C , D . Coimmunoprecipitation between Flag - Lrig1 and HA - TrkB ( C ) or between Flag - Lrig3 and HA - TrkB ( D ) overexpressed in HEK293 cells . Cell extracts were analysed by immunoprecipitation with anti - Flag antibodies followed by immunoblot ( IB ) with antibodies against HA . Reprobing of the same blots with anti - Flag antibodies is shown below . The bottom panel shows HA expression in total lysates . Data represent n = 3 independent experiments .

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C, D. Coimmunoprecipitation between Flag-Lrig1 and HA-TrkB (C) or between Flag-Lrig3 and HA-TrkB (D) overexpressed in HEK293 cells. Cell extracts were analysed by immunoprecipitation with anti-Flag antibodies followed by immunoblot (IB) with antibodies against HA. Reprobing of the same blots with anti-Flag antibodies is shown below. The bottom panel shows HA expression in total lysates. Data represent n=3 independent experiments.


E . Coimmunoprecipitation between HA - TrkB and Flag - Lrig1 or between HA - TrkB and HA - Lrig2 exogenously expressed in HEK293 cells . Cell extracts were analysed by IP with anti - pan - Trk antibodies followed by IB with antibodies against Flag or Lrig2 . Reprobing of the same blots with anti - TrkB antibodies is shown below . The bottom panels show Flag and Lrig2 expression in total lysates . Data represent n = 2 independent assays .

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E. Coimmunoprecipitation between HA-TrkB and Flag-Lrig1 or between HA-TrkB and HA-Lrig2 exogenously expressed in HEK293 cells. Cell extracts were analysed by IP with anti-pan-Trk antibodies followed by IB with antibodies against Flag or Lrig2. Reprobing of the same blots with anti-TrkB antibodies is shown below. The bottom panels show Flag and Lrig2 expression in total lysates. Data represent n=2 independent assays.


F . In vivo interaction between Lrig1 and TrkB . Coimmunoprecipitation between Lrig1 and TrkB receptor endogenously expressed from P15 rat hippocampal tissue extracts . Samples were equally divided in two parts and then the analysis was done by immunoprecipitation with control ( anti - HA epitope tag antibody ) or anti - Lrig1 antibodies , followed by immunoblotting with anti - TrkB antibody . Reprobing of the same blot with anti - Lrig1 antibody is also shown . Expression of TrkB in one aliquot of the starting material ( indicated as lysate ) is included . Arrow indicates the band of TrkB coimmunoprecipitated with anti - Lrig1 antibody . Similar results were obtained in n = 3 independent assays .

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F. In vivo interaction between Lrig1 and TrkB. Coimmunoprecipitation between Lrig1 and TrkB receptor endogenously expressed from P15 rat hippocampal tissue extracts. Samples were equally divided in two parts and then the analysis was done by immunoprecipitation with control (anti-HA epitope tag antibody) or anti-Lrig1 antibodies, followed by immunoblotting with anti-TrkB antibody. Reprobing of the same blot with anti-Lrig1 antibody is also shown. Expression of TrkB in one aliquot of the starting material (indicated as lysate) is included. Arrow indicates the band of TrkB coimmunoprecipitated with anti-Lrig1 antibody. Similar results were obtained in n=3 independent assays.


G . Ligand - dependent activation of TrkB ( p - TrkB ) was evaluated by transient transfection of HA - TrkB plasmid with either a control or a Flag - Lrig1 vector into HEK cells . After 36 h cells were serum - starved and stimulated with or without BDNF ( 30 ng / ml ) for 15 min . The level of TrkB activation ( p - TrkB ) was evaluated in total cell lysates by immunoblotting ( IB ) with a specific antibody that recognizes TrkB phosphorylated in tyrosine 705 ( pY705 ) . Reprobing of the same blot with anti - HA and anti - Flag antibodies is shown . Fold of p - TrkB change relative to total TrkB is indicated . Similar results were obtained in n = 3 independent assays .

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G. Ligand-dependent activation of TrkB (p-TrkB) was evaluated by transient transfection of HA-TrkB plasmid with either a control or a Flag- Lrig1 vector into HEK cells. After 36 h cells were serum-starved and stimulated with or without BDNF (30 ng/ml) for 15 min. The level of TrkB activation (p-TrkB) was evaluated in total cell lysates by immunoblotting (IB) with a specific antibody that recognizes TrkB phosphorylated in tyrosine 705 (pY705). Reprobing of the same blot with anti-HA and anti-Flag antibodies is shown. Fold of p-TrkB change relative to total TrkB is indicated. Similar results were obtained in n=3 independent assays.


H . TrkB ubiquitination was evaluated by transient transfection of HA - TrkB plasmid with either a control or a Flag - Lrig1 vector into MN1 cells . After 36 h , cells were serum - starved , pre - treated with the cell - permeable proteasome inhibitor MG - 132 ( 20 M ) and stimulated with BDNF for 15 min . Total lysates were immunoprecipitated with anti - HA antibodies followed by immunoblot ( IB ) with antibodies against ubiquitin . Reprobing of the same blot with anti - HA antibodies is also shown . TrkB activation ( p - TrkB ) was evaluated in cell lysates . Reprobing of the same blot with anti - TrkB and anti - Flag antibodies is also shown . Fold of p - TrkB ( p - Y705 ) change relative to total TrkB is indicated . Data represent n = 3 independent assays .

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H. TrkB ubiquitination was evaluated by transient transfection of HA-TrkB plasmid with either a control or a Flag-Lrig1 vector into MN1 cells. After 36 h, cells were serum-starved, pre-treated with the cell-permeable proteasome inhibitor MG-132 (20 M) and stimulated with BDNF for 15 min. Total lysates were immunoprecipitated with anti-HA antibodies followed by immunoblot (IB) with antibodies against ubiquitin. Reprobing of the same blot with anti-HA antibodies is also shown. TrkB activation (p-TrkB) was evaluated in cell lysates. Reprobing of the same blot with anti-TrkB and anti-Flag antibodies is also shown. Fold of p-TrkB (p-Y705) change relative to total TrkB is indicated. Data represent n=3 independent assays.


A . Immunoblot showing TrkB activation in hippocampal neurons cultured from Lrig1 heterozygous ( + / - ) and Lrig1 knockout ( - / - ) mice littermates treated in the absence or in the presence of BDNF ( 30 ng / ml ) for 30 minutes . Reprobing of the same blot with anti - ßIII - Tubulin is shown as loading control . B . Fold of TrkB activation relative to unstimulated control group ( phospho - TrkB at Tyr705 ) in hippocampal neurons cultured from Lrig1 ( + / + ; + / - ) and Lrig1 ( - / - ) mice treated in the absence or in the presence of BDNF ( 30 ng / ml ) for 30 minutes . Results are presented as mean SEM of n = 4 independent experiments . ( * p < 0 . 05 by Students t test ) .

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A. Immunoblot showing TrkB activation in hippocampal neurons cultured from Lrig1 heterozygous (+/-) and Lrig1 knockout (-/-) mice littermates treated in the absence or in the presence of BDNF (30 ng/ml) for 30 minutes. Reprobing of the same blot with anti- ßIII-Tubulin is shown as loading control.B. Fold of TrkB activation relative to unstimulated control group (phospho-TrkB at Tyr705) in hippocampal neurons cultured from Lrig1 (+/+; +/-) and Lrig1 (-/-) mice treated in the absence or in the presence of BDNF (30 ng/ml) for 30 minutes. Results are presented as mean SEM of n=4 independent experiments. (*p<0.05 by Students t test).


C . Immunoblot showing MAPK activation in hippocampal neurons cultured from Lrig1 wild - type ( + / + ) and Lrig1 - deficient ( - / - ) mice littermates treated in the absence or in the presence of BDNF ( 30 ng / ml ) for the indicated times . Reprobing of the same blot with anti - ßIII - Tubulin is shown as loading control . Fold of MAPK activation relative to tubulin is indicated . D . Fold of MAPK activation ( P - MAPK ) relative to untreated control group in hippocampal neurons cultured from Lrig1 ( + / + ; + / - ) and Lrig1 ( - / - ) mice treated in the absence or in the presence of BDNF ( 30 ng / ml ) for 30 minutes . Results are presented as mean SEM of n = 4 independent experiments . ( * p < 0 . 05 by Students t test ) .

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C. Immunoblot showing MAPK activation in hippocampal neurons cultured from Lrig1 wild-type (+/+) and Lrig1-deficient (-/-) mice littermates treated in the absence or in the presence of BDNF (30 ng/ml) for the indicated times. Reprobing of the same blot with anti- ßIII-Tubulin is shown as loading control. Fold of MAPK activation relative to tubulin is indicated.D. Fold of MAPK activation (P-MAPK) relative to untreated control group in hippocampal neurons cultured from Lrig1 (+/+; +/-) and Lrig1 (-/-) mice treated in the absence or in the presence of BDNF (30 ng/ml) for 30 minutes. Results are presented as mean SEM of n=4 independent experiments. (*p<0.05 by Students t test).


E . Representative images of mousehippocampal neurons transfected at 4 DIV with either GFP - expressing control vector or Lrig1shRNA - GFP plasmid . After transfection , neurons were cultured in the absence or in the presence of BDNF ( 25 ng / ml ) for 48 h ( 7 DIV ) . Arrows indicate branching points along the principal dendrite . Scale bar , 10 m .

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E. Representative images of mousehippocampal neurons transfected at 4 DIV with either GFP-expressing control vector or Lrig1shRNA-GFP plasmid. After transfection, neurons were cultured in the absence or in the presence of BDNF (25 ng/ml) for 48 h (7 DIV). Arrows indicate branching points along the principal dendrite. Scale bar, 10 m.


F - I . Quantification of primary dendrites ( F ) , secondary dendrites ( G ) , total branching ( H ) and branchig of the principal dendrite ( I ) of hippocampal neurons treated as indicated in E . The results are shown as individual values and the means of a representative assay . A total of 30 neurons were analyzed per experimental condition . Similar results were obtained in n = 3 independent experiments .

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F-I. Quantification of primary dendrites (F), secondary dendrites (G), total branching (H) and branchig of the principal dendrite (I) of hippocampal neurons treated as indicated in E. The results are shown as individual values and the means of a representative assay. A total of 30 neurons were analyzed per experimental condition. Similar results were obtained in n=3 independent experiments.


J . Lrig1 + / + and Lrig1 - / - hippocampal lysates from postnatal day 11 ( P11 ) mice were immunoblotted against phospho - TrkB ( p - TrkB ) at Tyr705 and total TrkB . Quantification ( mean SD ) of the p - TrkB / TrkB ratio between Lrig1 + / + ( n = 4 ) and Lrig1 - / - ( n = 3 ) hippocampi is also shown .

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J. Lrig1+/+ and Lrig1-/- hippocampal lysates from postnatal day 11 (P11) mice were immunoblotted against phospho-TrkB (p-TrkB) at Tyr705 and total TrkB. Quantification (mean SD) of the p-TrkB/TrkB ratio between Lrig1+/+ (n=4) and Lrig1-/- (n=3) hippocampi is also shown.


B , C , Analysis of GFP transfer after transplantation of Nrl : : GFP donor photoreceptors into wildtype C57BL / 6J ( B ) and Nrl - / - ( C ) recipient retinas after 14 ( wt , n = 9 ; Nrl - / - , n = 7 ) and 21 ( wt , n = 6 ; Nrl - / - , n = 4 ) and 90 ( wt , n = 5 ; Nrl - / - , n = 4 ) days . Tissue is stained for GFP and nuclei ( Hoechst ) . In this and all following figures , donor cells ( D ) are located above , and acceptor cells with GFP ( A ) are located below the outer limiting membrane ( white dashed line ) , which delimits the apical border of the outer nuclear layer ( ONL ) . In wildtype recipients , donor cells are typically located above the outer segment region ( between the grey dashed lines ) . In Nrl - / - recipients , donor cells are located in the inner segment ( IS ) region . Plotted is the ratio of the numbers of GFP + photoreceptors in the ONL ( acceptor photoreceptors ) and GFP + donor photoreceptors in the subretinal space ( SRS ) over time .

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B, C, Analysis of GFP transfer after transplantation of Nrl::GFP donor photoreceptors into wildtype C57BL/6J (B) and Nrl-/- (C) recipient retinas after 14 (wt, n=9; Nrl-/-, n=7) and 21 (wt, n=6; Nrl-/-, n=4) and 90 (wt, n=5; Nrl-/-, n=4) days. Tissue is stained for GFP and nuclei (Hoechst). In this and all following figures, donor cells (D) are located above, and acceptor cells with GFP (A) are located below the outer limiting membrane (white dashed line), which delimits the apical border of the outer nuclear layer (ONL). In wildtype recipients, donor cells are typically located above the outer segment region (between the grey dashed lines). In Nrl-/- recipients, donor cells are located in the inner segment (IS) region. Plotted is the ratio of the numbers of GFP+ photoreceptors in the ONL (acceptor photoreceptors) and GFP+ donor photoreceptors in the subretinal space (SRS) over time.


F , Co - transplanted P3 - 4 Crx : : Cre and P3 - 5 ROSAmT / mG donor photoreceptors into C57BL / 6J recipient retinas show Cre reporter gene induction in donor cells located in the SRS 14 days post - transplantation .

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F, Co-transplanted P3-4 Crx::Cre and P3-5 ROSAmT/mG donor photoreceptors into C57BL/6J recipient retinas show Cre reporter gene induction in donor cells located in the SRS 14 days post-transplantation.


A - C , Combined FISH and IHF to detect GFP ( A , B ) and GNAT1 ( C ) 21 days post - transplantation of Nrl : : GFP photoreceptors into C57BL / 6J ( A ) and Nrl - / - ( B , C ) recipients .

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A-C, Combined FISH and IHF to detect GFP (A, B) and GNAT1 (C) 21 days post-transplantation of Nrl::GFP photoreceptors into C57BL/6J (A) and Nrl-/- (B, C) recipients.


F - K , FISH and IHF for GFP and GFP transcripts in C57BL / 6J recipients transplanted with lentiviral - infected donor cells expressing cytoplasmic GFP ( Lenti - GFP ) ( F , I ) and nuclear - localized GFP ( Lenti - nls - GFP ) ( G , J ) . H , MT index for cytosolic ( Lenti - GFP , n = 5 ) and nuclear localized ( Lenti - nls - GFP , n = 3 ) . K , Quantification of transcripts in donor and acceptor photoreceptors . ( Lenti - GFP , n = 6 images from 3 transplanted animals ; Lenti - nls - GFP , n = 8 images from 4 transplanted animals ) .

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F-K, FISH and IHF for GFP and GFP transcripts in C57BL/6J recipients transplanted with lentiviral-infected donor cells expressing cytoplasmic GFP (Lenti -GFP) (F, I) and nuclear-localized GFP (Lenti-nls-GFP) (G, J). H, MT index for cytosolic (Lenti-GFP, n=5) and nuclear localized (Lenti-nls-GFP, n=3). K, Quantification of transcripts in donor and acceptor photoreceptors. (Lenti-GFP, n=6 images from 3 transplanted animals; Lenti-nls-GFP, n=8 images from 4 transplanted animals).


B - D ' ' , Higher magnification of regions in A ( indicated by yellow dashed boxes ) showing colocalization of ATP5B and Mito - DsRed - labelled mitochondria in the outer segments ( B ) , soma ( C ) , and the axon ( D ) of acceptor photoreceptors .

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B-D'', Higher magnification of regions in A (indicated by yellow dashed boxes) showing colocalization of ATP5B and Mito-DsRed-labelled mitochondria in the outer segments (B), soma (C), and the axon (D) of acceptor photoreceptors.


F , G , C57BL / 6J cells with mitochondria labelled with MitoTrackerTM - Green ( MTG ) or MitoTrackerTM - Red ( MTR ) exchange mitochondria after 3 DIV ( 0 DIV , n = 6 ; 3 DIV , n = 6 ) . White dashed line delimitates both cell somas and the protrusion connecting them ( F ) . No exchange is observed when the two photoreceptor populations are separated by a transwell ( n = 3 ) , nor in co - cultures of live MTG - labelled and lysed MTR - labelled cells ( n = 3 ) ( G ) .

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F, G, C57BL/6J cells with mitochondria labelled with MitoTrackerTM-Green (MTG) or MitoTrackerTM-Red (MTR) exchange mitochondria after 3 DIV (0 DIV, n=6; 3 DIV, n=6). White dashed line delimitates both cell somas and the protrusion connecting them (F). No exchange is observed when the two photoreceptor populations are separated by a transwell (n=3), nor in co-cultures of live MTG-labelled and lysed MTR-labelled cells (n=3) (G).


A , Maximum intensity projection of a confocal micrograph of a cryostat section from a Nrl - / - recipient retina 21 days after transplantation with Nrl : : GFP donor photoreceptors , showing protrusions ( pink arrows ) connecting donor and acceptor photoreceptors .

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A, Maximum intensity projection of a confocal micrograph of a cryostat section from a Nrl-/- recipient retina 21 days after transplantation with Nrl::GFP donor photoreceptors, showing protrusions (pink arrows) connecting donor and acceptor photoreceptors.


D , E , Left : 3D reconstruction of two - photon ( D ) and light sheet microscopy images of intact cleared ROSAmT / mG eye showing that GFP + - donor and - acceptor photoreceptors are in the same area . D , Right : A high magnification image showing GFP + - donor and - acceptor photoreceptors connected by protrusions ( pink arrows ) . E , Right : In detail , donor and acceptor photoreceptors also appear connected through cell protrusions ( pink arrow ) .

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D, E, Left: 3D reconstruction of two-photon (D) and light sheet microscopy images of intact cleared ROSAmT/mG eye showing that GFP+-donor and -acceptor photoreceptors are in the same area. D, Right: A high magnification image showing GFP+-donor and -acceptor photoreceptors connected by protrusions (pink arrows). E, Right: In detail, donor and acceptor photoreceptors also appear connected through cell protrusions (pink arrow).


A , Confocal image shows a Lenti - GFP infected donor photoreceptor ( yellow arrow ) connected to an acceptor photoreceptor . The donor protrusion ( pink arrow ) co - localizes with α - tubulin but not the outer segment marker GNAT1 .

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A, Confocal image shows a Lenti-GFP infected donor photoreceptor (yellow arrow) connected to an acceptor photoreceptor. The donor protrusion (pink arrow) co-localizes with α-tubulin but not the outer segment marker GNAT1.


B , Nrl : : GFP donor photoreceptors transplanted in a Prphr2 - / - recipient retina exhibit polarized Prphr2 expression ( yellow arrow ) on the apical side of the donor photoreceptor . Basal protrusions ( pink arrows ) indicate donor protrusions that contact the host retina .

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B, Nrl::GFP donor photoreceptors transplanted in a Prphr2-/- recipient retina exhibit polarized Prphr2 expression (yellow arrow) on the apical side of the donor photoreceptor. Basal protrusions (pink arrows) indicate donor protrusions that contact the host retina.


C , D , Expression of β - tubulin ( C ) , SV2 ( D ) and GFP in the protrusions connecting Lenti - GFP - infected C57BL / 6J donor photoreceptors with acceptor photoreceptors . Pink arrows point to the protrusions connecting donor and acceptor cells .

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C, D, Expression of β-tubulin (C), SV2 (D) and GFP in the protrusions connecting Lenti-GFP-infected C57BL/6J donor photoreceptors with acceptor photoreceptors. Pink arrows point to the protrusions connecting donor and acceptor cells.


A , B , Examples of live Nrl : : GFP and C57BL / 6J retinal cultures showing actin + ( SirActin ) and GFP + ( A ) and tubulin + ( ViaFluor ) and MitoTracker Green + ( MTG ) protrusions ( B ) connecting photoreceptors . DIC , differential interference contrast microscopy .

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A, B, Examples of live Nrl::GFP and C57BL/6J retinal cultures showing actin+ (SirActin) and GFP+ (A) and tubulin+ (ViaFluor) and MitoTracker Green+ (MTG) protrusions (B) connecting photoreceptors. DIC, differential interference contrast microscopy.


E , Time lapse imaging showing movement of MitoTracker Green + puncta in photoreceptors connected by a protrusion . White arrows point MTG + puncta inside of the protrusion during the time lapse imaging .

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E, Time lapse imaging showing movement of MitoTracker Green+ puncta in photoreceptors connected by a protrusion. White arrows point MTG+ puncta inside of the protrusion during the time lapse imaging.


C , Confocal microscopy image of cryostat sections of C57BL / 6J recipient retinas 21 days after transplant of C57BL / J6 photoreceptors infected with empty vector ( n = 5 ) , RHOA overexpression ( RHOA OE ) ( n = 7 ) or RAC1 dominant negative ( RAC1 DN ) ( n = 7 ) lentiviruses . Quantification of MT in retinas transplanted with control , RHOA and RAC1 DN lentiviruses .

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C, Confocal microscopy image of cryostat sections of C57BL/6J recipient retinas 21 days after transplant of C57BL/J6 photoreceptors infected with empty vector (n=5), RHOA overexpression (RHOA OE) (n=7) or RAC1 dominant negative (RAC1 DN) (n=7) lentiviruses. Quantification of MT in retinas transplanted with control, RHOA and RAC1 DN lentiviruses.


D , GFP staining of whole - mounted retinas after transplantation with control , RHOA and RAC1 DN lentivirus infected donor cells ( n = 4 per group ) . Quantification shows a significant reduction in the average protrusion length per cell and in the total protrusion length per cell after RHOA OE and RAC1 DN infection compared to the empty vector control .

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D, GFP staining of whole-mounted retinas after transplantation with control, RHOA and RAC1 DN lentivirus infected donor cells (n=4 per group). Quantification shows a significant reduction in the average protrusion length per cell and in the total protrusion length per cell after RHOA OE and RAC1 DN infection compared to the empty vector control.


A Genome - wide nucleosome maps were generated from the frequency distribution of the read midpoint positions of pair - end sequenced MNase - resistant fragments in the size range 138 - 161 bp ( spanning the nucleosome footprint ) . Figure shows a section of a nucleosome map taken from chromosome 12 derived from pluripotent iPSC ( pl - iPSC ) to show examples of nucleosomes selected as highly positioned , marked * .

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A Genome-wide nucleosome maps were generated from the frequency distribution of the read midpoint positions of pair-end sequenced MNase-resistant fragments in the size range 138-161 bp (spanning the nucleosome footprint). Figure shows a section of a nucleosome map taken from chromosome 12 derived from pluripotent iPSC (pl-iPSC) to show examples of nucleosomes selected as highly positioned, marked *.


B The distribution of positioned nucleosomes ( derived 138 - 161 bp size class ) and sub - nucleosome fragments ( derived 112 - 137 bp size class ) for pl - iPSC and iPSC - derived NPC ( NPC ) . Actual calculated numbers are presented above each column .

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B The distribution of positioned nucleosomes (derived 138-161 bp size class) and sub-nucleosome fragments (derived 112-137 bp size class) for pl-iPSC and iPSC-derived NPC (NPC). Actual calculated numbers are presented above each column.


D Average frequency distribution of nucleosome positions relative to each other . Data was aligned to each mapped nucleosome in the genome and plotted for a 600 bp window for pl - iPSC , NPC and the chronic myelogenous leukaemia ( Cml ) derived cell line , K562 . In all cases , a prominent single nucleosome is presented with only minor flanking peaks , indicating that the majority of nucleosomes do not occur as evenly spaced arrays in human cells .

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D Average frequency distribution of nucleosome positions relative to each other. Data was aligned to each mapped nucleosome in the genome and plotted for a 600 bp window for pl-iPSC, NPC and the chronic myelogenous leukaemia (Cml) derived cell line, K562. In all cases, a prominent single nucleosome is presented with only minor flanking peaks, indicating that the majority of nucleosomes do not occur as evenly spaced arrays in human cells.


E Distribution of nucleosome array sizes for pl - iPSC , NPC and K562 , calculated as the number of nucleosomes within a distance of 150 - 200 bp of each other . Bars show the number of nucleosomes arrayed as singletons ( 1 ) , pairs ( 2 ) , triplets ( 3 ) and greater than 3 ( > 3 ) , displayed as log10 values and the percentage distribution within a cell line is shown above the column .

E Distribution of nucleosome array sizes for pl-iPSC, NPC and K562, calculated as the number of nucleosomes within a distance of 150-200 bp of each other. Bars show the number of nucleosomes arrayed as singletons (1), pairs (2), triplets (3) and greater than 3 (>3), displayed as log10 values and the percentage distribution within a cell line is shown above the column.


A Bar chart showing the number of nucleosomes that map to selected chromatin states [ 20 ] . Active ( State 1 ) ; Insulator ( State 8 ) ; Repressed , Rep , ( State 12 ) and heterochromatin , Hetero ( State 13 ) , and to open chromatin ( based on ATAC - seq ) . Shown are the observed number of nucleosomes per chromatin state ( black ) and the expected number calculated assuming a random distribution across the genome ( grey ) .

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A Bar chart showing the number of nucleosomes that map to selected chromatin states [20]. Active (State 1); Insulator (State 8); Repressed, Rep, (State 12) and heterochromatin, Hetero (State 13), and to open chromatin (based on ATAC-seq). Shown are the observed number of nucleosomes per chromatin state (black) and the expected number calculated assuming a random distribution across the genome (grey).


A , B Nucleosome maps at and surrounding , the TSS region of the NANOG and GRIA1 genes in pl - iPSC ( upper , black ) and in NPC cells ( lower , grey ) . The TSS is indicated by a dashed line at chr 12 : 7 , 941 , 991 for NANOG and chr5 : 152 , 870 , 105 for GRIA1 . * indicates the position of a highly positioned nucleosome associated with the TSS of the active gene .

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A, B Nucleosome maps at and surrounding, the TSS region of the NANOG and GRIA1 genes in pl-iPSC (upper, black) and in NPC cells (lower, grey). The TSS is indicated by a dashed line at chr 12: 7,941,991 for NANOG and chr5: 152,870,105 for GRIA1. * indicates the position of a highly positioned nucleosome associated with the TSS of the active gene.


C Global frequency distribution of nucleosome distributions at TSS of protein - coding genes . Alignment of nucleosome maps centred on TSS using the non - redundant list of 66 , 047 mapped human protein - coding gene TSS . The features corresponding to the nucleosome free region ( NFR ) and - 1 to + 2 nucleosomes are marked .

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C Global frequency distribution of nucleosome distributions at TSS of protein-coding genes. Alignment of nucleosome maps centred on TSS using the non-redundant list of 66,047 mapped human protein-coding gene TSS. The features corresponding to the nucleosome free region (NFR) and -1 to +2 nucleosomes are marked.


D Genome - wide positioning of nucleosomes within ± 300 bp of a TSS of an active gene . The number of nucleosomes observed are plotted for both pl - iPSC and NPC cell types for gene sets selected either as uniquely expressed in pl - iPSC ( n = 3 , 833 ) or NPC ( n = 2 , 082 ) . If positioned nucleosomes are a strong predictor of gene activity , high numbers of nucleosomes would be expected for pluripotent - specific genes in pl - iPSC , but not NPC stages , and for NPC specific genes in NPC , but not pl - iPSC stages . No correlation between gene activity and positioned nucleosomes was observed .

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D Genome-wide positioning of nucleosomes within ± 300 bp of a TSS of an active gene. The number of nucleosomes observed are plotted for both pl-iPSC and NPC cell types for gene sets selected either as uniquely expressed in pl-iPSC (n=3,833) or NPC (n=2,082). If positioned nucleosomes are a strong predictor of gene activity, high numbers of nucleosomes would be expected for pluripotent-specific genes in pl-iPSC, but not NPC stages, and for NPC specific genes in NPC, but not pl-iPSC stages. No correlation between gene activity and positioned nucleosomes was observed.


A The average distribution of nucleosomes centred on the RE1 site ( n = 871 ) , spanning 2 Kb of flanking genomic sequence .

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A The average distribution of nucleosomes centred on the RE1 site (n=871), spanning 2 Kb of flanking genomic sequence.


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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 levels were normalized to Hprt and Gapdh mRNAs . n = 6 - 9 per group , P = 0 . 004 ( unpaired two - tailed Student ' s t - tests ) .

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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 − / − 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 .

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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 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 ) .

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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 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 .

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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 and eosin staining transverse heart cross - sections showing myocyte cross - sectional area ( 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 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 .

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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 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 ) .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 ) 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 .

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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 ) 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 .

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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 ) 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 ) 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 ) .

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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 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 .

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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 ) 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 .

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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 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 ) .

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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 ) 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 .

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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 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 .

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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 ) 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 . 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 .

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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 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 ) .

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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 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 .

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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 , 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 .

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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 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 .

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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 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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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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

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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 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 :

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

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

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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 analysis of expression of CaSR in human spinal tissue .

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


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 .

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

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


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 .

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

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


( 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 .

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(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 test . ( C ) Quantitative analysis of structural parameters of spinal ankylosis by μCT analysis . n = 5 per group , one ‐ way ANOVA , Bonferroni post hoc .

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(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 test .

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

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


( 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 .

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(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 and Immunofluorescence analyses of the spine specimen of PGIS model compare to baseline . n = 5 per group .

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(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 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 .

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(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 and Immunofluorescence analyses of the spine specimen of PGIS model at 30 Weeks compare to baseline . GP : Growth plate . n = 5 per group .

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

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(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 test . ( C ) Quantitative analysis of structural parameters of pathological new bone by μCT analysis . n = 9 per group , one ‐ way ANOVA , Bonferroni post hoc .

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(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 test .

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

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


( 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 .

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(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 and Immunofluorescence analyses of pathological new bone formation in hind paws . n = 9 per group .

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