Figure 5 ( A - C ) U2OS ATG2A / B double knock out ( DKO ) cells or DKO reconstituted with ATG2A - WT , - mLIR ( FCIL / AAAA ) or - mYFS ( YFS / AAA ) were starved for 2 h , fixed and stained for LC3B ( green ) or WIPI2 ( magenta ) , ( B ) ATG9A ( magenta ) or ( C ) GABARAP ( Magenta ) and imaged using a Zeiss 880 Airyscan super - resolution confocal microscope . Images are representatives of n = 3 independent experiments . Scale bars 10µm . Transmission electron micrographs of ATG2A / B DKO cells ( upper left ) , DKO + ATG2A - WT ( upper right ) , DKO + ATG2A - mLIR ( lower left ) and DKO + ATG2A - mYFS ( lower right ) starved ( EBSS ) for 2 h . Clustered small vesicles ( open arrowheads ; DKO and DKO - mLIR ) and autophagosomes ( closed arrow heads , DKO + ATG2A - WT and - mYFS ) indicated . Images are representative of n = 3 independent experiments . Scale bars 500nm . ER = endoplasmic reticulum ; N = Nucleus ; Mt = Mitochondria ; G = Golgi ; AP = Autophagosome .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A-C) U2OS ATG2A/B double knock out (DKO) cells or DKO reconstituted with ATG2A-WT, -mLIR (FCIL/AAAA) or -mYFS (YFS/AAA) were starved for 2 h, fixed and stained for LC3B (green) or WIPI2 (magenta), (B) ATG9A (magenta) or (C) GABARAP (Magenta) and imaged using a Zeiss 880 Airyscan super-resolution confocal microscope. Images are representatives of n=3 independent experiments. Scale bars 10µm.Transmission electron micrographs of ATG2A/B DKO cells (upper left), DKO + ATG2A-WT (upper right), DKO+ ATG2A-mLIR (lower left) and DKO+ATG2A-mYFS( lower right) starved (EBSS) for 2 h. Clustered small vesicles (open arrowheads; DKO and DKO-mLIR) and autophagosomes (closed arrow heads, DKO+ATG2A-WT and -mYFS) indicated. Images are representative of n=3 independent experiments. Scale bars 500nm. ER= endoplasmic reticulum; N = Nucleus; Mt = Mitochondria; G = Golgi; AP = Autophagosome.


Figure 1A Immunoblot analysis of overexpressed and ALFA - tagged CNX in CNX knockout cell lines clones # 1 and # 2 , which were used for mass spectrometry . Endogenous CNX level of wildtype HEK293T cells is shown as a control . C Volcano plots derived from LC - MS / MS analyses of ALFA - tagged CNX , immunoprecipitated in 1 % NP - 40 buffer from transfected CNX deficient HEK293T cells after DSSO crosslinking and compared to empty vector ( EV ) control co - IPs . Depicted is an enlarged section of the associated complete volcano plot in the upper right . Among the significantly enriched proteins , annotations are Uniprot gene names . CNX is shown in green . ER proteins with the GO - term annotation " endoplasmic reticulum " , which are localized to the ER lumen , are highlighted in blue and among those several known CNX interaction partners are indicated with their names . Membrane proteins are shown in red and those which are non - glycosylated are additionally labeled with their gene names . E Co - immunoprecipitations of endogenous CNX with FLAG - tagged Cx32 WT and its mutants including quantification and statistical analysis . Constructs were expressed in HEK293T cells . One representative immunoblot is shown . Monomers and dimers of Cx32 are indicated with brackets . All samples were normalized to WTF Immunofluorescence images of Cx32 and its mutants . COS - 7 cells were transiently transfected with the indicated FLAG - tagged Cx32 constructs and immunofluorescence microscopy was performed using anti PDI ( green ) as an ER marker . Detection of Cx32 was performed using anti FLAG antibodies and suitable labeled secondary antibodies ( red ) . Nuclei were stained with DAPI ( blue ) . All three channels are overlayed . Images are representative of cells from at least three different biological replicates . GJ denotes gap junctions observed at cell - cell junctions of cells transfected with WT Cx32 .

O B-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Immunoblot analysis of overexpressed and ALFA-tagged CNX in CNX knockout cell lines clones #1 and #2, which were used for mass spectrometry. Endogenous CNX level of wildtype HEK293T cells is shown as a control.C Volcano plots derived from LC-MS/MS analyses of ALFA-tagged CNX, immunoprecipitated in 1% NP-40 buffer from transfected CNX deficient HEK293T cells after DSSO crosslinking and compared to empty vector (EV) control co-IPs. Depicted is an enlarged section of the associated complete volcano plot in the upper right. Among the significantly enriched proteins, annotations are Uniprot gene names. CNX is shown in green. ER proteins with the GO-term annotation "endoplasmic reticulum", which are localized to the ER lumen, are highlighted in blue and among those several known CNX interaction partners are indicated with their names. Membrane proteins are shown in red and those which are non-glycosylated are additionally labeled with their gene names.E Co-immunoprecipitations of endogenous CNX with FLAG-tagged Cx32 WT and its mutants including quantification and statistical analysis. Constructs were expressed in HEK293T cells. One representative immunoblot is shown. Monomers and dimers of Cx32 are indicated with brackets. All samples were normalized to WTF Immunofluorescence images of Cx32 and its mutants. COS-7 cells were transiently transfected with the indicated FLAG-tagged Cx32 constructs and immunofluorescence microscopy was performed using anti PDI (green) as an ER marker. Detection of Cx32 was performed using anti FLAG antibodies and suitable labeled secondary antibodies (red). Nuclei were stained with DAPI (blue). All three channels are overlayed. Images are representative of cells from at least three different biological replicates. GJ denotes gap junctions observed at cell-cell junctions of cells transfected with WT Cx32.


Figure 2B Co - immunoprecipitations of endogenous CNX with the different CL - TMD constructs ( NVT glycosylation site mutated to QVT ) including quantification and statistical analysis . One representative immunoblot is shown . The asterisk ( * ) denotes a fraction of cleaved species as observed previously for similar constructs ( Behnke et al . , 2016 ) . All samples were normalized to the construct containing TMD1C Interaction analysis of Calnexin with the first transmembrane domain of Cx32 . Intracellular BMH crosslinking of transiently transfected V5 - tagged CNX with the transiently transfected R26C mutant of Cx32 - TMD1 . In the AA mutant of CNX , the two cysteines within the CNX TMD were replaced by alanines . The species at approx . 250 kDa are possibly CNX dimers . E COS - 7 cells were transiently transfected with the indicated constructs and immunofluorescence microscopy was performed using anti PDI ( green ) as an ER marker . Detection of minCNXTMD and both control constructs was carried out by mScarlet - I fluorescence ( red ) . Nuclei were stained with DAPI ( blue ) . Images are representative of cells from at least three different biological replicates . F Interaction of Cx32 - TMD1 with the minCNX system . MinCNXTMD constructs and controls ( see ( D ) ) were co - transfected with Cx32 - TMD1 into HEK293T cells . Interaction between the proteins was analyzed by co - immunoprecipitation experiments followed by immunoblotting .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B Co-immunoprecipitations of endogenous CNX with the different CL-TMD constructs (NVT glycosylation site mutated to QVT) including quantification and statistical analysis. One representative immunoblot is shown. The asterisk (*) denotes a fraction of cleaved species as observed previously for similar constructs (Behnke et al., 2016). All samples were normalized to the construct containing TMD1C Interaction analysis of Calnexin with the first transmembrane domain of Cx32. Intracellular BMH crosslinking of transiently transfected V5-tagged CNX with the transiently transfected R26C mutant of Cx32-TMD1. In the AA mutant of CNX, the two cysteines within the CNX TMD were replaced by alanines. The species at approx. 250 kDa are possibly CNX dimers.E COS-7 cells were transiently transfected with the indicated constructs and immunofluorescence microscopy was performed using anti PDI (green) as an ER marker. Detection of minCNXTMD and both control constructs was carried out by mScarlet-I fluorescence (red). Nuclei were stained with DAPI (blue). Images are representative of cells from at least three different biological replicates.F Interaction of Cx32-TMD1 with the minCNX system. MinCNXTMD constructs and controls (see (D)) were co-transfected with Cx32-TMD1 into HEK293T cells. Interaction between the proteins was analyzed by co-immunoprecipitation experiments followed by immunoblotting.


Figure 3B HEK293T cells were transiently transfected with plasmids expressing ConMem or the indicated variants , where either the first ( N1 ) or the second ( N2 ) NVT glycosylation motif was ablated ( N to Q mutation ) . Lysates were treated with or without EndoH or PNGaseF as indicated , and analyzed by immunoblotting . N - glycosylation occurs in the ER , complex glycosylation in the Golgi . C COS - 7 cells were transfected with the indicated constructs and fluorescence microscopy was performed using Sec61 mCherry ( red ) as an ER marker . ConMem , detected by sfGFP fluorescence ( green ) , localizes to the plasma membrane . Nuclei were stained with DAPI ( blue ) . Images are representative of cells from at least three different biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B HEK293T cells were transiently transfected with plasmids expressing ConMem or the indicated variants, where either the first (N1) or the second (N2) NVT glycosylation motif was ablated (N to Q mutation). Lysates were treated with or without EndoH or PNGaseF as indicated, and analyzed by immunoblotting. N-glycosylation occurs in the ER, complex glycosylation in the Golgi.C COS-7 cells were transfected with the indicated constructs and fluorescence microscopy was performed using Sec61 mCherry (red) as an ER marker. ConMem, detected by sfGFP fluorescence (green), localizes to the plasma membrane. Nuclei were stained with DAPI (blue). Images are representative of cells from at least three different biological replicates.


Figure 4B Representative blots from co - immunoprecipitation experiments from HEK293T cells transfected with the indicated ConMem constructs with or without ER - lumenal glycosylation site ( N1 ) ( mean ± SEM , N = 3 , * P value < 0 . 05 , two - tailed Student ' s t tests ) . Individual values were normalized to ConMem N1Q ( Val ) values that were set to 1 . C Interaction of ConMem N1Q variants as described in ( A ) with endogenous CNX ( mean ± SEM , N ≥ 5 , * P value < 0 . 05 , two - tailed Student ' s t tests ) . Individual values were normalized to ConMem N1Q ( Val ) values that were set to 1 . D Same as in ( C ) only that selected amino acids ( proline and arginine ) where shifted through the ConMem N1Q TMD segment to five different positions as shown in the illustration , revealing positional binding dependencies ( mean ± SEM , N ≥ 4 , * P value < 0 . 05 , two - tailed Student ' s t tests ) . Individual values were normalized to ConMem N1Q ( Val ) values that were set to 1 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B Representative blots from co-immunoprecipitation experiments from HEK293T cells transfected with the indicated ConMem constructs with or without ER-lumenal glycosylation site (N1) (mean ± SEM, N = 3, *P value < 0.05, two-tailed Student's t tests). Individual values were normalized to ConMem N1Q (Val) values that were set to 1.C Interaction of ConMem N1Q variants as described in (A) with endogenous CNX (mean ± SEM, N ≥ 5, *P value < 0.05, two-tailed Student's t tests ). Individual values were normalized to ConMem N1Q (Val) values that were set to 1. D Same as in (C) only that selected amino acids (proline and arginine) where shifted through the ConMem N1Q TMD segment to five different positions as shown in the illustration, revealing positional binding dependencies (mean ± SEM, N ≥ 4, *P value < 0.05, two-tailed Student's t tests). Individual values were normalized to ConMem N1Q (Val) values that were set to 1.


Figure 5D Interaction of non - glycosylated ConMem N1Q constructs with V5 - tagged CNX . In CNX variants , amino acids important for interaction as revealed in ( A ) were replaced by valine residues . HEK293T cells were transiently transfected with the indicated constructs and cell lysates and HA - immunoprecipitates were analyzed for HA - tagged ConMem N1Q and co - immunoprecipitating CNX mutants . E The same as in ( D ) for Cx32 - TMD1 F Analysis of mutations in the CNX TMD region and their effect on binding glycosylated ConMem . G The same as in ( D ) for Cx32 full - lengthH Interaction of Cx32 - TMD1 with CNX mutants , where amino acids not predicted to be important for interaction as shown in ( A ) were exchanged by valine residues . HEK293T cells were transiently transfected with the indicated constructs . I Shifting of the residues Y , T and L , most important vor CNX substrate interaction , and replacing these against valine residues , impacts interactions with Cx32 - TMD1 . HEK293T cells were transiently transfected with the indicated constructs .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5D Interaction of non-glycosylated ConMem N1Q constructs with V5-tagged CNX. In CNX variants, amino acids important for interaction as revealed in (A) were replaced by valine residues. HEK293T cells were transiently transfected with the indicated constructs and cell lysates and HA-immunoprecipitates were analyzed for HA-tagged ConMem N1Q and co-immunoprecipitating CNX mutants. E The same as in (D) for Cx32-TMD1 F Analysis of mutations in the CNX TMD region and their effect on binding glycosylated ConMem. G The same as in (D) for Cx32 full-lengthH Interaction of Cx32-TMD1 with CNX mutants, where amino acids not predicted to be important for interaction as shown in (A) were exchanged by valine residues. HEK293T cells were transiently transfected with the indicated constructs.I Shifting of the residues Y, T and L, most important vor CNX substrate interaction, and replacing these against valine residues, impacts interactions with Cx32-TMD1. HEK293T cells were transiently transfected with the indicated constructs.


Figure 6A Analysis of the interaction of the binding deficient CNX YTL TMD variant with Rhodopsin . CNX knockout HEK293T cells were transiently transfected with either N - terminal ALFA - tagged Rhodopsin alone or in combination with the indicated V5 - tagged CNX constructs . In the absence of any CNX , Rho is hardly detectable ( long exposure shown ) . Cell lysates and ALFA - immunoprecipitates were analyzed for Rhodopsin and co - immunoprecipitating CNX . B Analysis of Rhodopsin degradation and its dependency on CNX . CNX knockout HEK293T cells that were transfected with the indicated constructs were incubated with cycloheximide and lysates were collected at different timepoints . CNX WT and CNX YTL immunoblots are from the same blot ( dashed line ) , showing similar expression levels . Hsc70 was used as a loading control ( also from the same blot , dashed line ) . Quantifications are shown below representative

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A Analysis of the interaction of the binding deficient CNX YTL TMD variant with Rhodopsin. CNX knockout HEK293T cells were transiently transfected with either N-terminal ALFA-tagged Rhodopsin alone or in combination with the indicated V5-tagged CNX constructs. In the absence of any CNX, Rho is hardly detectable (long exposure shown). Cell lysates and ALFA-immunoprecipitates were analyzed for Rhodopsin and co-immunoprecipitating CNX.B Analysis of Rhodopsin degradation and its dependency on CNX. CNX knockout HEK293T cells that were transfected with the indicated constructs were incubated with cycloheximide and lysates were collected at different timepoints. CNX WT and CNX YTL immunoblots are from the same blot (dashed line), showing similar expression levels. Hsc70 was used as a loading control (also from the same blot, dashed line). Quantifications are shown below representative immunoblots


Figure 2 ( B ) Histogram displaying the SILAC ratios ( log2 ) of stress - induced phosphorylation sites in setup cdc55Δ ( upper panel ) and setup rts1Δ ( lower panel ) ( Hollenstein et al . , 2020 ) , stratified by their Hog1 - dependence ( grey area versus thick black line ) . ( D ) Line plot monitoring growth rate ( y - axis ) of indicated strains ( colored lines ) under different osmolarity conditions ( x - axis ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) Histogram displaying the SILAC ratios (log2) of stress-induced phosphorylation sites in setup cdc55Δ (upper panel) and setup rts1Δ (lower panel) (Hollenstein et al., 2020), stratified by their Hog1-dependence (grey area versus thick black line).(D) Line plot monitoring growth rate (y-axis) of indicated strains (colored lines) under different osmolarity conditions (x-axis).


Figure 4B ) Western - blot illustrating the depletion of AID - Cdc55 at various time points after addition of auxin ( upper panel ) ; Pgk2 serves as a negative control ( lower panel ) . ( D ) Transcriptional analysis of stress - induced expression of ESR genes CTT1 ( upper panel ) and PGM2 ( lower panel ) under different strain conditions ( i . e . presence / absence of IGO1 / 2 and induction of iAID - Cdc55 depletion ) . Measured time points after stress application are depicted on the x - axis , target gene mRNA levels are illustrated on the y - axis as fold over reference gene IPP1 , values are normalized relative to wild type without induction of iAID - Cdc55 depletion at 30 minutes post - stress . E ) Transcriptional analysis of stress - induced expression of ESR genes CTT1 ( upper panel ) and PGM2 ( lower panel ) at 30 minutes post - stress . Target gene mRNA levels are illustrated on the y - axis as fold over reference gene IPP1 , values are normalized relative to wild type without induction of iAID - Cdc55 depletion . dep . : Cdc55 depleted , n . d . : Cdc55 non - depleted .

O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B) Western-blot illustrating the depletion of AID-Cdc55 at various time points after addition of auxin (upper panel); Pgk2 serves as a negative control (lower panel).(D) Transcriptional analysis of stress-induced expression of ESR genes CTT1 (upper panel) and PGM2 (lower panel) under different strain conditions (i.e. presence/absence of IGO1/2 and induction of iAID-Cdc55 depletion). Measured time points after stress application are depicted on the x-axis, target gene mRNA levels are illustrated on the y-axis as fold over reference gene IPP1, values are normalized relative to wild type without induction of iAID-Cdc55 depletion at 30 minutes post-stress.E) Transcriptional analysis of stress-induced expression of ESR genes CTT1 (upper panel) and PGM2 (lower panel) at 30 minutes post-stress. Target gene mRNA levels are illustrated on the y-axis as fold over reference gene IPP1, values are normalized relative to wild type without induction of iAID-Cdc55 depletion. dep.: Cdc55 depleted, n.d.: Cdc55 non-depleted.


Figure 5 ( C ) Mobility shift assays monitoring phosphorylation - induced mobility changes of Gis1 ( two upper rows ) and Rph1 ( two lower rows ) upon hyperosmotic stress in wild type , igo1 ∆ igo2 ∆ and Cdc55 - depleted cells ( right - hand side ) . ( E ) Western blot phosphorylation shift assays of wild type Gis1 - myc ( upper panel ) and Rph1 - Flag ( lower panel ) , and mutated forms Gis1 - 5A - myc ( upper panel ) and Rph1 - 5A - Flag ( lower panel ) . The measurement was done using Phos - tag ( Kinoshita et al . , 2006 ) and at the indicated time points after exposure to hyperosmotic stress ( 0 . 5 M NaCl ) using Pgk1 protein levels as a negative control . The location of the mutated residues is shown in the respective schematics . Red boxes : demethylase associated domains ( JmjN and JmjC ) , blue box : zinc finger domain . ( F ) Transcriptional analysis of stress - induced expression of ESR genes CTT1 ( upper panel ) and PGM2 ( lower panel ) at 30 minutes post - stress across indicated strains expressing either wild type or point - mutated forms of Gis1 and Rph1 in a gis1 ∆ rph1 ∆ deletion background , with the empty vector as control .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(C) Mobility shift assays monitoring phosphorylation-induced mobility changes of Gis1 (two upper rows) and Rph1 (two lower rows) upon hyperosmotic stress in wild type, igo1∆igo2∆ and Cdc55-depleted cells (right-hand side).(E) Western blot phosphorylation shift assays of wild type Gis1-myc (upper panel) and Rph1-Flag (lower panel), and mutated forms Gis1-5A-myc (upper panel) and Rph1-5A-Flag (lower panel). The measurement was done using Phos-tag (Kinoshita et al., 2006) and at the indicated time points after exposure to hyperosmotic stress (0.5 M NaCl) using Pgk1 protein levels as a negative control. The location of the mutated residues is shown in the respective schematics. Red boxes: demethylase associated domains (JmjN and JmjC), blue box: zinc finger domain.(F) Transcriptional analysis of stress-induced expression of ESR genes CTT1 (upper panel) and PGM2 (lower panel) at 30 minutes post-stress across indicated strains expressing either wild type or point-mutated forms of Gis1 and Rph1 in a gis1∆rph1∆ deletion background, with the empty vector as control.


Figure 2A . Unified cell cycle map of unperturbed ( gray ) and serum - starved cells ( 1d : light blue , 7d : dark blue , N = 3007 cells ) . The proliferative cell cycle ( dotted gray line ) and the hypomitogenic arrest trajectory ( black dotted line ) are indicated on the map . Inset : Each treatment condition is shown individually on the unified map ( other conditions are shown in lighter gray ) . ( B ) Diffusion pseudotime , ( C ) p21 , ( D ) cyclin D1 , ( E ) DNA content of unperturbed ( left panels ) or serum - starved cells ( right panels ) are plotted on the map . ( F ) phospho / total RB of unperturbed ( left panels ) or serum - starved cells ( right panels ) are plotted on the map . G . Time - lapse imaging of cyclinD1 - mVenus intensity in unperturbed ( control , green ) and serum - starved cells ( blue ) . Cells were serum - starved for at least 8h prior to imaging . The solid line represents the population median and the shaded area indicates the 95 % confidence interval . N = 105 control cells and N = 111 starved cells . I . Median nuclear p27 abundance in serum - starved cells is plotted on the map .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O

Figure 2A. Unified cell cycle map of unperturbed (gray) and serum-starved cells (1d: light blue, 7d: dark blue, N = 3007 cells). The proliferative cell cycle (dotted gray line) and the hypomitogenic arrest trajectory (black dotted line) are indicated on the map. Inset: Each treatment condition is shown individually on the unified map (other conditions are shown in lighter gray).(B) Diffusion pseudotime, (C) p21, (D) cyclin D1, (E) DNA content of unperturbed (left panels) or serum-starved cells (right panels) are plotted on the map.(F) phospho/total RB of unperturbed (left panels) or serum-starved cells (right panels) are plotted on the map.G. Time-lapse imaging of cyclinD1-mVenus intensity in unperturbed (control, green) and serum-starved cells (blue). Cells were serum-starved for at least 8h prior to imaging. The solid line represents the population median and the shaded area indicates the 95% confidence interval. N = 105 control cells and N = 111 starved cells.I. Median nuclear p27 abundance in serum-starved cells is plotted on the map.


Figure 3Unified cell cycle map of unperturbed ( gray ) and etoposide - treated cells ( 1 μM ; 1d : light green , 2d : green , 3d : dark green , 4d : darker green - see inset , N = 4315 cells ) . The unperturbed cell cycle trajectory ( dotted gray line ) and two arrest trajectories ( into G02C and G04C ; black dotted lines ) are indicated on the map . Inset : Each condition is shown individually on the map ( other conditions are shown in lighter gray ) . B - E . ( B ) Diffusion pseudotime , ( C ) DNA content , ( D ) phospho / total RB and ( E ) p21 of unperturbed ( left panels ) or etoposide - treated cells ( right panels ) are plotted on the arrest architecture . Median nuclear values are shown . F . Time - lapse imaging of CDK2 activity ( DHB - mCherry , gray ) and p21 - YPet ( green ) intensity in etoposide - treated cells . Schematic shows the two arrest trajectories observed following etoposide treatment . Cells that successfully complete G2 ( " Mothers " , N = 32 cells ) but arrest following cell division ( " Daughters " , N = 45 cells ) are shown in the two leftmost panels . Cells that arrest in G2 ( N = 40 cells ) are shown in the rightmost panel . The solid lines represent population medians and the shaded area indicates the 95 % confidence interval .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Unified cell cycle map of unperturbed (gray) and etoposide-treated cells (1 μM; 1d: light green, 2d: green, 3d: dark green, 4d: darker green - see inset, N = 4315 cells). The unperturbed cell cycle trajectory (dotted gray line) and two arrest trajectories (into G02C and G04C; black dotted lines) are indicated on the map. Inset: Each condition is shown individually on the map (other conditions are shown in lighter gray).B-E. (B) Diffusion pseudotime, (C) DNA content, (D) phospho/total RB and (E) p21 of unperturbed (left panels) or etoposide-treated cells (right panels) are plotted on the arrest architecture. Median nuclear values are shown.F. Time-lapse imaging of CDK2 activity (DHB-mCherry, gray) and p21-YPet (green) intensity in etoposide-treated cells. Schematic shows the two arrest trajectories observed following etoposide treatment. Cells that successfully complete G2 ("Mothers", N = 32 cells) but arrest following cell division ("Daughters", N = 45 cells) are shown in the two leftmost panels. Cells that arrest in G2 (N = 40 cells) are shown in the rightmost panel. The solid lines represent population medians and the shaded area indicates the 95% confidence interval.


Figure 4A . 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 cellsB . 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 ) . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. 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 cellsB. 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).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.


Figure 5A . 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 cellsC - 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. 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 cellsC-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.


Figure 6D . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6D. 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.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.


Figure 1 ( E ) Weight and survival curves of neutrophil specific Nlrp3A350VneoR Gsdmd - / - mice . n ≥

O O B-PANEL_START O O O O O O O O O O O O O O O O O O

Figure 1(E) Weight and survival curves of neutrophil specific Nlrp3A350VneoR Gsdmd-/- mice. n≥18


Figure 3 ( 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

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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


Figure 4 ( 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

O O B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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 replicats


Figure 5 ( 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 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

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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 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 replicats


Figure 6 ( 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

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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 replicats


Figure 4 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.


Figure 5Panel 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Panel 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).


Figure 2 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.


Figure 4 ( 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 . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.(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.


Figure 5 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.


Figure 6 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.


Figure 1 ( 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 ( 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 . 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 . 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 .

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

Figure 1 (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  (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.  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. 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.


Figure 2 ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 (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. (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. (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.


Figure 3 ( 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 . ( 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 . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 (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.  (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. 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.


Figure 4 ( 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 . ( 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 . ( 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 . ( H ) H & E and SOFG staining of new bone in hind paws . Data information : CB : Cortical bone ; NB : New bone ( 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 . ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 (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.  (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.  (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.  (H) H&E and SOFG staining of new bone in hind paws. Data information: CB: Cortical bone; NB: New bone  (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.  (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


Figure 5 ( 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 . ( C ) RT - qPCR analysis of osteogenesis markers in hBMSC cells . n = 3 , one ‐ way ANOVA , Bonferroni post hoc . ( D ) Alizarin red staining of hBMSC cells . ( E ) 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 . ( 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 . ( 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 ( 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 ( 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 ( 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 . ( N ) Quantitative analysis of area of UF and CF . n = 5 , Student ' s t -

O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O

Figure 5 (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. (C) RT-qPCR analysis of osteogenesis markers in hBMSC cells. n=3, one‐way ANOVA, Bonferroni post hoc.  (D) Alizarin red staining of hBMSC cells. (E) 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. (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.  (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  (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  (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  (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. (N) Quantitative analysis of area of UF and CF. n=5, Student's t-test


Figure 6 ( 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 . ( 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 . ( 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 .

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

Figure 6 (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.  (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.  (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.


Figure 7 ( 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 ( 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 ( 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

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

Figure 7 (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  (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  (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


Figure 1Representative 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Representative 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.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.


Figure 2Western 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 . 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 .

O B-PANEL_START O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Western 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.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.


Figure 3Testicular 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 . Number of bivalents per nucleus . Frequencies of nuclei with 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 ) . Number of MLH1 foci per nucleus . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O B-PANEL_START O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O

Figure 3Testicular 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.Number of bivalents per nucleus.Frequencies of nuclei with 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).Number of MLH1 foci per nucleus.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.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.


Figure 4Immunofluorescence 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 . 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O

Figure 4Immunofluorescence 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.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.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.


Figure 5Western 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 . 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 .

O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Western 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.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.


Figure 6Co - 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 .

O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Co-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.