A . Arrest architecture of replicative stress . 2C and 4C arrest trajectories are shown , the regions that correspond to mitotic skipping and the senescent state are annotated and the arrest trajectory of 8C polyploid cells is indicated . N = 4315

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A. Arrest architecture of replicative stress. 2C and 4C arrest trajectories are shown, the regions that correspond to mitotic skipping and the senescent state are annotated and the arrest trajectory of 8C polyploid cells is indicated. N = 4315 cells


B . Progression of arrest states over time following etoposide treatment . Etoposide - treated cells ( upper panel : colored by condition , lower panel : colored by DNA content ) populating 2C , 4C , 8C and senescent arrest states at each day of treatment ( 1 - 4 d ) .

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B. Progression of arrest states over time following etoposide treatment. Etoposide-treated cells (upper panel: colored by condition, lower panel: colored by DNA content) populating 2C, 4C, 8C and senescent arrest states at each day of treatment (1-4 d).


C . Distribution of senescence - associated β - galactosidase ( SA - βgal ) activity in individual cells following etoposide treatment ( 1 μM , 0 - 4 d ) .

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C. Distribution of senescence-associated β-galactosidase (SA-βgal) activity in individual cells following etoposide treatment (1 μM, 0-4 d).


D - K . ( D ) GSK3β , ( E ) phospho ( Thr157 ) - p27 , ( F ) p27 , ( G ) CDK4 , ( H ) cyclin D1 , ( I ) cyclin E , ( J ) cell area and ( K ) DNA : cytoplasm ratio of etoposide - treated cells are plotted on the map . Median nuclear values are shown in D - I . Area indicated with a dotted line represents the senescent region of the map .

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D-K. (D) GSK3β, (E) phospho(Thr157)-p27, (F) p27, (G) CDK4, (H) cyclin D1, (I) cyclin E, (J) cell area and (K) DNA:cytoplasm ratio of etoposide-treated cells are plotted on the map. Median nuclear values are shown in D-I. Area indicated with a dotted line represents the senescent region of the map.


L - O . ( L ) Cyclin A , ( M ) cyclin B1 , ( N ) CDH1 and ( O ) SKP2 of etoposide - treated cells are plotted on the map . Median nuclear values are shown . Area indicated with a dotted line shows the trajectory of mitotic skipping and transition into senescence .

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L-O. (L) Cyclin A, (M) cyclin B1, (N) CDH1 and (O) SKP2 of etoposide-treated cells are plotted on the map. Median nuclear values are shown. Area indicated with a dotted line shows the trajectory of mitotic skipping and transition into senescence.


A . Distribution of senescence - associated β - galactosidase ( SA - βgal ) activity in individual cells following etoposide or palbociclib treatment ( 1 μM , 4 / 8 d ) . Control : N = 6050 cells , etop 4d : N = 409 cells , etop 8d : N = 732 cells , palbo 4d : N = 1837 cells , palbo 8d : N = 1677 cells . Statistical significance was determined using a one - way analysis of variance ( ANOVA ) with Sidak ' s post hoc test ( * * * p < 0 . 0001 ) .

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A. Distribution of senescence-associated β-galactosidase (SA-βgal) activity in individual cells following etoposide or palbociclib treatment (1 μM, 4/8 d). Control: N = 6050 cells, etop 4d: N = 409 cells, etop 8d: N = 732 cells, palbo 4d: N = 1837 cells, palbo 8d: N = 1677 cells. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Sidak's post hoc test (*** p < 0.0001).


B . Unified cell cycle map of the unperturbed ( gray ) , palbociclib - ( palbo , 1 μM , 4 / 8 d , blue ) and etoposide - ( etop , 1 μM , 4 / 8 d , green ) treated cells . Control : N = 10499 cells , etop : N = 2692 cells , palbo : N = 4931

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B. Unified cell cycle map of the unperturbed (gray), palbociclib- (palbo, 1 μM, 4/8 d, blue) and etoposide- (etop,1 μM, 4/8 d, green) treated cells. Control: N = 10499 cells, etop: N = 2692 cells, palbo: N = 4931 cells


C - K . ( C ) DNA content , ( D ) phospho / total RB , ( E ) p16 , ( F ) TGFβ1 , ( G ) IL - 6 , ( H ) IL - 8 , ( I ) phospho - p38 , ( J ) phospho - STAT3 and ( K ) phospho - STAT5 are plotted on the map . Median nuclear values are shown .

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C-K. (C) DNA content, (D) phospho/total RB, (E) p16, (F) TGFβ1, (G) IL-6, (H) IL-8, (I) phospho-p38, (J) phospho-STAT3 and (K) phospho-STAT5 are plotted on the map. Median nuclear values are shown.


D . Representative single - cell trace demonstrating mitotic skipping and endoreduplication following etoposide treatment ( 1 μM ) by time - lapse imaging . CDK2 activity ( DHB - mCherry , blue ) , cell cycle phase ( PCNA - mTq2 , S phase shown as gray shaded area ) and p21 - YPet intensity ( green ) are plotted versus time of etoposide treatment .

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D. Representative single-cell trace demonstrating mitotic skipping and endoreduplication following etoposide treatment (1 μM) by time-lapse imaging. CDK2 activity (DHB-mCherry, blue), cell cycle phase (PCNA-mTq2, S phase shown as gray shaded area) and p21-YPet intensity (green) are plotted versus time of etoposide treatment.


E . Cyclin D1 abundance ( left ) and the proportion of polyploid cells ( right ) , as measured by immunofluorescence and Hoechst staining , respectively , following siRNA - mediated knockdown of cyclin D in control and etoposide - treated cells . Boxplots show data from four independent replicates ( gray circles ) . Data information : Statistical significance in right panels was determined using a two - way analysis of variance ( ANOVA ) with Sidak ' s post hoc test ( * * * p < 0 . 001 , * p = 0 . 02 ) . boxes show the interquartile range , the whiskers indicate the full distribution of points and the central band represents the population .

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E. Cyclin D1 abundance (left) and the proportion of polyploid cells (right), as measured by immunofluorescence and Hoechst staining, respectively, following siRNA-mediated knockdown of cyclin D in control and etoposide-treated cells. Boxplots show data from four independent replicates (gray circles). Data information: Statistical significance in right panels was determined using a two-way analysis of variance (ANOVA) with Sidak's post hoc test (*** p < 0.001, * p = 0.02). boxes show the interquartile range, the whiskers indicate the full distribution of points and the central band represents the population.


F . Cyclin D1 abundance ( left ) and the proportion of polyploid cells ( right ) , as measured by immunofluorescence and Hoechst staining , respectively , following doxycycline ( dox ) - induced upregulation of cyclin D1 in control and etoposide - treated cells . Boxplots show data from six independent replicates ( gray circles ) . Data information : Statistical significance in right panels was determined using a two - way analysis of variance ( ANOVA ) with Sidak ' s post hoc test ( * * * p < 0 . 001 , * p = 0 . 02 ) . boxes show the interquartile range , the whiskers indicate the full distribution of points and the central band represents the population .

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F. Cyclin D1 abundance (left) and the proportion of polyploid cells (right), as measured by immunofluorescence and Hoechst staining, respectively, following doxycycline (dox)-induced upregulation of cyclin D1 in control and etoposide-treated cells. Boxplots show data from six independent replicates (gray circles). Data information: Statistical significance in right panels was determined using a two-way analysis of variance (ANOVA) with Sidak's post hoc test (*** p < 0.001, * p = 0.02). boxes show the interquartile range, the whiskers indicate the full distribution of points and the central band represents the population.


G . Cyclin A abundance ( left ) and the proportion of polyploid cells ( right ) , as measured by immunofluorescence and Hoechst staining , respectively , following doxycycline ( dox ) - induced upregulation of cyclin A2 in control and etoposide - treated cells . Boxplots show data from six independent replicates ( gray circles ) . Data information : Statistical significance in right panels was determined using a two - way analysis of variance ( ANOVA ) with Sidak ' s post hoc test ( * * * p < 0 . 001 , * p = 0 . 02 ) . boxes show the interquartile range , the whiskers indicate the full distribution of points and the central band represents the population .

G. Cyclin A abundance (left) and the proportion of polyploid cells (right), as measured by immunofluorescence and Hoechst staining, respectively, following doxycycline (dox)-induced upregulation of cyclin A2 in control and etoposide-treated cells. Boxplots show data from six independent replicates (gray circles). Data information: Statistical significance in right panels was determined using a two-way analysis of variance (ANOVA) with Sidak's post hoc test (*** p < 0.001, * p = 0.02). boxes show the interquartile range, the whiskers indicate the full distribution of points and the central band represents the population.


( E ) Weight and survival curves of neutrophil specific Nlrp3A350VneoR Gsdmd - / - mice . n ≥

(E) Weight and survival curves of neutrophil specific Nlrp3A350VneoR Gsdmd-/- mice. n≥18


( A , B ) MPO staining of liver issue used as a marker for myeloid cells including monocytes in both Nlrp3D301N - MRP8 and Nlrp3A350V - MRP8 mutant ( n ≥ 3 mice per genotype , representative images shown , bar indicates 250 μM ) . H & E staining of liver tissue with histologic scoring of liver inflammation ( bar indicates 100 μM , representative images are shown ) . TUNEL staining of liver tissue ( Representative images shown , bar indicates 250 μM ) . Data information : n ≥ 6 mice per genotype , significance determined by unpaired t test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) , data are expressed as mean + / − SD , dots represent individual biological

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(A,B) MPO staining of liver issue used as a marker for myeloid cells including monocytes in both Nlrp3D301N-MRP8 and Nlrp3A350V-MRP8 mutant (n≥3 mice per genotype, representative images shown, bar indicates 250 μM). H&E staining of liver tissue with histologic scoring of liver inflammation (bar indicates 100 μM, representative images are shown). TUNEL staining of liver tissue (Representative images shown, bar indicates 250 μM). Data information: n≥6 mice per genotype, significance determined by unpaired t test (* p < 0.05; ** p < 0.01; *** p < 0.001), data are expressed as mean +/− SD, dots represent individual biological replicats


( B , Protein expression in liver tissue of cleaved and mature forms of Casp1 ( p20 ) , IL - 1β and TNF - α ( 17kDa ) ( n = 3 mice per genotype for Western blots ) .

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(B, Protein expression in liver tissue of cleaved and mature forms of Casp1 (p20), IL-1β and TNF-α (17kDa) (n=3 mice per genotype for Western blots).


C ) Protein expression in liver tissue of cleaved and mature forms of Casp1 ( p20 ) , IL - 1β and TNF - α ( 17kDa ) Data information : n ≥ 6 mice per genotype , significance determined by unpaired t test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) , data are expressed as mean + / − SD , dots represent individual biological

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C) Protein expression in liver tissue of cleaved and mature forms of Casp1 (p20), IL-1β and TNF-α (17kDa) Data information: n≥6 mice per genotype, significance determined by unpaired t test (* p < 0.05; ** p < 0.01; *** p < 0.001), data are expressed as mean +/− SD, dots represent individual biological replicats


( A ) Immunohistochemical staining of F4 / 80 , a marker for resident macrophages , and mRNA expression of F4 / 80 in liver tissue . Data information : Representative images shown , bar indicates 250 μM for F4 / 80 n ≥ 6 mice per genotype , significance determined by unpaired t test ( * * p < 0 . 01 ; * * * p < 0 . 001 ) , data are expressed as mean + / − SD , dots represent individual biological

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(A) Immunohistochemical staining of F4/80, a marker for resident macrophages, and mRNA expression of F4/80 in liver tissue. Data information: Representative images shown, bar indicates 250 μM for F4/80 n≥6 mice per genotype, significance determined by unpaired t test (** p < 0.01; *** p < 0.001), data are expressed as mean +/− SD, dots represent individual biological replicats


( B ) Immunofluorescence staining of CLEC4F , a specific marker of Kupffer cells , and gene expression of CLEC4F in liver tissue . Data information : Representative images shown , bar indicates 100 μM for CLEC4F , n ≥ 6 mice per genotype , significance determined by unpaired t test ( * * p < 0 . 01 ; * * * p < 0 . 001 ) , data are expressed as mean + / − SD , dots represent individual biological

(B) Immunofluorescence staining of CLEC4F, a specific marker of Kupffer cells, and gene expression of CLEC4F in liver tissue. Data information: Representative images shown, bar indicates 100 μM for CLEC4F, n≥6 mice per genotype, significance determined by unpaired t test (** p < 0.01; *** p < 0.001), data are expressed as mean +/− SD, dots represent individual biological replicats


( C ) Immunohistochemical staining of Ly6C , a marker for pro - inflammatory macrophages , in liver tissue . Data information : Representative images shown , bar indicates 250 μM for Ly6C , n ≥ 6 mice per genotype , significance determined by unpaired t test ( * * p < 0 . 01 ; * * * p < 0 . 001 ) , data are expressed as mean + / − SD , dots represent individual biological

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(C) Immunohistochemical staining of Ly6C, a marker for pro-inflammatory macrophages, in liver tissue. Data information: Representative images shown, bar indicates 250 μM for Ly6C, n≥6 mice per genotype, significance determined by unpaired t test (** p < 0.01; *** p < 0.001), data are expressed as mean +/− SD, dots represent individual biological replicats


( A ) Collagen deposition assessed by Sirius red staining Data information : Representative images shown , bar indicates 250 μM , n ≥ 6 per genotype , significance determined by unpaired t test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) , data are expressed as mean + / − SD , dots represent individual biological

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(A) Collagen deposition assessed by Sirius red staining Data information: Representative images shown, bar indicates 250 μM, n≥6 per genotype, significance determined by unpaired t test (* p < 0.05; ** p < 0.01; *** p < 0.001), data are expressed as mean +/− SD, dots represent individual biological replicats


( C ) Protein expression of collagen 1a in whole liver lysate . n = 3 Data information : n ≥ 6 per genotype , significance determined by unpaired t test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) , data are expressed as mean + / − SD , dots represent individual biological

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(C) Protein expression of collagen 1a in whole liver lysate. n=3 Data information: n≥6 per genotype, significance determined by unpaired t test (* p < 0.05; ** p < 0.01; *** p < 0.001), data are expressed as mean +/− SD, dots represent individual biological replicats


( A - C ) Individuals in the neuropathology cohort were divided in groups of no significant primary pathology ( N = 31 ) , AD ( N = 36 ) , PD ( N = 13 ) , and others ( N = 8 ) , and analyzed together with N = 9 additional subjects with primary non - AD tauopathies ( PSP / CBD ) . Data is shown for plasma P - tau217 ( panel A ) , plaque density ( panel B ) , and tangle density ( panel C ) . Plasma P - tau217 was not elevated in PSP / CBD compared to subjects without significant primary pathology . AD , Alzheimer ' s disease ; CBD , corticobasal degeneration ; PSP , progressive supranuclear palsy ; PD , Parkinson ' s disease . The central band of the boxes are medians and the boxes show inter - quartile ranges . The whiskers are defined as the smallest ( largest ) observation greater ( less ) than or equal to the first ( third ) quartile minus ( plus ) 1 . 5 times the inter - quartile range .

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(A-C) Individuals in the neuropathology cohort were divided in groups of no significant primary pathology (N=31), AD (N=36), PD (N=13), and others (N=8), and analyzed together with N=9 additional subjects with primary non-AD tauopathies (PSP/CBD). Data is shown for plasma P-tau217 (panel A), plaque density (panel B), and tangle density (panel C). Plasma P-tau217 was not elevated in PSP/CBD compared to subjects without significant primary pathology. AD, Alzheimer's disease; CBD, corticobasal degeneration; PSP, progressive supranuclear palsy; PD, Parkinson's disease. The central band of the boxes are medians and the boxes show inter-quartile ranges. The whiskers are defined as the smallest (largest) observation greater (less) than or equal to the first (third) quartile minus (plus) 1.5 times the inter-quartile range.


Panel B shows associations between plasma P - tau217 and Aβ PET stratified by tertiles ( T ) of tau PET ( T1 : SUVR ≤ 1 . 05 , T2 : 1 . 05 < SUVR ≤ 1 . 11 , T3 : 1 . 11 < SUVR ≤ 3 . 03 ) . Panel C shows associations between plasma P - tau217 and tau PET stratified by tertiles ( T ) of Aβ PET ( T1 : SUVR ≤ 0 . 468 , T2 : 0 . 468 < SUVR ≤ 0 . 579 , T3 : 0 . 579 < SUVR ≤ 1 . 08 ) .

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Panel B shows associations between plasma P-tau217 and Aβ PET stratified by tertiles (T) of tau PET (T1: SUVR ≤ 1.05, T2: 1.05 < SUVR ≤ 1.11, T3: 1.11 < SUVR ≤ 3.03).   Panel C shows associations between plasma P-tau217 and tau PET stratified by tertiles (T) of Aβ PET (T1: SUVR ≤ 0.468, T2: 0.468 < SUVR ≤ 0.579, T3: 0.579 < SUVR ≤ 1.08).


( A ) . Cell growth assessment of ΔIndLon ( upper plot ) and ΔIndFtsH ( lower plot ) mutants grown under inducing ( blue ) or depleting conditions ( red ) . Growth was monitored by measuring DNA and protein biomass over time . The average from two independent biological replicates is shown .

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(A). Cell growth assessment of ΔIndLon (upper plot) and ΔIndFtsH (lower plot) mutants grown under inducing (blue) or depleting conditions (red). Growth was monitored by measuring DNA and protein biomass over time. The average from two independent biological replicates is shown.


( D ) . Role of Lon and FtsH under heat shock stress conditions . ΔIndLon and ΔIndFtsH mutants were grown under inducing ( Lon + or FtsH + ) or depleting conditions ( Lon + or FtsH + , 60h of depletion for both , Lon and FtsH ) , and then exposed at 45 ° C during 0 , 5 , 10 , 15 or 20 min . Then , growth after the heat treatment was monitored over time under inducing conditions by the 430 / 560 absorbance rate index that shows pH changes in the medium . The average from two independent biological replicates is shown for each condition .

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(D). Role of Lon and FtsH under heat shock stress conditions. ΔIndLon and ΔIndFtsH mutants were grown under inducing (Lon+ or FtsH+) or depleting conditions (Lon+ or FtsH+, 60h of depletion for both, Lon and FtsH), and then exposed at 45°C during 0, 5, 10, 15 or 20 min. Then, growth after the heat treatment was monitored over time under inducing conditions by the 430/560 absorbance rate index that shows pH changes in the medium. The average from two independent biological replicates is shown for each condition.


( C ) In vivo degradation assays of FtsA , FtsZ and DnaB . ΔIndLon mutants expressing the different N - terminal FLAG - tagged derivatives were grown in depleting conditions for 36h . Then , Lon expression was transiently induced for 3h before translation was blocked with gentamicin ( Gm ) . Samples were taken at the indicated time points after gentamicin treatment , and processed for immunoblot analysis using anti - FLAG antibodies . LC , loading control . As controls , non - induced cells were also treated with gentamicin and processed at the same time points . Immunoblots are representative of two independent experiments .

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(C) In vivo degradation assays of FtsA, FtsZ and DnaB. ΔIndLon mutants expressing the different N-terminal FLAG-tagged derivatives were grown in depleting conditions for 36h. Then, Lon expression was transiently induced for 3h before translation was blocked with gentamicin (Gm). Samples were taken at the indicated time points after gentamicin treatment, and processed for immunoblot analysis using anti-FLAG antibodies. LC, loading control. As controls, non-induced cells were also treated with gentamicin and processed at the same time points. Immunoblots are representative of two independent experiments.


( E and F ) Similar to panel B ( E ) and C ( F ) but for a C - terminal FLAG - tagged derivative of MPN201 as a representative of an unstable HsdS subunit .

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(E and F) Similar to panel B (E) and C (F) but for a C-terminal FLAG-tagged derivative of MPN201 as a representative of an unstable HsdS subunit.


( H ) Similar to panel B but for C - terminal FLAG - tagged derivatives of FtsH candidate substrates . Protein levels of FtsH and candidate substrates were assessed by immunoblot analysis using anti - FtsH and anti - FLAG antibodies . LC , loading control .

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(H) Similar to panel B but for C-terminal FLAG-tagged derivatives of FtsH candidate substrates. Protein levels of FtsH and candidate substrates were assessed by immunoblot analysis using anti-FtsH and anti-FLAG antibodies. LC, loading control.


( I ) Similar to panel C , but for FtsH candidate substrates . In this case , C - terminal FLAG - tagged derivatives were grown in depleting conditions for 60h before transient expression of FtsH for 3h and gentamicin treatment .

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(I) Similar to panel C, but for FtsH candidate substrates. In this case, C-terminal FLAG-tagged derivatives were grown in depleting conditions for 60h before transient expression of FtsH for 3h and gentamicin treatment.


( B ) Protein stability assessment of Lon substrates containing multiple or single mutations in putative Lon degrons . N - terminal FLAG - tagged derivatives with mutations shown in panel A were expressed in the ΔIndLon mutant . Protein levels were then determined by immunoblot using anti - Lon and anti - FLAG antibodies comparing inducing ( + ) or depleting ( - ) conditions ( 48h of depletion ) . LC , loading control .

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(B) Protein stability assessment of Lon substrates containing multiple or single mutations in putative Lon degrons. N-terminal FLAG-tagged derivatives with mutations shown in panel A were expressed in the ΔIndLon mutant. Protein levels were then determined by immunoblot using anti-Lon and anti-FLAG antibodies comparing inducing (+) or depleting (-) conditions (48h of depletion). LC, loading control.


( B ) Lon surveillance of truncated protein variants . As an example , an N - terminal FLAG - tagged derivative of MPN304 , which encodes a truncated variant of ArcA ( ArcA - Nt ) , was expressed in the ΔIndLon mutant , and its accumulation monitored over time under non - inducing conditions using anti - FLAG antibodies . Anti - Lon antibodies were used to monitor Lon depletion along the time course experiment ( western blot figure on left ) . MPN304 stability was also assessed by in vivo degradation experiments in Lon depleted mutants ( 36h of depletion ) , in which protein expression was blocked with gentamicin ( Gm ) during the indicated time points after transient induction of Lon for 3h . As control , samples after Gm treatment were also taken from non - induced samples . LC , loading control ( western blot figure on right ) . Immunoblots are representative of two independent experiments . A schematic representation of the arginine deiminase pathway , which is inactive in M . pneumoniae , and the estimated protein copy number of its components are also shown for the ΔIndLon mutant grown in inducing ( + ) or depleting ( - ) conditions .

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

(B) Lon surveillance of truncated protein variants. As an example, an N-terminal FLAG-tagged derivative of MPN304, which encodes a truncated variant of ArcA (ArcA-Nt), was expressed in the ΔIndLon mutant, and its accumulation monitored over time under non-inducing conditions using anti-FLAG antibodies. Anti-Lon antibodies were used to monitor Lon depletion along the time course experiment (western blot figure on left). MPN304 stability was also assessed by in vivo degradation experiments in Lon depleted mutants (36h of depletion), in which protein expression was blocked with gentamicin (Gm) during the indicated time points after transient induction of Lon for 3h. As control, samples after Gm treatment were also taken from non-induced samples. LC, loading control (western blot figure on right). Immunoblots are representative of two independent experiments. A schematic representation of the arginine deiminase pathway, which is inactive in M. pneumoniae, and the estimated protein copy number of its components are also shown for the ΔIndLon mutant grown in inducing (+) or depleting (-) conditions.


( E ) Protein quality control of terminal organelle assembly mediated by Lon . Top of panel E , a schematic representation of the genetic organization at the P65 operon after deleting the N - terminal region of the hmw2 gene in the ΔIndLon mutant . The expected PCR products for the deletion are also indicated . Below , an agarose electrophoresis gel showing the PCR validation of the intended deletion , and SDS - PAGE and immunoblot analyses monitoring the expression of specific terminal organelle proteins , whose stability depend on HMW2 , for ΔIndLon or ΔIndLon _ Δhmw2Nt cell lysates grown in inducing ( + ) or depleting ( - ) conditions ( 48h of depletion ) . LC , loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Protein quality control of terminal organelle assembly mediated by Lon. Top of panel E, a schematic representation of the genetic organization at the P65 operon after deleting the N-terminal region of the hmw2 gene in the ΔIndLon mutant. The expected PCR products for the deletion are also indicated. Below, an agarose electrophoresis gel showing the PCR validation of the intended deletion, and SDS-PAGE and immunoblot analyses monitoring the expression of specific terminal organelle proteins, whose stability depend on HMW2, for ΔIndLon or ΔIndLon_Δhmw2Nt cell lysates grown in inducing (+) or depleting (-) conditions (48h of depletion). LC, loading control.


( 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 :

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

O O O O O O O O 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) 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 .

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

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 .

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 .

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

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.


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. (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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 test . ( C ) Quantitative analysis of structural parameters of spinal ankylosis by μCT analysis . 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 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 test .

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

O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 test .

O O O O O O O O O O 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.


( 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 .

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

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


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and 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 O

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


( A ) μCT images of the spine in PGIS model ( 2D and 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 and SOFG staining 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 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 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 test .

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


( 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 .

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

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


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


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

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


( 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 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 analysis of area 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 staining of hBMSC cells for 11 days .

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


( B ) Quantification of Alizarin Red staining 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 analysis of osteogenesis markers in hBMSC cells . 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 .

(D) Alizarin red staining of hBMSC cells.


( E ) Quantification of Alizarin Red staining 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 analysis of osteogenesis markers in hBMSC cells . 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 analysis of phosphorylation of PLCγ protein levels in hBMSC cells .

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


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

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

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


( 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

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

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

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

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( 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

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(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 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

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

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

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


( 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 .

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

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(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 and Western Blot analyses of CaSR expressions in MC3T3 - E1 cells 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 and Western Blot analyses of CaSR expressions in MC3T3 - E1 cells 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 and Western Blot analyses of CaSR expressions in MC3T3 - E1 cells 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 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

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(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 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

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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 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 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

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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

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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 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

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Representative images of hematoxylin and eosin - stained testicular sections from the patient ( V : 3 ) and a man diagnosed with obstructive azoospermia , serving as the control . The magnified view of the boxed area is shown in the lower left corner of the image from the patient . The blue arrow indicates unaligned chromosomes in the representative metaphase cells . Scale bars , 50 μm .

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Representative images of hematoxylin and eosin-stained testicular sections from the patient (V:3) and a man diagnosed with obstructive azoospermia, serving as the control. The magnified view of the boxed area is shown in the lower left corner of the image from the patient. The blue arrow indicates unaligned chromosomes in the representative metaphase cells. Scale bars, 50 μm.


Immunofluorescence staining of testicular sections from control and patient V : 3 with antibodies against H3S10p ( red ) , a marker of metaphase cells . The nuclei were stained with Hoechst 33342 ( blue ) . The magnified view of the red box is shown in the lower left corner of the image from the patient .

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Immunofluorescence staining of testicular sections from control and patient V:3 with antibodies against H3S10p (red), a marker of metaphase cells. The nuclei were stained with Hoechst 33342 (blue). The magnified view of the red box is shown in the lower left corner of the image from the patient.


Sanger sequencing chromatograms showing the homozygous M1AP splicing mutation in patients and heterozygous mutation in their fertile brother . The arrowhead indicates the position of the mutation .

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Sanger sequencing chromatograms showing the homozygous M1AP splicing mutation in patients and heterozygous mutation in their fertile brother. The arrowhead indicates the position of the mutation.


Western blot analysis of M1AP expression in various tissues from adult wild - type ( WT ) mice . GAPDH served as the loading control .

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Western blot analysis of M1AP expression in various tissues from adult wild-type (WT) mice. GAPDH served as the loading control.


Western blot analysis of M1AP expression in testes from mice at different days postpartum ( dpp ) . GAPDH served as the loading control .

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Western blot analysis of M1AP expression in testes from mice at different days postpartum (dpp). GAPDH served as the loading control.


Immunofluorescence staining of M1AP ( green ) and SYCP3 ( red ) on spermatocyte spreads of WT or Spo11 - / - mice .

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Immunofluorescence staining of M1AP (green) and SYCP3 (red) on spermatocyte spreads of WT or Spo11-/- mice.


Quantification of M1AP foci in WT spermatocytes at the indicated stages . L , leptotene ; E / MZ , early / mid zygotene ; LZ , late zygotene ; EP , early pachytene ; M / LP , mid / late pachytene ; Z - like , zygotene - like .

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Quantification of M1AP foci in WT spermatocytes at the indicated stages. L, leptotene; E/MZ, early/mid zygotene; LZ, late zygotene; EP, early pachytene; M/LP, mid/late pachytene; Z-like, zygotene-like.


Testicular histology from 8 - week - old control and M1apKI / KI mice . The magnified view of the boxed area is shown in the lower left corner of the image of M1apKI / KI mice . The blue arrow indicates unaligned chromosomes in the representative metaphase cells .

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Testicular histology from 8-week-old control and M1apKI/KI mice. The magnified view of the boxed area is shown in the lower left corner of the image of M1apKI/KI mice. The blue arrow indicates unaligned chromosomes in the representative metaphase cells.


Meiotic metaphase I ( MMI ) spermatocytes stained with Giemsa . The arrows indicate univalent chromosomes .

Meiotic metaphase I (MMI) spermatocytes stained with Giemsa. The arrows indicate univalent chromosomes.


Frequencies of nuclei with univalents.


Frequencies of nuclei with only XY univalents , only autosome univalents , or both XY and autosome univalents .

Frequencies of nuclei with only XY univalents, only autosome univalents, or both XY and autosome univalents.


Representative spread spermatocytes stained for SYCP3 ( green ) and MLH1 ( red ) .

Representative spread spermatocytes stained for SYCP3 (green) and MLH1 (red).


Frequencies of autosomal chromosomes with 0 , 1 , and 2 or 3 MLH1 foci .

Frequencies of autosomal chromosomes with 0, 1, and 2 or 3 MLH1 foci.


Frequencies of nuclei with an MLH1 focus at the pseudoautosomal region ( PAR ) of XY chromosomes .

Frequencies of nuclei with an MLH1 focus at the pseudoautosomal region (PAR) of XY chromosomes.


Representative spread spermatocytes stained for the lateral element ( SYCP3 , green ) and the central element ( SIX6OS1 , red ) in early and mid / late pachynema . Miniaturized H1t staining ( white ) images are shown in the left lower corner of the overlay images . The arrows indicate the untouching XY chromosomes .

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Representative spread spermatocytes stained for the lateral element (SYCP3, green) and the central element (SIX6OS1, red) in early and mid/late pachynema. Miniaturized H1t staining (white) images are shown in the left lower corner of the overlay images. The arrows indicate the untouching XY chromosomes.


Quantification of nuclei with XY untouching in early and mid / late pachynema .

Quantification of nuclei with XY untouching in early and mid/late pachynema.


Immunofluorescence staining with antibodies against SYCP3 ( red ) and MSH4 ( green ) on spermatocyte spreads .

Immunofluorescence staining with antibodies against SYCP3 (red) and MSH4 (green) on spermatocyte spreads.


The mean number of MSH4 foci per nucleus in control and M1apKI / KI spermatocytes at the indicated stages .

The mean number of MSH4 foci per nucleus in control and M1apKI/KI spermatocytes at the indicated stages.


Immunofluorescence staining with antibodies against SYCP3 ( red ) and RAD51 ( green ) on spermatocyte spreads .

Immunofluorescence staining with antibodies against SYCP3 (red) and RAD51 (green) on spermatocyte spreads.


The mean number of RAD51 foci per nucleus in control and M1apKI / KI spermatocytes at the indicated stages .

The mean number of RAD51 foci per nucleus in control and M1apKI/KI spermatocytes at the indicated stages.


Immunofluorescence staining with antibodies against SYCP3 ( red ) and DMC1 ( green ) on spermatocyte spreads .

Immunofluorescence staining with antibodies against SYCP3 (red) and DMC1 (green) on spermatocyte spreads.


The mean number of DMC1 foci per nucleus in control and M1apKI / KI spermatocytes at the indicated stages .

The mean number of DMC1 foci per nucleus in control and M1apKI/KI spermatocytes at the indicated stages.


Western blot analysis of M1AP and TEX11 expression in fetal ovaries ( 16 . 5 days postcoitum ( dpc ) ) and testes ( 14 dpp ) from WT mice . GAPDH served as the loading control .

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Western blot analysis of M1AP and TEX11 expression in fetal ovaries (16.5 days postcoitum (dpc)) and testes (14 dpp) from WT mice. GAPDH served as the loading control.


Immunofluorescence staining with antibodies against SYCP3 ( red ) and MLH3 ( green ) on oocyte spreads from control and M1apKI / KI embryos ( 18 . 5 dpc ) . Number of MLH3 foci per cell in control and M1apKI / KI oocytes .

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Immunofluorescence staining with antibodies against SYCP3 (red) and MLH3 (green) on oocyte spreads from control and M1apKI/KI embryos (18.5 dpc). Number of MLH3 foci per cell in control and M1apKI/KI oocytes.


Representative haemotoxylin - stained ovarian sections of 5 - dpp - old control and M1apKI / KI mice . Follicle counts and total oocyte counts per ovary at 5 dpp .

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Representative haemotoxylin-stained ovarian sections of 5-dpp-old control and M1apKI/KI mice. Follicle counts and total oocyte counts per ovary at 5 dpp.


Co - IP using anti - M1AP antibody with whole - testis lysates of WT mice and M1ap - / - mice , followed by western blot analyses of M1AP , SHOC1 , and TEX11 .

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Co-IP using anti-M1AP antibody with whole-testis lysates of WT mice and M1ap-/- mice, followed by western blot analyses of M1AP, SHOC1, and TEX11.


Co - IP detecting the interaction between M1AP ( fused with an N - terminal MYC tag ) and each of the ZZS complex components ( fused with an N - terminal GFP tag ) exogenously expressed in HEK - 293T cells .

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Co-IP detecting the interaction between M1AP (fused with an N-terminal MYC tag) and each of the ZZS complex components (fused with an N-terminal GFP tag) exogenously expressed in HEK-293T cells.


Immunofluorescence staining of M1AP ( green ) and TEX11 ( red ) on the spermatocyte spreads of WT mice . Ratios of M1AP - TEX11 co - localizing foci to the M1AP total foci or TEX11 total foci at the indicated meiotic prophase substages . Each dot represents the ratio calculated from one nucleus .

Immunofluorescence staining of M1AP (green) and TEX11 (red) on the spermatocyte spreads of WT mice.   Ratios of M1AP-TEX11 co-localizing foci to the M1AP total foci or TEX11 total foci at the indicated meiotic prophase substages. Each dot represents the ratio calculated from one nucleus.


Immunofluorescence staining of M1AP ( green ) and DMC1 ( red ) on the spermatocyte spreads of WT mice . Ratios of M1AP - DMC1 co - localizing foci to the M1AP total foci or DMC1 total foci at the indicated meiotic prophase substages . Each dot represents the ratio calculated from one nucleus .

Immunofluorescence staining of M1AP (green) and DMC1 (red) on the spermatocyte spreads of WT mice.   Ratios of M1AP-DMC1 co-localizing foci to the M1AP total foci or DMC1 total foci at the indicated meiotic prophase substages. Each dot represents the ratio calculated from one nucleus.


Immunofluorescence staining of M1AP ( green ) and RPA2 ( red ) on the spermatocyte spreads of WT mice . Ratios of M1AP - RPA2 co - localizing foci to the M1AP total foci or RPA2 total foci at the indicated meiotic prophase substages . Each dot represents the ratio calculated from one nucleus .

Immunofluorescence staining of M1AP (green) and RPA2 (red) on the spermatocyte spreads of WT mice.   Ratios of M1AP-RPA2 co-localizing foci to the M1AP total foci or RPA2 total foci at the indicated meiotic prophase substages. Each dot represents the ratio calculated from one nucleus.


Immunofluorescence staining of TEX11 ( green ) and SYCP3 ( red ) on the spermatocyte spreads of control and M1apKI / KI testes . The mean number of TEX11 foci per cell in control and M1apKI / KI spermatocytes . Frequencies of nuclei with TEX11 foci detected at the PAR in spread early pachytene spermatocytes with touching XY chromosomes .

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Immunofluorescence staining of TEX11 (green) and SYCP3 (red) on the spermatocyte spreads of control and M1apKI/KI testes.   The mean number of TEX11 foci per cell in control and M1apKI/KI spermatocytes.   Frequencies of nuclei with TEX11 foci detected at the PAR in spread early pachytene spermatocytes with touching XY chromosomes.


A . Western blot analysis of mTORC1 activity in C2C12 - Vevtor ( VC ) and Aster - C KO ( KO ) cells in complete medium ( CM ) and in response to nutrient starvation or stimulation with amino acids for 30 min . B and C . Statistical analysis of phosphorylated level of S6K ( B ) and 4E - BP1 ( C ) in C2C12 - Vector and Aster - C KO cells in CM , under nutrient starvation ( - AA ) and in response to stimulation with amino acids ( + AA ) using ImageJ software . Data are represented as mean ± SD ( n = 3 biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 by Student ' s t test .

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A. Western blot analysis of mTORC1 activity in C2C12-Vevtor (VC) and Aster-C KO (KO) cells in complete medium (CM) and in response to nutrient starvation or stimulation with amino acids for 30 min. B and C. Statistical analysis of phosphorylated level of S6K (B) and 4E-BP1 (C) in C2C12-Vector and Aster-C KO cells in CM, under nutrient starvation (-AA) and in response to stimulation with amino acids (+AA) using ImageJ software. Data are represented as mean ± SD (n=3 biological replicates). *p < 0.05, **p<0.01 by Student's t test.


D . Immunostaining of endogenous mTOR and LAMP1 in C2C12 - Vector and Aster - C KO cells under nutrient starvation ( - AA ) and in response to AA stimulation ( + AA ) . LAMP1 was immunostained as a lysosome marker . Arrows highlight the co - localization of mTOR with LAMP1 . Scale bar , 40 μm ( Row 1 ) and 10 μm ( Rows 2 , 3 , 4 ) .

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D. Immunostaining of endogenous mTOR and LAMP1 in C2C12-Vector and Aster-C KO cells under nutrient starvation (-AA) and in response to AA stimulation (+AA). LAMP1 was immunostained as a lysosome marker. Arrows highlight the co-localization of mTOR with LAMP1. Scale bar, 40 μm (Row 1) and 10 μm (Rows 2, 3, 4).


E . Statistical analysis of the Pearson ' s correlation coefficient of mTOR and LAMP1 in Figure 1F ( n = 12 - 15 cells per group ) . Data are represented as mean ± SD . * * * p < 0 . 001 by one way ANOVA .

E. Statistical analysis of the Pearson's correlation coefficient of mTOR and LAMP1 in Figure 1F (n=12-15 cells per group). Data are represented as mean ± SD. ***p<0.001 by one way ANOVA.


F . Western blot analysis of mTORC1 activity in Aster - C KO cells stably re - expressing exogenous GFP - Aster - C under nutrient starvation and AA stimulation . Aster - C KO cells were transfected with GFP - Aster - C and selected with G418 for 2 weeks .

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F. Western blot analysis of mTORC1 activity in Aster-C KO cells stably re-expressing exogenous GFP-Aster-C under nutrient starvation and AA stimulation. Aster-C KO cells were transfected with GFP-Aster-C and selected with G418 for 2 weeks.


G . Immunostaining of endogenous mTOR and LAMP1 in C2C12 - Vector and Aster - C KO cells under cholesterol depletion by treatment with 0 . 5 % MCD for 2 hrs ( - CHOL ) or cholesterol ( 50 µM ) re - stimulation for 1 hr ( + CHOL ) . Arrows highlight the co - localization of mTOR with LAMP1 . Scale bar , 40 μm ( Row 1 ) and 10 μm ( Rows 2 , 3 , 4 ) .

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G. Immunostaining of endogenous mTOR and LAMP1 in C2C12-Vector and Aster-C KO cells under cholesterol depletion by treatment with 0.5% MCD for 2 hrs (-CHOL) or cholesterol (50 µM) re-stimulation for 1 hr (+CHOL). Arrows highlight the co-localization of mTOR with LAMP1. Scale bar, 40 μm (Row 1) and 10 μm (Rows 2, 3, 4).


H . Western blot analysis of mTORC1 activity in C2C12 - Vector and Aster - C KO cells in CM , under starvation , cholesterol depletion by treatment with 0 . 5 % MCD for 2 hrs or cholesterol ( 50 µM ) re - stimulation for the indicated time .

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H. Western blot analysis of mTORC1 activity in C2C12-Vector and Aster-C KO cells in CM, under starvation, cholesterol depletion by treatment with 0.5% MCD for 2 hrs or cholesterol (50 µM) re-stimulation for the indicated time.


A . Confocal image analysis depicting the co - localization of Aster - C with the ER in C2C12 cells transiently expressing GFP - Aster - C and DsRed - ER under starvation and in response to AA stimulation . Scale bar , 5 μm .

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A. Confocal image analysis depicting the co-localization of Aster-C with the ER in C2C12 cells transiently expressing GFP-Aster-C and DsRed-ER under starvation and in response to AA stimulation. Scale bar, 5 μm.


B . Subcellular fractionation analysis of mTOR , GATOR2 complex and Aster - C localization in HEK293T cells transiently expressing FLAG - Aster - C under starvation and in response to AA stimulation . LAMP1 , Sec63 and GAPDH were used as lysosomal ( Lyso ) , rough ER ( RER ) and cytosol ( Cyto ) protein markers , respectively . Microsomal fraction ( Micro ) was also used in the blot .

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B. Subcellular fractionation analysis of mTOR, GATOR2 complex and Aster-C localization in HEK293T cells transiently expressing FLAG-Aster-C under starvation and in response to AA stimulation. LAMP1, Sec63 and GAPDH were used as lysosomal (Lyso), rough ER (RER) and cytosol (Cyto) protein markers, respectively. Microsomal fraction (Micro) was also used in the blot.


C . Subcellular fractionation analysis of mTOR and GATOR2 complex in C2C12 - Vector and Aster - C KO cells under starvation and in response to AA stimulation . LAMP1 , Sec63 and GAPDH were used as lysosomal ( Lyso ) , rough ER ( RER ) and cytosol ( Cyto ) protein markers , respectively .

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C. Subcellular fractionation analysis of mTOR and GATOR2 complex in C2C12-Vector and Aster-C KO cells under starvation and in response to AA stimulation. LAMP1, Sec63 and GAPDH were used as lysosomal (Lyso), rough ER (RER) and cytosol (Cyto) protein markers, respectively.


D . Co - IP analysis of the interaction of Aster - C with exogenous mTOR in response to starvation and AA or insulin stimulation in HEK293T cells transiently expressing the indicated proteins . Anti - Myc antibody was used for immunoprecipitation .

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D. Co-IP analysis of the interaction of Aster-C with exogenous mTOR in response to starvation and AA or insulin stimulation in HEK293T cells transiently expressing the indicated proteins. Anti-Myc antibody was used for immunoprecipitation.


E . Co - IP analysis of the interaction of Aster - C with endogenous mTOR and the GATOR2 complex using FLAG - Sestrin2 ( SESN2 ) as a positive control in HEK293T cells transiently expressing the indicated proteins . Anti - FLAG antibody was used for immunoprecipitation .

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E. Co-IP analysis of the interaction of Aster-C with endogenous mTOR and the GATOR2 complex using FLAG-Sestrin2 (SESN2) as a positive control in HEK293T cells transiently expressing the indicated proteins. Anti-FLAG antibody was used for immunoprecipitation.


F . Co - IP analysis of the interaction of Aster - C with mTOR , the GATOR2 complex , GATOR1 complex and the Ragulator - Rag complex using FLAG - DEPDC5 as a positive control in HEK293T cells transiently expressing the indicated proteins . Anti - FLAG antibody was used for immunoprecipitation . Black arrow indicates the RagC protein .

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F. Co-IP analysis of the interaction of Aster-C with mTOR, the GATOR2 complex, GATOR1 complex and the Ragulator-Rag complex using FLAG-DEPDC5 as a positive control in HEK293T cells transiently expressing the indicated proteins. Anti-FLAG antibody was used for immunoprecipitation. Black arrow indicates the RagC protein.


A . Co - IP analysis of the interaction of Aster - C with COPA and COPG in response to starvation and AA stimulation using FLAG - DEPDC5 as a negative control in HEK293T cells transiently expressing the indicated proteins . Anti - FLAG antibody was used for immunoprecipitation . Black arrow indicates the COPG protein .

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A. Co-IP analysis of the interaction of Aster-C with COPA and COPG in response to starvation and AA stimulation using FLAG-DEPDC5 as a negative control in HEK293T cells transiently expressing the indicated proteins. Anti-FLAG antibody was used for immunoprecipitation. Black arrow indicates the COPG protein.


B . Co - IP analysis of the interaction of COPA with mTOR in HEK293T cells transiently expressing GFP - COPA in response to starvation and AA stimulation . Anti - GFP antibody was used for immunoprecipitation . Black arrow indicates the COPG protein .

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B. Co-IP analysis of the interaction of COPA with mTOR in HEK293T cells transiently expressing GFP-COPA in response to starvation and AA stimulation. Anti-GFP antibody was used for immunoprecipitation. Black arrow indicates the COPG protein.


C . Co - IP analysis of the interaction of COPA with mTOR in C2C12 - Vector and Aster - C KO cells in response to starvation and AA stimulation . Anti - COPA antibody was used for immunoprecipitation .

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C. Co-IP analysis of the interaction of COPA with mTOR in C2C12-Vector and Aster-C KO cells in response to starvation and AA stimulation. Anti-COPA antibody was used for immunoprecipitation.


D . Confocal imaging analysis depicting the co - localization of YFP - mTOR ( yellow ) with GFP - COPA ( green ) and lysosomes ( red ) in live C2C12 - Vector and Aster - C KO cells in response to nutrient starvation and re - stimulation by AA . Arrows highlight co - localization of mTOR , COPA and lysosomes . Scale bar , 20 μm . E and F . Statistical analysis of the Pearson ' s correlation coefficient of mTOR / Lysosome ( E ) and COPA / Lysosome ( F ) in Figure 3D ( n = 10 - 12 cells per group ) . Data are represented as mean ± SD . * * * p < 0 . 001 by one - way ANOVA .

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D. Confocal imaging analysis depicting the co-localization of YFP-mTOR (yellow) with GFP-COPA (green) and lysosomes (red) in live C2C12-Vector and Aster-C KO cells in response to nutrient starvation and re-stimulation by AA. Arrows highlight co-localization of mTOR, COPA and lysosomes. Scale bar, 20 μm. E and F. Statistical analysis of the Pearson's correlation coefficient of mTOR/Lysosome (E) and COPA/Lysosome (F) in Figure 3D (n=10-12 cells per group). Data are represented as mean ± SD. ***p<0.001 by one-way ANOVA.


Time - lapse confocal imaging analysis of the co - localization of YFP - mTOR ( red ) with GFP - COPA ( green ) and lysosomes ( blue ) in live C2C12 cells under nutrient starvation ( 0 min ) , and in response to AA re - stimulation for indicated time ( 0 . 5 min , 2 min , 6 min and 8 min ) . Arrows highlight COPA puncta that were co - localized with mTOR or COPA puncta alone prior to their association with mTOR and lysosomes . . Scale bar , 5 μm .

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Time-lapse confocal imaging analysis of the co-localization of YFP-mTOR (red) with GFP-COPA (green) and lysosomes (blue) in live C2C12 cells under nutrient starvation (0 min), and in response to AA re-stimulation for indicated time (0.5 min, 2 min, 6 min and 8 min). Arrows highlight COPA puncta that were co-localized with mTOR or COPA puncta alone prior to their association with mTOR and lysosomes.. Scale bar, 5 μm.


A . Confocal imaging analysis depicting the lysosomal association of mTOR in response to AA stimulation in the presence or absence of BFA ( 10 μM ) or Exo - 2 ( 10 μM ) in live C2C12 cells transiently expressing YFP - mTOR and stained with Lysotracker Red . Scale bar , 20 μm .

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A. Confocal imaging analysis depicting the lysosomal association of mTOR in response to AA stimulation in the presence or absence of BFA (10 μM) or Exo-2 (10 μM) in live C2C12 cells transiently expressing YFP-mTOR and stained with Lysotracker Red. Scale bar, 20 μm.


Western blot analysis of mTORC1 activity in C2C12 cells in CM , in response to nutrient starvation and AA stimulation in the presence or absence of BFA ( 10 μM ) ( B )

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Western blot analysis of mTORC1 activity in C2C12 cells in CM, in response to nutrient starvation and AA stimulation in the presence or absence of BFA (10 μM) (B)


C . Western blot analysis of mTORC1 activity in C2C12 cells in CM , in response to nutrient starvation and AA stimulation in the presence or absence of Exo - 2 ( 10 μM ) ( C ) .

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C. Western blot analysis of mTORC1 activity in C2C12 cells in CM, in response to nutrient starvation and AA stimulation in the presence or absence of Exo-2 (10 μM) (C).


D . Western blot analysis of mTORC1 activity in C2C12 - Vector and ARF1 KO cells in CM , in response to nutrient starvation and AA stimulation .

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D. Western blot analysis of mTORC1 activity in C2C12-Vector and ARF1 KO cells in CM, in response to nutrient starvation and AA stimulation.


E . Confocal imaging analysis depicting COPA puncta ( green ) formation and co - localization with lysosomes ( red ) in live C2C12 cells transiently expressing GFP - COPA and stained with Lysotracker Red in response to nutrient starvation and AA stimulation in the presence or absence of BFA ( 10 μM ) or Exo - 2 ( 10 μM ) . Arrows highlight co - localization of COPA with lysosomes . Scale bar , 5 μm .

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E. Confocal imaging analysis depicting COPA puncta (green) formation and co-localization with lysosomes (red) in live C2C12 cells transiently expressing GFP-COPA and stained with Lysotracker Red in response to nutrient starvation and AA stimulation in the presence or absence of BFA (10 μM) or Exo-2 (10 μM). Arrows highlight co-localization of COPA with lysosomes. Scale bar, 5 μm.


F . Statistical analysis of the Pearson ' s correlation coefficient of COPA and lysosomes in Figure 5E ( n = 10 - 15 cells per group ) . Data are represented as mean ± SD . * * * p < 0 . 001 by one - way ANOVA .

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F. Statistical analysis of the Pearson's correlation coefficient of COPA and lysosomes in Figure 5E (n=10-15 cells per group). Data are represented as mean ± SD. ***p<0.001 by one-way ANOVA.


A . Co - IP analysis of the interaction of Aster - C with COPA , MYH10 , WDR24 and Mios in HEK293T cells transiently expressing the indicated proteins in response to treatment with brefeldin A ( BFA , 10 μM ) and / or blebbistatin ( BBS , 10 μM ) , inhibitors of ARFs and MYH10 , respectively . Anti - FLAG antibody was used for immunoprecipitation .

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A. Co-IP analysis of the interaction of Aster-C with COPA, MYH10, WDR24 and Mios in HEK293T cells transiently expressing the indicated proteins in response to treatment with brefeldin A (BFA, 10 μM) and/or blebbistatin (BBS, 10 μM), inhibitors of ARFs and MYH10, respectively. Anti-FLAG antibody was used for immunoprecipitation.


B . Western blot analysis of mTORC1 activity in wild type ( WT ) and MYH10 KO MEF cells cultured in CM , or in response to starvation and AA stimulation .

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B. Western blot analysis of mTORC1 activity in wild type (WT) and MYH10 KO MEF cells cultured in CM, or in response to starvation and AA stimulation.


C . Western blot analysis of mTORC1 activity in MYH10 KO MEF cells with stably re - expressed exogenous mCherry - MYH10 in response to starvation and AA stimulation . MYH10 KO MEF cells were transfected with mCherry - MYH10 and selected with G418 for 2 weeks .

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C. Western blot analysis of mTORC1 activity in MYH10 KO MEF cells with stably re-expressed exogenous mCherry-MYH10 in response to starvation and AA stimulation. MYH10 KO MEF cells were transfected with mCherry-MYH10 and selected with G418 for 2 weeks.


D . Confocal imaging analysis depicting COPA puncta ( green ) formation and co - localization with mTOR ( red ) in live C2C12 cells transiently expression GFP - COPA and YFP - mTOR in response to nutrient starvation in the presence or absence of BBS ( 10 µM ) . Arrows highlight the co - localization of mTOR with COPA . Scale bar , 20 μm .

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D. Confocal imaging analysis depicting COPA puncta (green) formation and co-localization with mTOR (red) in live C2C12 cells transiently expression GFP-COPA and YFP-mTOR in response to nutrient starvation in the presence or absence of BBS (10 µM). Arrows highlight the co-localization of mTOR with COPA. Scale bar, 20 μm.


E . Statistical analysis of the Pearson ' s correlation coefficient of mTOR and COPA in Figure 6D ( n = 10 - 12 cells per group ) . Data are represented as mean ± SD . * * * p < 0 . 001 by student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Statistical analysis of the Pearson's correlation coefficient of mTOR and COPA in Figure 6D (n=10-12 cells per group). Data are represented as mean ± SD. ***p<0.001 by student's t test.


A . Confocal imaging analysis depicting mTOR puncta ( green ) formation on actin filaments ( red ) in COS - 7 cells transiently expressing YFP - mTOR in response to AA stimulation in the presence or absence of latrunculin B ( LA - B , 100 nM ) . Actin filaments were stained using BODIPY ™ 558 / 568 - Phalloidin . Arrows indicated the association of mTOR with actin filaments . Scale bar , 5 μm .

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A. Confocal imaging analysis depicting mTOR puncta (green) formation on actin filaments (red) in COS-7 cells transiently expressing YFP-mTOR in response to AA stimulation in the presence or absence of latrunculin B (LA-B, 100 nM). Actin filaments were stained using BODIPY™ 558/568-Phalloidin. Arrows indicated the association of mTOR with actin filaments. Scale bar, 5 μm.


B . Immunostaining of endogenous mTOR and LAMP1 in C2C12 cells under amino acids starvation ( - AA ) , or in response to AA stimulation ( + AA ) in the presence or absence of LA - B ( 100 nM ) . Scale bar , 20 μm .

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B. Immunostaining of endogenous mTOR and LAMP1 in C2C12 cells under amino acids starvation (-AA), or in response to AA stimulation (+AA) in the presence or absence of LA-B (100 nM). Scale bar, 20 μm.


C . Western blot analysis of mTORC1 activity in C2C12 cells in CM , in response to nutrient starvation and AA stimulation in the presence or absence of LA - B ( 100 nM ) .

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C. Western blot analysis of mTORC1 activity in C2C12 cells in CM, in response to nutrient starvation and AA stimulation in the presence or absence of LA-B (100 nM).


D . Co - IP analysis of the interaction of Aster - C with COPA and MYH10 in the presence or absence of LA - B ( 100 nM ) or cytochalasin D ( Cyto - D , 250 nM ) in HEK293T cells transiently expressing FLAG - Aster - C . Anti - FLAG antibody was used for immunoprecipitation .

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D. Co-IP analysis of the interaction of Aster-C with COPA and MYH10 in the presence or absence of LA-B (100 nM) or cytochalasin D (Cyto-D, 250 nM) in HEK293T cells transiently expressing FLAG-Aster-C. Anti-FLAG antibody was used for immunoprecipitation.


A , Genome browser images of ChIP - seq for SAGA proteins before and after induction of ER stress at ER stress response genes CHOP , ERP70 , and GRP78 . ATF4 / Xbp1s binding sites are from publicly available datasets ( GEO accessions GSE69304 and GSE49952 ) .

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A, Genome browser images of ChIP-seq for SAGA proteins before and after induction of ER stress at ER stress response genes CHOP, ERP70, and GRP78. ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).


C , Mean ChIP - seq signal for USP22 , GCN5 , and ATXN7L3 surrounding gene promoters from B . Nonparametric Mann - Whitney analysis of 7 bins ( grey shade ) surrounding maximal peak in Thaps treated conditions are reported .

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C, Mean ChIP-seq signal for USP22, GCN5, and ATXN7L3 surrounding gene promoters from B. Nonparametric Mann-Whitney analysis of 7 bins (grey shade) surrounding maximal peak in Thaps treated conditions are reported.


A , HCT116 cells were treated with 100 nM Thapsigargin for 24 hrs . Cells were stained with dye recognizing cleaved Caspases 3 and 7 and imaged every 4 hours . Scale bar = 400 µm . B , Quantification of apoptotic populations as indicated by Cas3 / 7 fluorescence from A . Three independent experiments are represented as mean ± SEM , with significance calculated by Two - way ANOVA between conditions over time ( f ( 3 ) = 50 . 530 , p < 0 . 001 ) ; significant pairwise comparisons by Tukey ' s HSD post - hoc assessment are indicated by * . C

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A, HCT116 cells were treated with 100 nM Thapsigargin for 24 hrs. Cells were stained with dye recognizing cleaved Caspases 3 and 7 and imaged every 4 hours. Scale bar = 400 µm. B, Quantification of apoptotic populations as indicated by Cas3/7 fluorescence from A. Three independent experiments are represented as mean ± SEM, with significance calculated by Two-way ANOVA between conditions over time (f(3)=50.530, p<0.001); significant pairwise comparisons by Tukey's HSD post-hoc assessment are indicated by *. C


A , HCT116 cells were treated with 100 nM Thapsigargin for the indicated times in the presence or absence of shRNA targeting USP22 . Cells were harvested and whole - cell lysate was subjected to immunoblotting with the indicated antibodies .

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A, HCT116 cells were treated with 100 nM Thapsigargin for the indicated times in the presence or absence of shRNA targeting USP22. Cells were harvested and whole-cell lysate was subjected to immunoblotting with the indicated antibodies.


A , Genome browser images of ChIP - seq for Pol II and Ub - H2B before and after induction of ER stress , with and without USP22 knockdown , at ER stress response genes CHOP , ERP70 , and GRP78 . ATF4 / Xbp1s binding sites are from publicly available datasets ( GEO accessions GSE69304 and GSE49952 ) .

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A, Genome browser images of ChIP-seq for Pol II and Ub-H2B before and after induction of ER stress, with and without USP22 knockdown, at ER stress response genes CHOP, ERP70, and GRP78. ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).