, one‐way ANOVA, Bonferroni post hoc.


( C ) RT - qPCR Western Blot analyses of CaSR expressions in MC3T3 - E1 treated with IL - 17A . n = 3 , one ‐ way ANOVA , Bonferroni post hoc .

(C) RT-qPCR and Western Blot analyses of CaSR expressions in MC3T3-E1 cells treated with IL-17A. n=3, one‐way ANOVA, Bonferroni post hoc.


( D ) RT - qPCR Western Blot analyses of CaSR expressions in MC3T3 - E1 treated with IL - 22 . n = 3 , one ‐ way ANOVA , Bonferroni post hoc .

(D) RT-qPCR and Western Blot analyses of CaSR expressions in MC3T3-E1 cells treated with IL-22. n=3, one‐way ANOVA, Bonferroni post hoc.


( E ) RT - qPCR Western Blot analyses of CaSR expressions in MC3T3 - E1 treated with IL - 23 . n = 3 , one ‐ way ANOVA , Bonferroni post hoc .

(E) RT-qPCR and Western Blot analyses of CaSR expressions in MC3T3-E1 cells treated with IL-23. n=3, one‐way ANOVA, Bonferroni post hoc.


( A ) Immunofluorescence of spine in PGIS mice . Quantitative of CaSR + p - p65 + CaSR + p - Stat3 + number in pathological new bone formation of PGIS model . n = 5 , one ‐ way ANOVA , Bonferroni post hoc . Data information : AF : Annulus fibrosus ; GP : Growth plate ; NB : New bone

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

(A) Immunofluorescence analysis of spine in PGIS mice. Quantitative analysis of CaSR+ p-p65+ cells and CaSR+ p-Stat3+ cells number in pathological new bone formation of PGIS model. n = 5, one‐way ANOVA, Bonferroni post hoc. Data information: AF: Annulus fibrosus; GP: Growth plate; NB: New bone


( A ) Immunofluorescence of spine in PGIS mice . Quantitative of CaSR + p - p65 + CaSR + p - Stat3 + number in pathological new bone formation of PGIS model . n = 5 , one ‐ way ANOVA , Bonferroni post hoc . Data information : AF : Annulus fibrosus ; GP : Growth plate ; NB : New

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


( B ) Immunofluorescence of pathological new bone of hind paws in DBA / 1 mice . Quantitative of CaSR + p - p65 + CaSR + p - Stat3 + number in pathological new bone formation sites . n = 9 , one ‐ way ANOVA , Bonferroni post hoc . CB : Cortical bone ; NB : New bone

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(B) Immunofluorescence analysis of pathological new bone of hind paws in DBA/1 mice. Quantitative analysis of CaSR+ p-p65+ cells and CaSR+ p-Stat3+ cells number in pathological new bone formation sites. n = 9, one‐way ANOVA, Bonferroni post hoc. CB: Cortical bone; NB: New bone


( B ) Immunofluorescence of pathological new bone of hind paws in DBA / 1 mice . Quantitative of CaSR + p - p65 + CaSR + p - Stat3 + number in pathological new bone formation sites . n = 9 , one ‐ way ANOVA , Bonferroni post hoc . CB : Cortical bone ; NB : New

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( C ) Immunofluorescence of Achilles tendon enthesis compartment in SMTS model . Quantitative of CaSR + p - p65 + CaSR + p - stat3 + number in PCT . n = 5 , Student ' s t - . UF : Uncalcified fibrocartilage ; CF : Calcified fibrocartilage

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(C) Immunofluorescence analysis of Achilles tendon enthesis compartment in SMTS model. Quantitative analysis of CaSR+ p-p65+ cells and CaSR+ p-stat3+ cells number in PCT. n=5, Student's t-test. UF: Uncalcified fibrocartilage; CF: Calcified fibrocartilage


( C ) Immunofluorescence of Achilles tendon enthesis compartment in SMTS model . Quantitative of CaSR + p - p65 + CaSR + p - stat3 + number in PCT . n = 5 , Student ' s t - . UF : Uncalcified fibrocartilage ; CF : Calcified

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Three sex chromosome axes could be detected in 2 - month - old XUsp26 + XUsp26 - Y spermatocytes . Immunofluorescence of SYCP3 ( red ) SYCP1 ( green ) in ( J ) , ATR ( red ) SYCP3 ( green ) in ( K ) was performed in 2 - month - old WT XUsp26 + XUsp26 - Y spermatocytes . Nuclei were stained with DAPI ( blue ) . arrows indicate Y chromosome . arrowheads indicate X chromosome .

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Three sex chromosome axes could be detected in the 2-month-old XUsp26+XUsp26-Y spermatocytes. Immunofluorescence analysis of SYCP3 (red) and SYCP1 (green) in (J), ATR (red) and SYCP3 (green) in (K) was performed in 2-month-old WT and XUsp26+XUsp26-Y spermatocytes. Nuclei were stained with DAPI (blue).The arrows indicate the Y chromosome. The arrowheads indicate the X chromosome.


D Histological of seminiferous tubules caudal epididymis of 6 - month - old Usp26 + / Y Usp26 - / Y mice .

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D Histological analysis of the seminiferous tubules and caudal epididymis of the 6-month-old Usp26+/Y and Usp26-/Y mice.


F deletion of Usp26 causes pachytene meiotic division arrest . Representative PAS - hematoxylin in 6 - month - old Usp26 + / Y Usp26 - / Y seminiferous tubules . Parafﬁn sections from Usp26 + / Y Usp26 - / Y testes were stained with PAS - hematoxylin . P : pachytene spermatocyte , D : diplotene spermatocyte , rST : round spermatid , eST : elongating spermatid , M : meiotic spermatocyte , aP : apoptotic pachytene spermatocytes , aM : abnormal meiotic divisions .

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F The deletion of Usp26 causes pachytene and meiotic division arrest. Representative PAS-hematoxylin staining in 6-month-old Usp26+/Y and Usp26-/Y seminiferous tubules. Parafﬁn sections from Usp26+/Y and Usp26-/Y testes were stained with PAS-hematoxylin. P: pachytene spermatocyte, D: diplotene spermatocyte, rST: round spermatid, eST: elongating spermatid, M: meiotic spermatocyte, aP: apoptotic pachytene spermatocytes, aM: abnormal meiotic divisions.


E X Y chromosomes were unpaired in Usp26 - / Y spermatocytes . Immunofluorescence of Chr X - FISH ( green ) , Chr Y - FISH ( red ) , SYCP3 ( ) was performed in 6 - month - old Usp26 + / Y Usp26 - / Y spermatocytes . arrowheads indicate X chromosome . F of unpaired X Y chromosomes in 6 - month - old Usp26 + / Y Usp26 - / Y mice ( n = 5 independent experiments ) . Red dots indicate Usp26 + / Y mice green dots indicate Usp26 - / Y mice .

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E The X and Y chromosomes were unpaired in Usp26-/Y spermatocytes. Immunofluorescence analysis of Chr X-FISH (green), Chr Y-FISH (red), and SYCP3 (white) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. The arrowheads indicate the X chromosome. F Quantification of unpaired X and Y chromosomes in 6-month-old Usp26+/Y and Usp26-/Y mice (n= 5 independent experiments). Red dots indicate Usp26+/Y mice and green dots indicate Usp26-/Y mice.


Sex chromosome recombination was perturbed in 6 - month - old Usp26 - / Y mice . Immunofluorescence of SYCP3 ( green ) , MRE11 ( red ) ( G ) ; was performed in 6 - month - old Usp26 + / Y Usp26 - / Y spermatocytes . Nuclei were stained with DAPI ( blue ) . arrows indicate sex chromosomes .

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Sex chromosome recombination was perturbed in 6-month-old Usp26-/Y mice. Immunofluorescence analysis of SYCP3 (green), MRE11 (red) (G); was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrows indicate the sex chromosomes.


Sex chromosome recombination was perturbed in 6 - month - old Usp26 - / Y mice . Immunofluorescence of ; SYCP3 ( green ) , γH2AX ( red ) ( H ) ; SYCP3 ( green ) , ATR ( red ) , p - ATM ( pink ) ( I ) was performed in 6 - month - old Usp26 + / Y Usp26 - / Y spermatocytes . Nuclei were stained with DAPI ( blue ) . arrows indicate sex chromosomes .

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Sex chromosome recombination was perturbed in 6-month-old Usp26-/Y mice. Immunofluorescence analysis of ; SYCP3 (green), γH2AX (red) (H); SYCP3 (green), ATR (red), p-ATM (pink) (I) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrows indicate the sex chromosomes.


L Crossover on sex chromosomes was impaired in 6 - month - old Usp26 - / Y mice . Immunofluorescence of SYCP3 ( green ) , MLH1 ( red ) , ATR ( ) was performed in 6 - month - old Usp26 + / Y Usp26 - / Y spermatocytes . Nuclei were stained with DAPI ( blue ) .

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L Crossover on sex chromosomes was impaired in 6-month-old Usp26-/Y mice. Immunofluorescence analysis of SYCP3 (green), MLH1 (red), and ATR (white) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue).


D Representative TUNEL results in Usp26 + / Y Usp26 - / Y testes . Parafﬁn sections from 6 - month - old Usp26 + / Y Usp26 - / Y testes were stained with TUNEL ( green ) DAPI ( blue ) to show dead in stage IV , VIII XII tubules with pachytene metaphase spermatocytes , respectively . arrowheads indicate laggard chromosomes . P : pachytene spermatocytes , rSt : round spermatid , spz : spermatozoa , M : meiotic spermatocyte , aP : apoptotic pachytene spermatocytes , aM : abnormal meiotic divisions , arSt : apoptotic round spermatid .

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D Representative TUNEL results in Usp26+/Y and Usp26-/Y testes. Parafﬁn sections from 6-month-old Usp26+/Y and Usp26-/Y testes were stained with TUNEL (green) and DAPI (blue) to show dead cells in stage IV, VIII and XII tubules with pachytene and metaphase spermatocytes, respectively. The arrowheads indicate the laggard chromosomes. P: pachytene spermatocytes, rSt: round spermatid, spz: spermatozoa, M: meiotic spermatocyte, aP: apoptotic pachytene spermatocytes, aM: abnormal meiotic divisions, arSt: apoptotic round spermatid.


E level of three known SAC was reduced in 2 - month - old 6 - month - old Usp26 − / Y mouse testes . Immunoblotting of MAD2 , BUBR1 , PLK1 USP26 was performed in 2 - month - old 6 - month - old Usp26 + / Y Usp26 - / Y testes . H3Ser10p Tub served as loading control .

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E The protein level of three known SAC proteins was reduced in 2-month-old and 6-month-old Usp26−/Y mouse testes. Immunoblotting of MAD2, BUBR1, PLK1 and USP26 was performed in 2-month-old and 6-month-old Usp26+/Y and Usp26-/Y testes. H3Ser10p and Tub served as loading control.


F Usp26 - deficient mice produced XY aneuploid round spermatids . FISH of Chr X ( green ) Chr Y ( red ) was performed in 2 - month - old 6 - month - old Usp26 + / Y Usp26 − / Y round spermatids . Nuclei were stained with DAPI ( blue ) . arrows indicate Y chromosome , arrowheads indicate X chromosome . G of different types of round spermatids in 2 - month - old Usp26 + / Y , Usp26 - / Y mice ( n = 3 independent experiments ) 6 - month - old Usp26 + / Y ( n = 3 independent experiments ) , Usp26 - / Y mice ( n = 4 independent experiments ) . P = 0 . 0026 for XY spermatozoa in 6 - month - old Usp26 + / Y Usp26 − / Y mice . P = 0 . 0216 for O spermatozoa in 6 - month - old Usp26 + / Y Usp26 − / Y mice . H Usp26 - deficient mice produced XY aneuploid spermatozoa . FISH of Chr X ( green ) Chr Y ( red ) was performed in 2 - month - old 6 - month - old Usp26 + / Y Usp26 − / Y spermatozoa . Nuclei were stained with DAPI ( blue ) . arrows indicate Y chromosome , arrowheads indicate X chromosome . I of different types of spermatozoa in 2 - month - old Usp26 + / Y , Usp26 - / Y mice ( n = 3 independent experiments ) 6 - month - old Usp26 + / Y , Usp26 - / Y mice ( n = 5 independent ex experiments ) . P = 0 . 0195 for XY spermatozoa in 6 - month - old Usp26 + / Y Usp26 − / Y mice . Data are presented as means ± SD .

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F Usp26-deficient mice produced XY aneuploid round spermatids. FISH analysis of Chr X (green) and Chr Y (red) was performed in 2-month-old and 6-month-old Usp26+/Y and Usp26−/Y round spermatids. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome. G Quantification of different types of round spermatids in 2-month-old Usp26 +/Y, Usp26-/Y mice (n= 3 independent experiments) and 6-month-old Usp26+/Y (n= 3 independent experiments), Usp26-/Y mice (n= 4 independent experiments). P=0.0026 for XY spermatozoa in 6-month-old Usp26+/Y and Usp26−/Y mice. P=0.0216 for O spermatozoa in 6-month-old Usp26+/Y and Usp26−/Y mice. H Usp26-deficient mice produced XY aneuploid spermatozoa. FISH assay of Chr X (green) and Chr Y (red) was performed in 2-month-old and 6-month-old Usp26+/Y and Usp26−/Y spermatozoa. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome. I Quantification of different types of spermatozoa in 2-month-old Usp26+/Y, Usp26-/Y mice (n= 3 independent experiments) and 6-month-old Usp26+/Y, Usp26-/Y mice (n= 5 independent ex experiments). P=0.0195 for XY spermatozoa in 6-month-old Usp26+/Y and Usp26−/Y mice. Data are presented as means ± SD.


X Y chromosomes were unpaired in Tex11 - / Y spermatocytes . Immunofluorescence of SYCP3 ( green ) , TRF1 ( red ) , ATR ( ) ( D ) ; SYCP3 ( green ) , MLH1 ( red ) , ATR ( ) ( E ) was performed in in Tex11 + / Y Tex11 - / Y spermatocytes . Nuclei were stained with DAPI ( blue ) . arrowheads indicate X chromosome .

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The X and Y chromosomes were unpaired in Tex11-/Y spermatocytes. Immunofluorescence analysis of SYCP3 (green), TRF1 (red), ATR (white) (D); SYCP3 (green), MLH1 (red), ATR (white) (E) was performed in in Tex11+/Y and Tex11-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrowheads indicate the X chromosome.


A , B of different types of spermatozoa in fertile men with or without USP26 mutations . spermatozoa from fertile men without USP26 mutations ( n = 10 independent experiments ) ; fertile men contained USP26 c . 463C > T mutation ( sample was analyzed in three repeated experiments ) , USP26 c . 125T > C mutation ( n = 2 independent experiments , one sample was analyzed in two repeated experiments ) , USP26 c . 1044T > A mutation ( n = 5 independent experiments ) , USP26 c . 370 _ 371insACA / 494T > C / 1423C > T mutation ( n = 10 independent experiments ) were collected to perform Chr X Chr Y FISH . P - values are shown for comparison to fertile men without USP26 mutations .

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A, B Quantification of different types of spermatozoa in fertile men with or without USP26 mutations. The spermatozoa from fertile men without USP26 mutations (n = 10 independent experiments); fertile men contained the USP26 c.463C>T mutation (The sample was analyzed in three repeated experiments), the USP26 c.125T>C mutation (n = 2 independent experiments, one sample was analyzed in two repeated experiments), the USP26 c.1044T>A mutation (n = 5 independent experiments), and the USP26 c.370_371insACA/494T>C/1423C>T mutation (n = 10 independent experiments) were collected to perform Chr X and Chr Y FISH. P-values are shown for comparison to fertile men without USP26 mutations.


C fertile men with USP26 mutated haplotype produced XY O aneuploid spermatozoa . FISH of Chr X ( green ) Chr Y ( red ) was performed in fertile men with or without USP26 mutated haplotype . Nuclei were stained with DAPI ( blue ) . arrows indicate Y chromosome , arrowheads indicate X chromosome .

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C The fertile men with the USP26 mutated haplotype produced XY and O aneuploid spermatozoa. FISH assay of Chr X (green) and Chr Y (red) was performed in fertile men with or without the USP26 mutated haplotype. Nuclei were stained with DAPI (blue). The arrows indicate the Y chromosome, and the arrowheads indicate the X chromosome.


Survival rates of control Cdc50a cKO mice after oil or 4 - OHT injection ( n = 5 for each group , P * * * = 0 . 0003 ) .

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Survival rates of control and Cdc50a cKO mice after oil or 4-OHT injection (n = 5 for each group, P*** = 0.0003).


Bar graphs showing audiogenic seizure scores following auditory stimuli ( 5 , 10 , 15 , 20 , 40 kHz or noise ( WN ) at 70 , 80 , 90 , or 100 dB ) in control ( E ) Cdc50a cKO ( F ) mice ( n = 8 for each group ) .

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Bar graphs showing audiogenic seizure scores following auditory stimuli (5, 10, 15, 20, 40 kHz or white noise (WN) at 70, 80, 90, or 100 dB) in control (E) and Cdc50a cKO (F) mice (n = 8 for each group).


Representative confocal z - stack showing cFos ( magenta ) - positive neurons from control Cdc50a cKO mice ( CTX : cortex , HP : hippocampus , IC : inferior colliculus ) . Scale bar , 20 μm . Bar graphs showing relative of cFos - positive in brains of control Cdc50a cKO mice ( n = 5 for each group , * * P = 0 . 0030 , n . s . , not significant ) .

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Representative confocal z-stack images showing cFos (magenta)-positive neurons from control and Cdc50a cKO mice (CTX: cortex, HP: hippocampus, and IC: inferior colliculus). Scale bar, 20 μm. Bar graphs showing the relative area of cFos-positive cells in the brains of control and Cdc50a cKO mice (n = 5 for each group, **P = 0.0030, n.s., not significant).


Schematic diagram of experiment . Representative confocal z - stack of PSD95 ( green ) - VGLUT1 ( red ) - positive excitatory ( A ) Gephyrin ( green ) - VGAT ( magenta ) - positive inhibitory ( B ) synapses in CTX , HP , IC ( CTX : cortex , HP : hippocampus , IC : inferior colliculus ) of control Cdc50a cKO mice . dashed represents where high magnified insert was taken from . Scale bar , 20 μm .

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Schematic diagram of the experiment. Representative confocal z-stack images of PSD95 (green)- and VGLUT1 (red)-positive excitatory (A) and Gephyrin (green)- and VGAT (magenta)-positive inhibitory (B) synapses in the CTX, HP, and IC (CTX: cortex, HP: hippocampus, and IC: inferior colliculus) of control and Cdc50a cKO mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm.


Representative current clamp traces from IC of control Cdc50a cKO mice ; + 100 pA ( upper ) + 400 pA ( lower ) currents were injected into control mice ( left ) Cdc50a cKO mice ( right ) for 500 ms .

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Representative current clamp traces from the IC of control and Cdc50a cKO mice; +100 pA (upper) and +400 pA (lower) currents were injected into control mice (left) and Cdc50a cKO mice (right) for 500 ms.


bar graphs show relative percentages of neurons showing PS exposure only in soma ( left ) overall PS exposure ( right ) ( n = 30 - 40 for each group ) in control Cdc50a cKO mice .

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The bar graphs show the relative percentages of neurons showing PS exposure only in the soma (left) and overall PS exposure (right) (n = 30-40 cells for each group) in control and Cdc50a cKO mice.


Schematic diagram of imaging of neuronal somas proximal distal dendrites of neurons in CA1 region of HP . Representative confocal z - stack showing Gephyrin - positive inhibitory post - synapses in somas ( red ) proximal ( green ) distal dendrites ( cyan ) of neurons in CA1 region of HP in control Cdc50a cKO mice . Scale bar , 20 μm . Bar graphs showing number of Gephyrin - positive inhibitory post - synapses in somas proximal distal dendrites of neurons in CA1 of HP in control Cdc50a cKO mice ( n = 4 for each group , P * * * = 0 . 0002 , P * * * * < 0 . 0001 ) .

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Schematic diagram of imaging of the neuronal somas and proximal and distal dendrites of neurons in the CA1 region of the HP. Representative confocal z-stack images showing Gephyrin-positive inhibitory post-synapses in the somas (red) and proximal (green) and distal dendrites (cyan) of neurons in the CA1 region of the HP in control and Cdc50a cKO mice. Scale bar, 20 μm. Bar graphs showing the number of Gephyrin-positive inhibitory post-synapses in the somas and proximal and distal dendrites of neurons in the CA1 of the HP in control and Cdc50a cKO mice (n = 4 for each group, P*** = 0.0002, P**** < 0.0001).


Representative confocal z - stack colocalization of Gephyrin ( green ) , IBA1 ( blue ) , CD68 ( red ) in IC of control Cdc50a cKO mice . Scale bar , 20 μm . Bar graphs showing relative colocalization of Gephyrin CD68 in IC of control Cdc50a cKO mice ( n = 3 for each group , * * P = 0 . 0024 ) .

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Representative confocal z-stack and colocalization images of Gephyrin (green), IBA1 (blue), and CD68 (red) in the IC of control and Cdc50a cKO mice. Scale bar, 20 μm. Bar graphs showing the relative colocalization area of Gephyrin and CD68 in the IC of control and Cdc50a cKO mice (n = 3 for each group, **P = 0.0024).