I : loss of SP1 expression is associated with a decrease in SWI / SNF occupancy of SP1 promoter an increase in H3K27me3 . Data represents ChIP - qPCR following 8 - hours or 18 - hours of 100nM mithramycin exposure immunoprecipitation of H3K27me3 ( 8 - hour , P = 0 . 009 ; 18 - hour P = 0 . 0001 ) ChIP quantitation is input ( ng of immunoprecipitated / input * 100 ) determined by absolute quantitation of sheared relative to a standard curve . Data represents mean with standard deviation derived from three independent experiments . P - values were determined by one - way ANOVA using Dunnet for multiple comparisons .

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I: The loss of SP1 expression is associated with a decrease in SWI/SNF occupancy of the SP1 promoter and an increase in H3K27me3. Data represents ChIP-qPCR analysis following 8-hours or 18-hours of 100nM mithramycin exposure and immunoprecipitation of H3K27me3 (8-hour, P= 0.009; 18-hour P=0.0001) ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons.


A : Heatmaps depicting ATAC - seq global structure following 8 - hours ( middle ) 18 - hours ( right ) 100nM MMA treatment . A 2kb window is centered on TSS . accessibility clusters at TSS does not change globally relative to solvent . of genes that gain accessibility or reduce accessibility on right .

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A: Heatmaps depicting ATAC-seq global chromatin structure following 8-hours (middle) and 18-hours (right) 100nM MMA treatment. A 2kb window is centered on the TSS. Chromatin accessibility clusters at the TSS and does not change globally relative to solvent. Quantification of genes that gain accessibility or reduce accessibility on the right.


E : IGV tracks of rhabdoid genes previously identified to be occupied by non - canonical SWI / SNF ( Michel et al . , 2018 ) . ID3 JUND3 decrease in H3K27ac occupancy accessibility following exposure to mithramycin for 8 - hours or 18 - hours .

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E: IGV tracks of rhabdoid tumor genes previously identified to be occupied by non-canonical SWI/SNF (Michel et al., 2018). ID3 and JUND3 decrease in H3K27ac occupancy and chromatin accessibility following exposure to mithramycin for 8-hours or 18-hours.


F : IGV tracks of rhabdoid genes previously identified to gain H3K27me3 upon SWI / SNF loss ( Erkek et al . , 2019 ) . CDK6 CDK2 decrease in H3K27ac occupancy accessibility following exposure to 8 - hours 18 - hours mithramycin treatment .

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F: IGV tracks of rhabdoid tumor genes previously identified to gain H3K27me3 upon SWI/SNF loss (Erkek et al., 2019). CDK6 and CDK2 decrease in H3K27ac occupancy and chromatin accessibility following exposure to 8-hours and 18-hours mithramycin treatment.


G : IGV tracks of genes identified to have an increase in accessibility , H3K27ac gene expression following mithramycin exposure . BMP1 ADIPOR1 play crucial roles in bone adipogenic differentiation , respectively .

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G: IGV tracks of genes identified to have an increase in accessibility, H3K27ac and gene expression following mithramycin exposure. BMP1 and ADIPOR1 play crucial roles in bone and adipogenic differentiation, respectively.


A : Immunohistochemistry recapitulates biochemistry described in vitro for mithramycin . G401 sections at 20X magnification stained with H & E , caspase 3 ( CC3 ; apoptosis ) or H3K27me3 . A marked increase in CC3 that correlates with H3K27me3 is seen only in mice treated with continuous infusion schedule but not vehicle . Scale bar ( lower left ) : 50μm .

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A: Immunohistochemistry analysis recapitulates the biochemistry described in vitro for mithramycin. G401 tumor sections at 20X magnification stained with H&E, cleaved caspase 3 (CC3; apoptosis) or H3K27me3. A marked increase in CC3 that correlates with H3K27me3 staining is seen only in mice treated with the continuous infusion schedule but not vehicle. Scale bar (lower left): 50μm.


B : Prolonged durable response cure of mice bearing G401 xenografts treated with 30mg / kg EC8042 administered continuously over 72 - hours . Treatment duration indicated by gray shaded box . Asterisk indicate an sacrificed due to unknown causes not related to progression or drug toxicity ( see text ) .

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B: Prolonged durable response and cure of mice bearing G401 xenografts treated with 30mg/kg EC8042 administered continuously over 72-hours. Treatment duration indicated by gray shaded box. Asterisk indicate an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity (see text).


C : Kaplan - Meier survival curves indicating extended survival for mice bearing established G401 xenografts treated with 3 - day continuous infusions of EC8042 in ( B ) . shaded box indicates duration of treatment . Asterisk indicate an sacrificed due to unknown causes not related to progression or drug toxicity ( see text ) .

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C: Kaplan-Meier survival curves indicating extended survival for mice bearing established G401 xenografts treated with the 3-day continuous infusions of EC8042 in (B). The shaded box indicates the duration of treatment. Asterisk indicate an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity (see text).


D : 20X image of section of G401 treated tumors stained for H3K27me3 , caspase 3 , γH2AX . sections compare vehicle to treatment started on day 1 with 30 mg / kg of EC8042 administered continuously for 72 - hours ( 3 - day pump ) . H3K27me3 increases correlates with apoptosis ( CC3 ) , however induction of CC3 is modest . γH2AX does not increase with treatment indicating damage is not responsible for these effects . Scale bar ( lower left ) : 50μm .

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D: 20X image of section of G401 treated tumors stained for H3K27me3, cleaved caspase 3, and γH2AX. The sections compare vehicle to treatment started on day 1 with 30 mg/kg of EC8042 administered continuously for 72-hours (3-day pump). H3K27me3 increases and correlates with apoptosis (CC3), however induction of CC3 is modest. γH2AX staining does not increase with treatment indicating DNA damage is not responsible for these effects. Scale bar (lower left): 50μm.


Immunohistochemistry of H & E stains from G401 xenograft tumors on 1 , 3 , 7 - days after treatment with vehicle or 3 - day EC8042 infusion . EC8042 treated xenograft tumors exhibit evidence of mesenchymal differentiation compared to vehicle . microCT of xenograft tumors on 7 - days after treatment exhibit enhanced calcification compared to vehicle . scale bar ( lower left ) : 100μm .

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Immunohistochemistry analysis of H&E stains from G401 xenograft tumors on 1, 3, and 7-days after treatment with vehicle or 3-day EC8042 infusion. EC8042 treated xenograft tumors exhibit evidence of mesenchymal differentiation compared to vehicle. microCT analysis of xenograft tumors on 7-days after treatment exhibit enhanced calcification compared to vehicle. IHC scale bar (lower left): 100μm.


B . RNAseq data are plotted in log2 fold change ( FC ) of protrusions over bodies against adjusted - log10 false discovery rate ( FDR ) ( n = 2 replicates , average FC values are represented ) . horizontal dashed marks FDR ( q ) = 0 . 05 threshold ; vertical dashed lines mark FC = 0 . 625 ( left ) or FC = 1 . 6 ( right ) thresholds .

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B. RNAseq data are plotted in log2 fold change (FC) levels of protrusions over cell bodies against adjusted -log10 false discovery rate (FDR) (n=2 replicates, average FC values are represented). The horizontal dashed line marks the FDR (q) = 0.05 threshold; vertical dashed lines mark the FC = 0.625 (left) or FC = 1.6 (right) thresholds.


C . Heat map represents k - means clustering of transcript log2 FC ( protrusions over bodies ) extracted from RNAseq datasets published elsewhere . corresponding HUVEC FC are shown in parallel .

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C. Heat map represents the k-means clustering of transcript log2 FC levels (protrusions over cell bodies) extracted from RNAseq datasets published elsewhere. The corresponding HUVEC FC levels are shown in parallel.


D . smFISH co - detection of k 5 mRNAs GAPDH in subconfluent motile HUVECs . arrows indicate orientation of localisation ; yellow dashed lines outline borders ; red circles highlight smFISH spots ; scale bars = 20

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D. smFISH co-detection of k 5 mRNAs and GAPDH in subconfluent motile HUVECs. arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 20 µm


E . Polarisation Index ( PI ) of k 5 mRNAs GAPDH in co - detected in HUVECs ( n ≥ 28 ; * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; Wilcoxon ) .

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E. Polarisation Index (PI) of k 5 mRNAs and GAPDH in co-detected in HUVECs (n≥28 cells; **P<0.01, ***P<0.001, ****P<0.0001; Wilcoxon test).


F . k 5 mRNAs PIs plotted against respective GAPDH PIs . slope of coloured lines represents average k 5 mRNA / GAPDH PI ; dashed grey represents a 1 : 1 ( n ≥ 28 ) .

F. k 5 mRNAs PIs plotted against respective GAPDH PIs. The slope of the coloured lines represents the average k 5 mRNA/GAPDH PI ratio; the dashed grey line represents a 1:1 ratio (n≥28 cells).


G . Top : distribution pattern of mRNAs clustered in k 2 , k 5 , k 7 GAPDH . Bottom : smFISH co - detection of exemplar k7 / k 2 mRNAs GAPDH in subconfluent motile HUVECs . arrows indicate orientation of localisation ; yellow dashed lines outline borders ; red circles highlight smFISH spots ; scale bars = 20

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G. Top: distribution pattern of mRNAs clustered in k 2, k 5, k 7 and GAPDH. Bottom: smFISH co-detection of exemplar k7 / k 2 mRNAs and GAPDH in subconfluent motile HUVECs. arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 20 µm


H . PIs of k 7 mRNAs GAPDH co - detected in HUVECs ( n ≥ 25 ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; paired t ) .

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H. PIs of k 7 mRNAs and GAPDH co-detected in HUVECs (n≥25 cells; *P<0.05, **P<0.01, ***P<0.001; paired t test).


I . PIs of k 2 mRNAs GAPDH co - detected in HUVECs ( n ≥ 19 ; * P < 0 . 05 ; Wilcoxon ) .

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I. PIs of k 2 mRNAs and GAPDH co-detected in HUVECs (n≥19 cells; *P<0.05; Wilcoxon test).


E . Left : representative subconfluent motile co - transfected with plasmids expressing Lyn - mCherry , MCP - GFPnls 24xMS2 - RAB13 3 ' UTR or 24xMS2 . Right : of with MCP - GFP localised to protrusions when co - transfected with full or deletion versions of RAB13 3 ' UTR ( n ≥ 3 experiments ) . arrowheads indicate non - localisation of MCP - GFPnls ; arrows indicate orientation of localisation ; yellow dashed lines outline borders ; scale bars = 20 µm For each k 5 mRNA 3 ' UTR , a diagram of full 3 ' UTR positions of motif is shown together with respective RNAseq mapped reads from HUVEC protrusions ; black arrowheads indicate predicted polyadenylation

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E. Left: representative subconfluent motile cells co-transfected with plasmids expressing Lyn-mCherry, MCP-GFPnls and 24xMS2-RAB13 3'UTR or 24xMS2. Right: percentage of cells with MCP-GFP localised to protrusions when co-transfected with full length or deletion versions of RAB13 3'UTR (n≥3 experiments). white arrowheads indicate non-nuclear localisation of MCP-GFPnls; arrows indicate the orientation of RNA localisation; yellow dashed lines outline cell borders; scale bars = 20 µm For each k 5 mRNA 3'UTR, a diagram of the full length 3'UTR and the positions of the RNA motif is shown together with the respective RNAseq mapped reads from HUVEC protrusions; black arrowheads indicate predicted polyadenylation sites


F . Left : representative co - transfected with plasmids expressing Lyn - mCherry , MCP - GFPnls 24xMS2 - k 5 3 ' UTRs . Right : of with MCP - GFP localised to protrusions when co - transfected with full or deletion versions of k 5 3 ' UTR ( n ≥ 3 experiments ) . arrowheads indicate non - localisation of MCP - GFPnls ; arrows indicate orientation of localisation ; yellow dashed lines outline borders ; scale bars = 20 µm For each k 5 mRNA 3 ' UTR , a diagram of full 3 ' UTR positions of motif is shown together with respective RNAseq mapped reads from HUVEC protrusions ; black arrowheads indicate predicted polyadenylation

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F. Left: representative cells co-transfected with plasmids expressing Lyn-mCherry, MCP-GFPnls and 24xMS2-k 5 3'UTRs. Right: percentage of cells with MCP-GFP localised to protrusions when co-transfected with full length or deletion versions of k 5 3'UTR (n≥3 experiments). white arrowheads indicate non-nuclear localisation of MCP-GFPnls; arrows indicate the orientation of RNA localisation; yellow dashed lines outline cell borders; scale bars = 20 µm For each k 5 mRNA 3'UTR, a diagram of the full length 3'UTR and the positions of the RNA motif is shown together with the respective RNAseq mapped reads from HUVEC protrusions; black arrowheads indicate predicted polyadenylation sites


B . Representative genotyping PCR demonstrates band shift in ∆ LE HUVECs .

B. Representative genotyping PCR demonstrates the band size shift in ∆LE HUVECs.


D . Wt ∆ LE HUVEC RNAseq mapped reads depicting RAB13 exon usage .

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D. Wt and ∆LE HUVEC RNAseq mapped reads depicting RAB13 exon usage.


E . of RAB13 mRNA smFISH spot number in Wt ∆ LE HUVECs ( n = 3 experiments ; ns : not significant ; unpaired t ) .

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E. Quantification of RAB13 mRNA smFISH spot number in Wt and ∆LE HUVECs (n=3 experiments; ns: not significant; unpaired t test).


F . Number of RAB13 mRNA smFISH spots plotted against respective Polarisation Index ( PI ) ( n = 29 ; ns : not significant ; linear regression ) .

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F. Number of RAB13 mRNA smFISH spots plotted against the respective Polarisation Index (PI) (n=29 cells; ns: not significant; linear regression).


G . Left : representative Western blotting ( WB ) of Wt ∆ LE HUVECs . Right : densitometry of WB data ( n = 3 samples ; ns : not significant ; unpaired t ) .

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G. Left: representative Western blotting (WB) of Wt and ∆LE HUVECs. Right: densitometry analysis of WB data (n=3 samples; ns: not significant; unpaired t test).


H . smFISH co - detection of RAB13 control GAPDH in Wt ∆ LE motile HUVECs cultured under subconfluent conditions . Arrows indicate orientation of localisation ; yellow dashed lines outline borders ; red circles highlight smFISH spots ; scale bars = 20 µm ( H ) . Bar charts are presented as means ± s . d .

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H. smFISH co-detection of RAB13 and control GAPDH in Wt and ∆LE motile HUVECs cultured under subconfluent conditions. Arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 20 µm (H). Bar charts are presented as means±s.d.


I . PI of RAB13 GAPDH co - detected in Wt ∆ LE HUVECs ( n = 29 ; P * * * < 0 . 001 , ns : not significant ; Mann - Whitney ) .

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I. PI of RAB13 and GAPDH co-detected in Wt and ∆LE HUVECs (n=29 cells; P***<0.001, ns: not significant; Mann-Whitney test).


J . RAB13 PI plotted against respective GAPDH PI . slope of coloured lines represents average RAB13 / GAPDH PI ; dashed grey represents a 1 : 1 ( n = 29 ) .

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J. RAB13 PI plotted against respective GAPDH PI. The slope of the coloured lines represents the average RAB13/GAPDH PI ratio; the dashed grey line represents a 1:1 ratio (n=29 cells).


A . Representative time - lapse microscopy of a bEnd . 3 co - transfected with plasmids expressing Lyn - mCherry , MCP - GFPnls 24xMS2 - RAB13 3 ' UTR . Arrowheads indicate

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A. Representative time-lapse microscopy of a bEnd.3 cell co-transfected with plasmids expressing Lyn-mCherry, MCP-GFPnls and 24xMS2-RAB13 3'UTR. Arrowheads indicate filopodia


B . Frequency of newly formed filopodia formed within 5 µm intervals relative to nearest MCP - GFPnls particle or a randomised ( ctrl ) position ( n = 99 filopodia ; * * P < 0 . 01 , ns : not significant ; Pearson r correlation ) .

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B. Frequency of newly formed filopodia formed within 5 µm intervals relative to the nearest MCP-GFPnls particle or a randomised (ctrl) position (n=99 filopodia; **P<0.01, ns: not significant; Pearson r correlation).


C . of newly - formed filopodia to MCP - GFPnls or a ctrl position plotted against filopodia duration ( n = 99 filopodia ; * P < 0 . 05 , ns : not significant ; Spearman r correlation ) .

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C. Distance of newly-formed filopodia to MCP-GFPnls or a ctrl position plotted against filopodia duration (n=99 filopodia; *P<0.05, ns: not significant; Spearman r correlation).


D . Wt ∆ LE HUVECs co - cultured on fibroblast monolayers . Endothelial were identified either with an antibody against endothelial marker PECAM - 1 ( left ) or through expression of a nucleofected plasmid encoding cytoskeletal marker Lifeact - GFP ( right ) . Arrowheads indicate

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D. Wt and ∆LE HUVECs co-cultured on fibroblast monolayers. Endothelial cells were identified either with an antibody against the endothelial cell marker PECAM-1 (left) or through expression of a nucleofected plasmid encoding the cytoskeletal marker Lifeact-GFP (right). Arrowheads indicate filopodia


E . Number of filopodia detected in co - cultured HUVECs ( n = 30 ; * * P < 0 . 01 ; unpaired t )

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E. Number of filopodia detected in co-cultured HUVECs (n=30 cells; **P<0.01; unpaired t test)


F . Number of filopodia detected in co - cultured HUVECs within 12 µm intervals relative to distal tip ( n = 30 ; * * P < 0 . 01 , P * * * < 0 . 001 , ns : not significant ; one - way ANOVA with Bonferroni ' s correction )

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F. Number of filopodia detected in co-cultured HUVECs within 12 µm intervals relative to cell distal tip (n=30 cells; **P<0.01, P***<0.001, ns: not significant; one-way ANOVA with Bonferroni's correction)


G . Number of filopodia detected in clones of co - cultured HUVECs within 12 µm intervals relative to distal tip .

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G. Number of filopodia detected in individual clones of co-cultured HUVECs within 12 µm intervals relative to cell distal tip.


B . Representative Puro - PLA experiments detecting newly synthesised RAB13 in HUVEC protrusions present in lower side of Transwell membranes . Puro : puromycin ; Aniso : anisomycin ; 1ary Abs : antibodies . arrowheads indicate Puro - PLA ; yellow dashed lines outline protrusion borders ( B ) ;

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B. Representative Puro-PLA experiments detecting newly synthesised RAB13 in HUVEC protrusions present in the lower side of Transwell membranes. Puro: puromycin; Aniso: anisomycin; 1ary Abs: primary antibodies. white arrowheads indicate Puro-PLA punctae; yellow dashed lines outline protrusion borders (B);


C . of RAB13 Puro - PLA normalised to protrusion ( n ≥ 40 protrusions ; * P < 0 . 05 , * * * * P < 0 . 0001 ; Kruskal - Wallis with Dunn ' s correction ) .

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C. Quantification of RAB13 Puro-PLA punctae normalised to protrusion area (n≥40 protrusions; *P<0.05, ****P<0.0001; Kruskal-Wallis test with Dunn's correction).


D . Representative RAB13 IF on migrating HUVECs .

D. Representative RAB13 IF assay on migrating HUVECs.


E . Left : representative Western blotting ( WB ) of siRNA transfected HUVECs . Right : densitometry of WB data ( n = 3 samples ; * P < 0 . 05 ; paired t ) .

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E. Left: representative Western blotting (WB) of siRNA transfected HUVECs. Right: densitometry analysis of WB data (n=3 samples; *P<0.05; paired t test).


F . Control ( ctrl ) RAB13 siRNA - treated HUVECs co - cultured on fibroblast monolayers . Endothelial were identified with an antibody against endothelial marker PECAM - 1 . black arrowheads indicate filopodia ( F )

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F. Control (ctrl) and RAB13 siRNA-treated HUVECs co-cultured on fibroblast monolayers. Endothelial cells were identified with an antibody against the endothelial cell marker PECAM-1. black arrowheads indicate filopodia (F)


G . Number of filopodia detected in co - cultured HUVECs ( n ≥ 35 ; * * P < 0 . 01 ; unpaired t ) .

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G. Number of filopodia detected in co-cultured HUVECs (n≥35 cells; **P<0.01; unpaired t test).


H . Number of filopodia detected in co - cultured HUVECs within 12 µm intervals relative to distal tip ( n ≥ 35 ; * P < 0 . 05 , ns : not significant ; Kruskal - Wallis with Dunn ' s correction ) .

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H. Number of filopodia detected in co-cultured HUVECs within 12 µm intervals relative to cell distal tip (n≥35 cells; *P<0.05, ns: not significant; Kruskal-Wallis test with Dunn's correction).


A . Left : Tg ( fli1ep : MCP - GFPnls ) zebrafish at 26 hours post fertilisation ( hpf ) displaying vascular - specific expression of MCP - GFPnls . Inset shows expression of MCP - GFPnls in intersomitic vessels ( ISVs ) sprouting from dorsal aorta ( DA ) . Middle : scheme depicts in vivo MS2 system strategy with fli1 enhancer / promoter ( fli1ep ) - driven expression of reporter constructs , simultaneous translation of Lyn - mCherry reporter binding of MCP - GFPnls to 24xMS2 - rab13 3 ' UTR . Right : scheme illustrates ISV expressing Lyn - mCherry imaged in panels B - D . TC : tip ; SC : stalk . Arrowheads indicate non - localisation of MCP - GFPnls ; arrows indicate direction of ISV sprouting ; yellow dashed lines outline ISV ( A )

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A. Left: Tg(fli1ep:MCP-GFPnls) zebrafish embryo at 26 hours post fertilisation (hpf) displaying vascular-specific expression of MCP-GFPnls. Inset shows the nuclear expression of MCP-GFPnls in the intersomitic vessels (ISVs) sprouting from the dorsal aorta (DA). Middle: scheme depicts the in vivo MS2 system strategy with fli1 enhancer/promoter (fli1ep)-driven expression of reporter constructs, simultaneous translation of Lyn-mCherry reporter and binding of MCP-GFPnls to 24xMS2-rab13 3'UTR. Right: scheme illustrates ISV cells expressing Lyn-mCherry imaged in panels B-D. TC: tip cell; SC: stalk cell. Arrowheads indicate non-nuclear localisation of MCP-GFPnls; arrows indicate direction of ISV sprouting; yellow dashed lines outline ISV (A)


( B - D ) Time - lapse microscopy of Tg ( fli1ep : MCP - GFPnls ) tip ( C ) stalk ( B , D ) displaying mosaic expression of Lyn - mCherry - 24xMS2 - rab13 3 ' UTR in ISV . Arrowheads indicate non - localisation of MCP - GFPnls ; arrows indicate direction of ISV sprouting ; yellow dashed lines outline ISV ( B - D )

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(B-D) Time-lapse microscopy of Tg(fli1ep:MCP-GFPnls) tip (C) and stalk (B,D) cells displaying mosaic expression of Lyn-mCherry-24xMS2-rab13 3'UTR in ISV cells. Arrowheads indicate non-nuclear localisation of MCP-GFPnls; arrows indicate direction of ISV sprouting; yellow dashed lines outline ISV cell (B-D) borders


B . Representative genotyping PCR demonstrates band shift in zebrafish harbouring a ∆ 482 rab13 3 ' UTR . Asterisk marks a heteroduplex formed between Wt ∆ 482 rab13 3 ' UTR PCR amplicons .

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B. Representative genotyping PCR demonstrates the band size shift in zebrafish harbouring a ∆482 rab13 3'UTR. Asterisk marks a heteroduplex formed between Wt and ∆482 rab13 3'UTR PCR amplicons.


D . RNAseq mapped reads depicting rab13 exon usage in Tg ( kdrl : EGFP ) rab13 + / + rab13 ∆ 3 ' UTR / ∆ 3 ' UTR zebrafish . Coloured lines indicate SNPs .

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D. RNAseq mapped reads depicting rab13 exon usage in Tg(kdrl:EGFP) rab13+/+ and rab13∆3'UTR /∆3'UTR zebrafish embryos. Coloured lines indicate SNPs.


E . qPCR of rab13 mRNA in 26 - 28 hpf clutch - matched sibling ( n ≥ 9 ; ns : not significant ; Kruskal - Wallis with Dunn ' s correction ) . : + / + , + / ∆ ∆ / ∆ represent Tg ( kdrl : EGFP ) rab13 + / + , rab13 + / ∆ 3 ' UTR rab13 ∆ 3 ' UTR / ∆ 3 ' UTR ,

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E. qPCR analysis of rab13 mRNA levels in individual 26-28 hpf clutch-matched sibling embryos (n≥9 embryos; ns: not significant; Kruskal-Wallis test with Dunn's correction). : +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3'UTR and rab13∆3'UTR /∆3'UTR embryos, respectively


F . smFISH detection of rab13 kdr mRNA in cultured GFP - expressing endothelial extracted from 48 hpf Tg ( kdrl : EGFP ) rab13 + / + rab13 ∆ 3 ' UTR / ∆ 3 ' UTR zebrafish . Arrows indicate orientation of localisation ; yellow dashed lines outline borders ; red circles highlight smFISH spots ; scale bars = 10 µm ( F ) .

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F. smFISH detection of rab13 and kdr mRNA in cultured GFP-expressing endothelial cells extracted from 48 hpf Tg(kdrl:EGFP) rab13+/+ and rab13∆3'UTR /∆3'UTR zebrafish embryos. Arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 10 µm (F).


G . Polarisation Index ( PI ) of rab13 kdr detected by smFISH in zebrafish ( n ≥ 8 ; * * P < 0 . 01 , ns : not significant ; one - way ANOVA with Bonferroni ' s correction ) . : + / + , + / ∆ ∆ / ∆ represent Tg ( kdrl : EGFP ) rab13 + / + , rab13 + / ∆ 3 ' UTR rab13 ∆ 3 ' UTR / ∆ 3 ' UTR ,

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G. Polarisation Index (PI) of rab13 and kdr detected by smFISH in individual zebrafish cells (n≥8 cells; **P<0.01, ns: not significant; one-way ANOVA with Bonferroni's correction). : +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3'UTR and rab13∆3'UTR /∆3'UTR embryos, respectively


H . rab13 PI plotted against respective kdr PI . slope of coloured lines represents average rab13 / kdr PI ; dashed grey represents a 1 : 1 ( n ≥ 8 ) . : + / + , + / ∆ ∆ / ∆ represent Tg ( kdrl : EGFP ) rab13 + / + , rab13 + / ∆ 3 ' UTR rab13 ∆ 3 ' UTR / ∆ 3 ' UTR ,

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

H. rab13 PI plotted against respective kdr PI. The slope of the coloured lines represents the average rab13/kdr PI ratio; the dashed grey line represents a 1:1 ratio (n≥8 cells). : +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3'UTR and rab13∆3'UTR /∆3'UTR embryos, respectively


A . Time - lapse confocal microscopy of representative Tg ( kdrl : EGFP ) rab13 + / + rab13 ∆ 3 ' UTR / ∆ 3 ' UTR . DLAV : dorsal longitudinal anastomotic vessel ; HM : horizontal myoseptum . T0 = 25 hpf ; arrowheads indicate extra branches emerging from main ISVs at HM position ; scale bars = 50 µm ( A ) .

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A. Time-lapse confocal microscopy of representative Tg(kdrl:EGFP) rab13+/+ and rab13∆3'UTR /∆3'UTR embryos. DLAV: dorsal longitudinal anastomotic vessel; HM: horizontal myoseptum. T0= 25 hpf; arrowheads indicate extra branches emerging from the main ISVs at the HM position; scale bars = 50 µm (A).


B . Frequency of ISV ectopic branching occurring at HM ( n = 4 experiments ; * P < 0 . 05 ; one - way ANOVA with Bonferroni ' s correction ) . + / + , + / ∆ ∆ / ∆ represent Tg ( kdrl : EGFP ) rab13 + / + , rab13 + / ∆ 3 ' UTR rab13 ∆ 3 ' UTR / ∆ 3 ' UTR , respectively ( B ) .

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B. Frequency of ISV ectopic branching occurring at the HM (n=4 experiments; *P<0.05; one-way ANOVA with Bonferroni's correction). +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3'UTR and rab13∆3'UTR /∆3'UTR embryos, respectively (B).


( a - d ) Rotenone ( Rot ) increased number of GFP - LC3 level of co - localization with mitochondria ( arrows ) in SH - SY5Y ( a - c ; 1 μM ) cortical neurons ( d ; 250 nM ) , quantified in Fig . 3f Supplementary Fig . S1b , c . Veh , vehicle .

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(a-d) Rotenone (Rot) increased the number of GFP-LC3 puncta and the level of co-localization with mitochondria (arrows) in SH-SY5Y cells (a-c; 1 μM) and primary cortical neurons (d; 250 nM), quantified in Fig. 3f and Supplementary Fig. S1b,c. Veh, vehicle.


( e , f ) Rotenone increased delivery of MitoTracker Green - stained mitochondria ( mt ) to LysoTracker Red ( LTR ) - stained lysosomes , inhibited by siRNA knockdown of ATG7 or LC3 in SH - SY5Y . Ctrl , control . Right : RNAi knockdown .

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(e,f) Rotenone increased delivery of MitoTracker Green-stained mitochondria (mt) to LysoTracker Red (LTR)-stained lysosomes, inhibited by siRNA knockdown of ATG7 or LC3 in SH-SY5Y cells. Ctrl, control. Right: RNAi knockdown.


( g ) Rotenone decreased IMM ( COXIV ) , OMM ( TOM40 ) matrix ( MnSOD ) expression in neurons , reversed by bafilomycin ( Baf ) inhibition of autolysosomal degradation , quantified in Supplementary Fig . S1e . Mean ± s . d . of n = 7 independent experiments for b , c , n = 3 independent experiments for f , g ( see Supplementary Table S4 for statistics source data ) ; * P0 . 05 versus vehicle control ; † P0 . 05 versus Rot / control siRNA . Scale bars , 10 μm . See Supplementary Fig . S7 for uncropped blots .

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

(g) Rotenone decreased IMM (COXIV), OMM (TOM40) and matrix (MnSOD) protein expression levels in primary neurons, reversed by bafilomycin (Baf) inhibition of autolysosomal degradation, quantified in Supplementary Fig. S1e. Mean ± s.d. of n = 7 independent experiments for b,c, and n = 3 independent experiments for f,g (see Supplementary Table S4 for statistics source data); *P0.05 versus vehicle control; †P0.05 versus Rot/control siRNA. Scale bars, 10 μm. See Supplementary Fig. S7 for uncropped blots.


( a , b ) IMM OMM fractions isolated from neurons following indicated treatments were lipid extracted for LC - MS . ( a ) MS spectra demonstrated increased cardiolipin ( CL ) content of OMM after rotenone ( Rot ) treatment , with diversification of cluster distribution to 7 clusters exhibited by IMM . Veh , vehicle . ( b ) Pie charts showing CL distribution between IMM OMM fractions from toxin - treated neurons , normalized to mitochondrial lipid phosphorus .

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(a,b) IMM and OMM fractions isolated from primary neurons following the indicated treatments were lipid extracted for LC-MS analysis. (a) MS spectra demonstrated increased cardiolipin (CL) content of the OMM after rotenone (Rot) treatment, with diversification of the cluster distribution to the 7 clusters exhibited by the IMM. Veh, vehicle. (b) Pie charts showing the CL distribution between IMM and OMM fractions from toxin-treated neurons, normalized to mitochondrial lipid phosphorus.


( c , d ) Treatment with rotenone caused significant increases in PLA2 - hydrolysable ( surface accessible ) CL assessed by LC - MS in cortical neurons ( c ; 250 nM ) SH - SY5Y ( d ; 1 μM ) .

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(c,d) Treatment with rotenone caused significant increases in PLA2-hydrolysable (surface accessible) CL assessed by LC-MS in primary cortical neurons (c; 250 nM) and SH-SY5Y cells (d; 1 μM).


( e , f ) Treatment with 6 - OHDA CCCP increased surface exposure of CL probed with annexin V in SH - SY5Y ( e , 120 μM ) parkin - expressing HeLa ( f , 20 μM ) , respectively . Mean ± s . d . of n = 3 independent experiments for c - f ( see Supplementary Table S4 for statistics source data ) ; * P0 . 05 versus vehicle control .

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(e,f) Treatment with 6-OHDA and CCCP increased surface exposure of CL probed with annexin V in SH-SY5Y (e, 120 μM) and parkin-expressing HeLa cells (f, 20 μM), respectively. Mean ± s.d. of n = 3 independent experiments for c-f (see Supplementary Table S4 for statistics source data); *P0.05 versus vehicle control.


( a - e ) SH - SY5Y expressing control ( Ctrl ) or scramblase - 3 siRNA # 508 ( PLS3 siRNA ) for 72 h were stained with MitoTracker Green FM , a transmembrane potential - independent dye , treated with rotenone ( Rot ; 1 μM ) . Veh , vehicle . ( a ) Anionic phospholipids exposed to outer surface of isolated mitochondria were quantified by flow cytometry for Alexa 647 - annexin V binding .

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(a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (a) Anionic phospholipids exposed to the outer surface of isolated mitochondria were quantified by flow cytometry for Alexa 647-annexin V binding.


( a - e ) SH - SY5Y expressing control ( Ctrl ) or scramblase - 3 siRNA # 508 ( PLS3 siRNA ) for 72 h were stained with MitoTracker Green FM , a transmembrane potential - independent dye , treated with rotenone ( Rot ; 1 μM ) . Veh , vehicle . ( b - d ) PLS3 knockdown had no effect on rotenone - induced autophagy ( b ) , but decreased mitophagy in SH - SY5Y treated with rotenone ( c ; 1 μM ) or 6 - OHDA ( d ; 120 μM ) .

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(a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (b-d) PLS3 knockdown had no effect on rotenone-induced autophagy (b), but decreased mitophagy in SH-SY5Y cells treated with rotenone (c; 1 μM) or 6-OHDA (d; 120 μM).


( a - e ) SH - SY5Y expressing control ( Ctrl ) or scramblase - 3 siRNA # 508 ( PLS3 siRNA ) for 72 h were stained with MitoTracker Green FM , a transmembrane potential - independent dye , treated with rotenone ( Rot ; 1 μM ) . Veh , vehicle . ( d , e ) effects of PLS3 siRNA were recapitulated using a second siRNA # 433 ( d , Supplementary Fig . S3b , g ) , reversed by transfection with an RNAi - resistant mouse PLS3 vector ( e ) . Inset : PLS3 overexpression .

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(a-e) SH-SY5Y cells expressing control (Ctrl) or scramblase-3 siRNA #508 (PLS3 siRNA) for 72 h were stained with MitoTracker Green FM, a transmembrane potential-independent dye, and treated with rotenone (Rot; 1 μM). Veh, vehicle. (d,e) The effects of PLS3 siRNA were recapitulated using a second siRNA #433 (d, Supplementary Fig. S3b,g), and reversed by transfection with an RNAi-resistant mouse PLS3 vector (e). Inset: PLS3 overexpression.


( f - i ) neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone ( 250 nM ) analysed for autophagy ( Supplementary Fig . S1b ) or mitophagy by either co - localization ( f )

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(f-i) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone (250 nM) and analysed for autophagy (Supplementary Fig. S1b) or mitophagy by either co-localization analysis (f)


( f - i ) neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone ( 250 nM ) analysed for autophagy ( Supplementary Fig . S1b ) or mitophagy by either co - localization ( f ) , or with mitochondrial immunoblotting ( g ) densitometry ( h , i Supplementary Fig . S4c ) . Knockdown efficiencies are shown in Supplementary Figs S3 S4 . Mean ± s . d . of n = 4 independent experiments for b , c , n = 3 independent experiments for a , d - f , h - i ( see Supplementary Table S4 for statistics source data ) ; * P0 . 05 versus vehicle ; † P0 . 05 versus toxin - treated control siRNA ; * * P0 . 05 versus Rot / PLS3 siRNA . See Supplementary Fig . S7 for uncropped blots .

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(f-i) Primary neurons transfected for 72 h with CLS siRNA or scrambled control siRNA were treated with rotenone (250 nM) and analysed for autophagy (Supplementary Fig. S1b) or mitophagy by either co-localization analysis (f), or with mitochondrial protein immunoblotting (g) and densitometry (h,i and Supplementary Fig. S4c). Knockdown efficiencies are shown in Supplementary Figs S3 and S4. Mean ± s.d. of n = 4 independent experiments for b,c, and n = 3 independent experiments for a,d-f,h-i (see Supplementary Table S4 for statistics source data); *P0.05 versus vehicle; †P0.05 versus toxin-treated control siRNA; **P0.05 versus Rot/PLS3 siRNA. See Supplementary Fig. S7 for uncropped blots.


( a ) Western blot showed beclin 1 cleavage products ( Mr49 K ) were not observed in neurons exposed to mitophagy - inducing sublethal concentrations of rotenone ( 250 nM × 2 h : Rot low ) or STS ( 100 nM × 2 h ) . However , beclin 1 was on exposure to a lethal dose of rotenone ( 1 mM × 24 h : Rot high ) . Veh , vehicle ; CTD , C - terminal domain .

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(a) Western blot analysis showed beclin 1 cleavage products (Mr49 K) were not observed in primary neurons exposed to mitophagy-inducing sublethal concentrations of rotenone (250 nM×2 h: Rot low) or STS (100 nM×2 h). However, beclin 1 was cleaved on exposure to a lethal dose of rotenone (1 mM×24 h: Rot high). Veh, vehicle; CTD, C-terminal domain.


( b , c ) Likewise , SH - SY5Y did not show beclin 1 cleavage on exposure to rotenone ( b ) or 6 - OHDA ( c ; duplicate lanes ) . Data are representative of 2 - 3 independent experiments per toxin .

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(b,c) Likewise, SH-SY5Y cells did not show beclin 1 cleavage on exposure to rotenone (b) or 6-OHDA (c; duplicate lanes). Data are representative of 2-3 independent experiments per toxin.


( d ) lysates from stable PINK1 - Flag - expressing SH - SY5Y were treated with 6 - OHDA ( 120 μM ) , rotenone ( 1 μM ) or FCCP ( 2 μM ) , analysed by Flag immunoblot for PINK1 - Flag .

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(d) Cell lysates from stable PINK1-Flag-expressing SH-SY5Y cells were treated with 6-OHDA (120 μM), rotenone (1 μM) or FCCP (2 μM), and analysed by Flag immunoblot for PINK1-Flag levels.


( e ) SH - SY5Y transfected with HA - parkin were treated with indicated toxins , immunolabelled for HA ( green ) mitochondrial p60 antigen ( red ) analysed for HA - parkin redistribution to mitochondria . FCCP elicited translocation of parkin to mitochondria ( arrows ) , which was not seen with other mitophagy stimuli .

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(e) SH-SY5Y cells transfected with HA-parkin were treated with the indicated toxins, immunolabelled for HA (green) and mitochondrial p60 antigen (red) and analysed for HA-parkin redistribution to mitochondria. FCCP elicited translocation of parkin to mitochondria (arrows), which was not seen with the other mitophagy stimuli.


( f ) mitochondrial pellets were prepared from SH - SY5Y treated with vehicle ( V ) , 6 - OHDA ( O , 120 μM ) or rotenone ( R , 1 μM ) . Gels were loaded with or mitochondrial , immunoblotted for p62 / SQSMT1 , indicated fractionation markers .

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(f) Cytosolic and mitochondrial pellets were prepared from SH-SY5Y cells treated with vehicle (V), 6-OHDA (O, 120 μM) or rotenone (R, 1 μM). Gels were loaded with cytosolic or mitochondrial proteins, and immunoblotted for p62/SQSMT1, and the indicated fractionation markers.


( g , h ) Confocal of p62 / SQSMT1 distribution in SH - SY5Y ( g ) in cortical neurons ( h ) that were co - transfected with mCherry - tagged p62 / SQSMT1 mitochondrially targeted GFP . Note recruitment of p62 to large mitochondrial ( g , yellow , arrows ) in FCCP - treated , which was not observed in control or rotenone - treated . Scale bars , 10 μm . See Supplementary Fig . S7 for uncropped blots .

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(g,h) Confocal analysis of p62/SQSMT1 distribution in SH-SY5Y cells (g) and in primary cortical neurons (h) that were co-transfected with mCherry-tagged p62/SQSMT1 and mitochondrially targeted GFP. Note recruitment of p62 to large mitochondrial aggregates (g, yellow, arrows) in FCCP-treated cells, which was not observed in control or rotenone-treated cells. Scale bars, 10 μm. See Supplementary Fig. S7 for uncropped blots.


( a ) Recombinant LC3 incubated with tetraoleoyl - CL ( TOCL ) or dioleoylphosphatidylcholine ( DOPC ) liposomes at indicated molar ratios was analysed by blue native PAGE ; liposome binding impaired gel entry of .

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(a) Recombinant LC3 incubated with tetraoleoyl-CL (TOCL) or dioleoylphosphatidylcholine (DOPC) liposomes at the indicated molar ratios was analysed by blue native PAGE; liposome binding impaired the gel entry of proteins.


( b ) Titration of phospholipids ( for example , TOCL ) to LC3 was performed to evaluate that prevents 50 % of LC3 from entering gels ( IC50 ) as an index of relative affinity . Bottom left inset : representative gel for TOCL / LC3 = 12 ; top right inset : comparison of IC50 values for TOCL versus dioleoyl - phosphatidic acid ( DOPA ) tetralinoleoyl - CL ( TLCL ) versus monolyso - trilinoleoyl - CL ( lyso - CL ) . IC50 for DOPG was > 15 . * P0 . 05 versus TOCL ; † P0 . 05 versus TLCL .

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(b) Titration of phospholipids (for example, TOCL) to LC3 was performed to evaluate the ratio that prevents 50% of the LC3 from entering the gels (IC50) as an index of relative affinity. Bottom left inset: representative gel for TOCL/LC3 = 12; top right inset: comparison of IC50 values for TOCL versus dioleoyl-phosphatidic acid (DOPA) and tetralinoleoyl-CL (TLCL) versus monolyso-trilinoleoyl-CL (lyso-CL). The IC50 for DOPG was >15. *P0.05 versus TOCL; †P0.05 versus TLCL.


( f - j ) arrowhead in e shows position of N - terminal truncation used to create GFP - LC3 deletion mutants , which were analysed for GFP - LC3 formation ( f , g , i ) participation in mitophagy ( h , j ) in response to indicated stimuli . Rot , rotenone ; Veh , vehicle .

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(f-j) The arrowhead in e shows the position of the N-terminal truncation used to create GFP-LC3 deletion mutants, which were analysed for GFP-LC3 puncta formation (f,g,i) and participation in mitophagy (h,j) in response to the indicated stimuli. Rot, rotenone; Veh, vehicle.


( e , k ) Dual point mutations were prepared on basis of predicted CL contact residues ( e ) , analysed for participation in mitophagy elicited by Rot or 6 - OHDA ( k ) . Inset : expression of GFP - LC3 plasmids ( uncropped blots in Supplementary Fig . S7 ) . UT , untransfected . Mean ± s . d . of n = 3 independent experiments for f - h , n = 4 independent experiments for i , j ( see Supplementary Table S4 for statistics source data ) ; * P0 . 05 versus respective vehicle . † P0 . 05 versus toxin - treated wild type ( WT ) .

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(e,k) Dual point mutations were prepared on the basis of predicted CL contact residues (e), and analysed for participation in mitophagy elicited by Rot or 6-OHDA (k). Inset: expression of GFP-LC3 plasmids (uncropped blots in Supplementary Fig. S7). UT, untransfected. Mean ± s.d. of n = 3 independent experiments for f-h, and n = 4 independent experiments for i,j (see Supplementary Table S4 for statistics source data); *P0.05 versus respective vehicle. †P0.05 versus toxin-treated wild type (WT).


D . Time course of pyrene actin polymerization showing influence of different SPIN90 constructs in reactions containing 50 nM Bos taurus Arp2 / 3 3 μM 15 % pyrene labeled actin . E . Plot of maximum polymerization rate versus of SPIN90 for reactions shown in ( D )

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D. Time course of pyrene actin polymerization showing the influence of different SPIN90 constructs in reactions containing 50 nM Bos taurus Arp2/3 complex and 3 μM 15% pyrene labeled actin. E. Plot of maximum polymerization rate versus concentration of SPIN90 for reactions shown in (D)


F . Plot of maximum polymerization rate versus of SPIN90 for pyrene actin polymerization assays containing 2 . 5 μM SPIN90 , 500 nM Arp2 / 3 , 3 μM actin indicated of SPIN90 ARM domain construct

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F. Plot of maximum polymerization rate versus concentration of SPIN90 for pyrene actin polymerization assays containing 2.5 μM SPIN90, 500 nM Arp2/3 complex, 3 μM actin and the indicated concentration of the SPIN90 ARM domain construct


G . Time courses of pyrene actin polymerization as described in panel ( D ) , except influence of SPIN90 middle segment is tested on its own or in presence of 351 - 722 construct

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G. Time courses of pyrene actin polymerization as described in panel (D), except the influence of the SPIN90 middle segment is tested on its own or in the presence of the 351-722 construct


B . Time courses of pyrene actin polymerization showing influence of mutations on ability of SPIN90 ( 269 - 722 ) to activate Arp2 / 3 . Reactions contained 3 μM 15 % pyrene labeled actin , 50 nM Bos taurus Arp2 / 3 , 20 μM wild type or mutant SPIN90 ( 269 - 722 ) . C . Plot of maximum polymerization rate versus of SPIN90 ( 269 - 722 ) for reactions as shown in panel ( B )

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B. Time courses of pyrene actin polymerization showing the influence of mutations on the ability of SPIN90(269-722) to activate Arp2/3 complex. Reactions contained 3 μM 15% pyrene labeled actin, 50 nM Bos taurus Arp2/3 complex, and 20 μM wild type or mutant SPIN90(269-722). C. Plot of maximum polymerization rate versus concentration of SPIN90(269-722) for reactions as shown in panel (B)


A . Plot of maximum polymerization rate versus of small molecule Arp2 / 3 inhibitor CK - 666 . Reactions contained 3 μM 15 % pyrene labeled actin , 50 nM Bos taurus Arp2 / 3 indicated concentrations of CK - 666

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A. Plot of maximum polymerization rate versus concentration of the small molecule Arp2/3 complex inhibitor CK-666. Reactions contained 3 μM 15% pyrene labeled actin, 50 nM Bos taurus Arp2/3 complex and the indicated concentrations of CK-666


B . SDS - PAGE gel showing results of actin filament copelleting . Actin filaments at 3 . 5 μM were used to co - pellet 0 . 6 μM Bos taurus Arp2 / 3 in presence or absence of indicated concentrations of SPIN90 ( 269 - 722 ) ( S : Soluble fraction , P : Pellet ) C . Plot of co - pelleting data as shown in ( B ) , run in quadruplicate with error bars showing standard

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B. SDS-PAGE gel showing results of actin filament copelleting assay. Actin filaments at 3.5 μM were used to co-pellet 0.6 μM Bos taurus Arp2/3 complex in the presence or absence of the indicated concentrations of SPIN90(269-722) (S: Soluble fraction, P: Pellet) C. Plot of co-pelleting data as shown in (B), run in quadruplicate with error bars showing the standard deviation


A ) Principal component ( PCA ) of - seq data derived from 54 single in 8 developmental stages . X axis shows first ( PC1 ) y axis second ( PC2 ) principal component . variance explained by each component is indicated in parenthesis . Morphological stages are differentially

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A) Principal component analysis (PCA) of RNA-seq data derived from 54 single embryos in 8 developmental stages. X axis shows the first (PC1) and y axis the second (PC2) principal component. The variance explained by each component is indicated in parenthesis. Morphological stages are differentially colored


B ) of X - chromosomal over autosomal median transcript abundances ( X / A ) of preblastoderm ( PB ) , in 7 developmental stages . Each data point is a single . A of 1 represents balanced transcript between X chromosome autosomes ( dotted horizontal ) .

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B) Ratio of X-chromosomal over autosomal median transcript abundances (X/A) of preblastoderm (PB), female and male embryos in 7 developmental stages. Each data point is a single embryo. A ratio of 1 represents balanced transcript levels between the X chromosome and autosomes (dotted horizontal line).


C ) Log2 Fold Change ( log2FC ) of - seq between ( F ) ( M ) of expressed genes on X chromosome ( X , left , total = 1515 ) on autosomes ( A , right , total = 7451 ) in 7 developmental stages .

C) Log2 Fold Change (log2FC) of RNA-seq between female (F) and male (M) embryos of expressed genes on X chromosome (X, left, total=1515) and on autosomes (A, right, total=7451) in 7 developmental stages.


D ) Log2 Fold Change ( log2FC ) of - seq between ( F ) ( M ) of X - chromosomal genes which are constitutively ( left , total = 303 ) or developmentally expressed ( right , total = 303 ) in 7 developmental stages . P - values ( pval ) are calculated by one - sample Wilcoxon signed rank to assess median log2FC values against zero ( balanced expression ) ; ns : non - significant ( p - value > 0 . 001 ) .

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D) Log2 Fold Change (log2FC) of RNA-seq between female (F) and male (M) embryos of X- chromosomal genes which are constitutively (left, total=303) or developmentally expressed (right, total=303) in 7 developmental stages. P-values (pval) are calculated by one-sample Wilcoxon signed rank test to assess the median log2FC values against zero (balanced expression); ns: non-significant (p-value > 0.001).


A ) Example genomic region of MSL2 MNase - ChIP - seq profiles in early ( stage 5 - 8 , orange ) late ( stage 13 - 15 , purple ) mixed - sex . binding is defined as sub - nucleosomal fragments ( 10 - 130 bp ) , while binding as mono - nucleosomal fragments ( 130 - 220 bp ) . Called peaks / regions are represented as boxes above tracks HAS below . Late - appearing regions are marked by a plus ( + ) sign . Graph represents pooled IP / Input signal of 3 biological replicates .

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A) Example genomic region of MSL2 MNase-ChIP-seq profiles in early (stage 5-8, orange) and late (stage 13-15, purple) mixed-sex embryos. DNA binding is defined as sub-nucleosomal fragments (10 - 130 bp), while chromatin binding as mono-nucleosomal fragments (130 - 220 bp). Called peaks/regions are represented as boxes above the tracks and HAS below. Late-appearing regions are marked by a plus (+) sign. Graph represents pooled IP/Input signal of 3 biological replicates.


B ) Average composite plots of sub - nucleosomal MSL2 MNase - ChIP - seq ( ) centered at PionX ( left ) or HAS ( right ) in early late mixed - sex . Total number of PionX = 56 HAS = 247 . Graph represents average IP / Input signal of 3 biological replicates . Shaded indicates 95 % confidence interval across sites .

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B) Average composite plots of sub-nucleosomal MSL2 MNase-ChIP-seq (DNA) centered at PionX (left) or HAS (right) in early and late mixed-sex embryos. Total number of PionX = 56 and HAS = 247. Graph represents average IP/Input signal of 3 biological replicates. Shaded area indicates the 95% confidence interval across sites.


C ) Average composite plots of mono - nucleosomal MSL2 ChIP - seq ( ) centered at PionX ( left ) or HAS ( right ) in early late mixed - sex .

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C) Average composite plots of mono-nucleosomal MSL2 ChIP-seq (Chromatin) centered at PionX (left) or HAS (right) in early and late mixed-sex embryos.


B ) Average composite plots of MSL2 [ ] profiles ( stage 5 - 8 , orange stage 13 - 15 , purple ) centered at MSL2 peaks which do not overlap ( ) or overlap MSL2 regions ( + ) , MSL2 regions which do not overlap MSL2 peaks ( ) . Graphs represent average IP / Input signal of 3 biological replicates . Shaded indicates 95 % confidence interval across sites .

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B) Average composite plots of MSL2 [Chromatin] profiles (stage 5-8, orange and stage 13-15, purple) centered at MSL2 DNA peaks which do not overlap (DNA) or overlap MSL2 chromatin regions (DNA + Chromatin), and MSL2 chromatin regions which do not overlap MSL2 DNA peaks (Chromatin). Graphs represent average IP/Input signal of 3 biological replicates. Shaded area indicates the 95% confidence interval across sites.


C ) Average composite plots of MSL2 [ ] profiles centered at MSL2 regions called in both early late ( left ) or only in late stages ( right ) . Graphs represent average IP / Input signal of 3 biological replicates . Shaded indicates 95 % confidence interval across regions . Total number of early = 212 late = 87 .

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C) Average composite plots of MSL2 [Chromatin] profiles centered at MSL2 chromatin regions called in both early and late (left) or only in late stages (right). Graphs represent average IP/Input signal of 3 biological replicates. Shaded area indicates the 95% confidence interval across regions. Total number of early = 212 and late = 87.


D ) Average composite plots of MOF [ ] profiles centered at MSL2 regions called in both early late ( left ) or only in late stages ( right ) .

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D) Average composite plots of MOF [Chromatin] profiles centered at MSL2 chromatin regions called in both early and late (left) or only in late stages (right).


A ) Example genomic region of binding by MSL2 ( ) , binding of MSL2 ( Chrom . ) , MOF H4K16ac MNase - ChIP - seq profiles in early ( stage 5 - 8 , orange ) late ( stage 13 - 15 , purple ) mixed - sex . Called peaks / regions are represented as boxes above tracks . Graph represents pooled IP / Input signal of 3 biological replicates .

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A) Example genomic region of DNA binding by MSL2 (DNA), and chromatin binding of MSL2 (Chrom.), MOF and H4K16ac MNase-ChIP-seq profiles in early (stage 5-8, orange) and late (stage 13-15, purple) mixed-sex embryos. Called peaks/regions are represented as boxes above the tracks. Graph represents pooled IP/Input signal of 3 biological replicates.


B ) distribution of X - chromosomal MOF H4K16ac regions , which are present in both early late . P - value ( Wilcoxon rank sum ) is indicated . Total number of regions are 459 , 443 , 854 400 , respectively .

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B) Size distribution of X-chromosomal MOF and H4K16ac regions, which are present in both early and late embryos. P-value (Wilcoxon rank sum test) is indicated. Total number of regions are 459, 443, 854 and 400, respectively.


C ) Average composite plots of MOF MNase - ChIP - seq centered at Transcription Start Site ( TSS ) or Transcription Termination Site ( TTS ) in early or late for X - chromosomal genes ( total = 1515 ) . Graph represents average IP / Input signal of 3 biological replicates . Shaded indicates 95 % confidence interval across sites .

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C) Average composite plots of MOF MNase-ChIP-seq centered at Transcription Start Site (TSS) or Transcription Termination Site (TTS) in early or late embryos for X-chromosomal genes (total=1515). Graph represents average IP/Input signal of 3 biological replicates. Shaded area indicates the 95% confidence interval across sites.


D ) Average composite plots of H4K16ac MNase - ChIP - seq centered at TSS or TTS in early or late for X - chromosomal genes ( total = 1515 ) .

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D) Average composite plots of H4K16ac MNase-ChIP-seq centered at TSS or TTS in early or late embryos for X-chromosomal genes (total=1515).


A ) Scatterplot comparing X - chromosomal genic H4K16ac MNase - ChIP - seq in early ( stage 5 - 8 ) late ( stage 13 - 15 ) mixed - sex . Genes are grouped by k - means clustering ( k = 3 ) . numbers of genes are cluster 1 = 979 , cluster 2 = 364 cluster 3 = 172 . B ) Log2 Fold Change ( log2FC ) of - seq between ( F ) ( M ) at genes grouped as in A ( cluster 1 - 3 ) .

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A) Scatterplot comparing X-chromosomal genic H4K16ac MNase-ChIP-seq levels in early (stage 5-8) and late (stage 13-15) mixed-sex embryos. Genes are grouped by k-means clustering (k=3). The numbers of genes are cluster 1 = 979, cluster 2 = 364 and cluster 3 = 172. B) Log2 Fold Change (log2FC) of RNA-seq between female (F) and male (M) embryos at genes grouped as in A (cluster 1-3).


C ) Log10 between middle of a gene its nearest functional MSL2 peak ( cluster 1 - 3 ) . Red indicates median to nearest peak .

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C) Log10 distance between the middle of a gene and its nearest functional MSL2 DNA peak (cluster 1-3). Red line indicates median distance to the nearest peak.


A ‐ F ′ Confocal of wild ‐ type ( A , C , E ) mtm ‐ 3 ( tm4475 ) ( B , D , F ) at 1 . 5 ‐ fold stage stained by DAPI ( A - F ) anti ‐ SQST ‐ 1 ( A ′ , B ′ ) , anti ‐ SEPA ‐ 1 ( C ′ , D ′ ) or anti ‐ LGG ‐ 1 ( E ′ , F ′ ) antibodies . Scale bars : 5 μm .

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A‐F′ Confocal fluorescent images of wild‐type (A, C, E) and mtm‐3(tm4475) (B, D, F) embryos at the 1.5‐fold stage stained by DAPI (A-F) and anti‐SQST‐1 (A′, B′), anti‐SEPA‐1 (C′, D′) or anti‐LGG‐1 (E′, F′) antibodies. Scale bars: 5 μm.


G Western blot of LGG ‐ 1 ‐ I LGG ‐ 1 - II ( lipidated ) in wild type , epg ‐ 6 , mtm ‐ 3 .

G Western blot analysis of LGG‐1‐I and LGG‐1-II (lipidated) in wild type, epg‐6, and mtm‐3.


H mtm ‐ 3 epg ‐ 6 mutant L1 larvae have shortened mean maximum life span in absence of food . At least 300 were scored each day .

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H mtm‐3 and epg‐6 mutant L1 larvae have shortened mean and maximum life span in the absence of food. At least 300 animals were scored each day.


A - C of wild ‐ type ( 1 . 5 ‐ fold stage ) expressing GFP : : MTM ‐ 3 ( A ) , GFP : : MTM ‐ 3 ( C459S ) ( B ) , or GFP : : MTM ‐ 3 ( C459S , del PH ‐ G ) ( C ) .

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A-C Fluorescent images of wild‐type embryos (1.5‐fold stage) expressing GFP::MTM‐3 (A), GFP::MTM‐3(C459S) (B), or GFP::MTM‐3(C459S, del PH‐G) (C).


D ‐ G ″ Confocal of wild ‐ type ( D ‐ D ″ ) , mtm ‐ 3 ( E ‐ E ″ ) , atg ‐ 2 ( F ‐ F ″ ) , vps ‐ 34 ( G ‐ G ″ ) at 200 ‐ stage expressing GFP : : MTM ‐ 3 ( C459S ) stained by anti ‐ LGG ‐ 1 antibody . Insets show magnified views . Arrows indicate overlapping GFP LGG ‐ 1 . Scale bars : 5 μm .

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D‐G″ Confocal fluorescent images of wild‐type (D‐D″), mtm‐3 (E‐E″), atg‐2 (F‐F″), and vps‐34 (G‐G″) embryos at the 200‐cell stage expressing GFP::MTM‐3(C459S) and stained by anti‐LGG‐1 antibody. Insets show magnified views. Arrows indicate overlapping GFP and LGG‐1 puncta. Scale bars: 5 μm.


H , I Number of GFP : : MTM ‐ 3 ( C459S ) ‐ positive co ‐ localization of GFP : : MTM ‐ 3 ( C459S ) ‐ LGG ‐ 1 ‐ positive in various strains [ mtm ‐ 3 ( tm4475 ) , atg ‐ 2 ( bp576 ) , lgg ‐ 1 ( bp500 ) , vps ‐ 34 ( h797 ) ] . At least 10 ( I ) 15 ( H ) were scored per . Data are shown as mean ± SD . Data from different mutant backgrounds were compared with wild type . * * P 0 . 0001 ; N . S . : not statistically different .

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H, I Number of GFP::MTM‐3(C459S)‐positive puncta and co‐localization of GFP::MTM‐3(C459S)‐ and LGG‐1‐positive puncta in various strains [mtm‐3(tm4475), atg‐2(bp576), lgg‐1(bp500), vps‐34(h797)]. At least 10 (I) and 15 (H) embryos were scored per strain. Data are shown as mean ± SD. Data from different mutant backgrounds were compared with wild type. **P 0.0001; N.S.: not statistically different.


J MTM ‐ 3 but not MTM ‐ 3 ( C459S ) exhibits phosphatase activity toward PtdIns3P PtdIns ( 3 , 5 ) P2 in vitro .

J MTM‐3 but not MTM‐3(C459S) exhibits phosphatase activity toward PtdIns3P and PtdIns(3,5)P2 in vitro.


A - K Confocal of 200 ‐ ‐ stage from indicated strains stained by both anti ‐ SEPA ‐ 1 anti ‐ LGG ‐ 1 antibodies . Insets show magnified views . Arrows indicate overlapping SEPA ‐ 1 LGG ‐ 1 . wild type contains no visible SEPA ‐ 1 at this stage is therefore not shown . Scale bars : 5 μm .

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A-K Confocal fluorescent images of 200‐cell‐stage embryos from the indicated strains stained by both anti‐SEPA‐1 and anti‐LGG‐1 antibodies. Insets show magnified views. Arrows indicate overlapping SEPA‐1 and LGG‐1 puncta. The wild type contains no visible SEPA‐1 aggregates at this stage and is therefore not shown. Scale bars: 5 μm.


L of SEPA ‐ 1 co ‐ localizing with LGG ‐ 1 in each . At least 10 were scored per . Data are shown as mean ± SD . * * P 0 . 0001 ; N . S . : not statistically different .

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

L The percentage of SEPA‐1 puncta co‐localizing with LGG‐1 in each strain. At least 10 embryos were scored per strain. Data are shown as mean ± SD. **P 0.0001; N.S.: not statistically different.


A - G Confocal of 1 . 5 ‐ fold ‐ stage from indicated strains expressing both SQST ‐ 1 : : GFP NUC ‐ 1 : : mCHERRY . Insets show magnified views . Arrows indicate overlapping or closely associated GFP mCHERRY . Scale bars : 5 μm . wild type contains no visible SQST ‐ 1 : : GFP at this stage is therefore not shown .

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A-G Confocal fluorescent images of 1.5‐fold‐stage embryos from the indicated strains expressing both SQST‐1::GFP and NUC‐1::mCHERRY. Insets show magnified views. Arrows indicate overlapping or closely associated GFP and mCHERRY. Scale bars: 5 μm. The wild type contains no visible SQST‐1::GFP puncta at this stage and is therefore not shown.


H , I of SQST ‐ 1 : : GFP ‐ positive that overlap with ( H ) or are close to ( I ) NUC ‐ 1 : : mCHERRY ‐ positive structures in each . At least 10 were scored per . Data are shown as mean ± SD . * * P 0 . 0001 ; N . S . : not statistically different .

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

H, I The percentage of SQST‐1::GFP‐positive puncta that overlap with (H) or are close to (I) NUC‐1::mCHERRY‐positive structures in each strain. At least 10 embryos were scored per strain. Data are shown as mean ± SD. **P 0.0001; N.S.: not statistically different.


J - L of in wild type ( I ) mtm ‐ 3 ( tm4475 ) ( J , K ) . Double ‐ autophagosomes ( arrows ) were observed in mtm ‐ 3 but not wild ‐ type . Scale bars : 500 nm .

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J-L TEM analysis of embryos in wild type (I) and mtm‐3(tm4475) (J, K). Double‐membrane autophagosomes (arrows) were observed in mtm‐3 but not wild‐type embryos. Scale bars: 500 nm.


A - H of at 4 ‐ fold stage in indicated strains expressing ATG ‐ 18 : : GFP ( A - D ) , ATG ‐ 18 ( FKKG ) : : GFP ( E , F ) , or ATG ‐ 18 ( FTTG ) : : GFP ( G , H ) .

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A-H Fluorescent images of embryos at the 4‐fold stage in the indicated strains expressing ATG‐18::GFP (A-D), ATG‐18(FKKG)::GFP (E, F), or ATG‐18(FTTG)::GFP (G, H).


J Number of wild ‐ type or mutated ATG ‐ 18 : : GFP in each . At least 15 were scored per ( mean ± SD ) . * * P 0 . 0001 ; N . S : not statistically different .

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J Number of wild‐type or mutated ATG‐18::GFP puncta in each strain. At least 15 embryos were scored per strain (mean ± SD). **P 0.0001; N.S: not statistically different.


I Confocal of 4 ‐ fold mtm ‐ 3 expressing ATG ‐ 18 : : GFP ( I ′ ) stained by DAPI ( I ) anti ‐ LGG ‐ 1 ( I ″ ) antibody . Insets show magnified views . Arrows indicate ATG ‐ 18 labeled by anti ‐ LGG ‐ 1 antibody . Scale bars : 5 μm .

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I Confocal fluorescent images of 4‐fold mtm‐3 embryos expressing ATG‐18::GFP (I′) and stained by DAPI (I) and anti‐LGG‐1 (I″) antibody. Insets show magnified views. Arrows indicate ATG‐18 puncta labeled by anti‐LGG‐1 antibody. Scale bars: 5 μm.


( A ) Microcomputed tomography ( micro - CT ) of proximal femur from 12 - week - old WT Sigmar1 gKO mice . Scale bars , 1 mm . ( B ) of bone volume per tissue volume ( BV / TV ) , trabecular number ( Tb . N ) , trabecular separation ( Tb . Sp ) , trabecular thickness ( Tb . Th ) , cortical region BV / TV ( Ct . BV / TV ) cortical thickness ( Ct . Th , mm ) ( n = 5 biological replicates ) . Data information : results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . Unpaired two - Student ' s t - ( B was used for statistical .

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(A) Microcomputed tomography (micro-CT) images of the proximal femur from 12-week-old male WT and Sigmar1 gKO mice. Scale bars, 1 mm. (B) Quantification of bone volume per tissue volume (BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp), trabecular thickness (Tb. Th), cortical region BV/TV (Ct. BV/TV) and cortical thickness (Ct. Th, mm) (n = 5 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. Unpaired two-tailed Student's t-test (B was used for statistical analysis.


( C ) Coronal of fifth lumbar spine . Scale Bars , 1 mm . ( D ) of trabecular bone parameters of lumbar spine ( n = 5 biological replicates ) . Data information : results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . Unpaired two - Student ' s t - D , was used for statistical .

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(C) Coronal images of the fifth lumbar spine. Scale Bars, 1 mm. (D) Quantification of trabecular bone parameters of lumbar spine (n = 5 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. Unpaired two-tailed Student's t-test D, was used for statistical analysis.


( E ) TRAP of tibias from WT Sigmar1 gKO mice . Scale bars , 50 μm . ( F G ) of osteoclast number per bone surface ( N . Oc / BS ) of osteoclast surface per bone surface ( Oc . S / BS ) ( n = 6 biological replicates ) . Data information : results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . Unpaired two - Student ' s t - F - G , was used for statistical .

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(E) TRAP staining of tibias from male WT and Sigmar1 gKO mice. Scale bars, 50 μm. (F and G) Quantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 6 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. Unpaired two-tailed Student's t-test F-G, was used for statistical analysis.


( H I ) Representative quantitative of calcein double labeling . Scale bars , 20 μm . ( n = 6 biological replicates ) Data information : results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . Unpaired two - Student ' s t - , I was used for statistical .

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

(H and I) Representative images and quantitative analysis of calcein double labeling. Scale bars, 20 μm. (n = 6 biological replicates) Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. Unpaired two-tailed Student's t-test , I was used for statistical analysis.


( A ) Micro - CT of proximal femur from WT or Sigmar1 gKO mice that received sham or ovariectomy surgery for 6 . Scale bars , 1 mm . ( B ) of bone volume per tissue volume ( BV / TV ) , trabecular number ( Tb . N ) , trabecular separation ( Tb . Sp ) trabecular thickness ( Tb . Th ) ( n = 5 biological replicates ) . Data information : results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons ( B were used for statistical .

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

(A) Micro-CT images of the proximal femur from female WT or Sigmar1 gKO mice that received sham or ovariectomy surgery for 6 weeks. Scale bars, 1 mm. (B) Quantification of bone volume per tissue volume (BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp) and trabecular thickness (Tb. Th) (n = 5 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test (B were used for statistical analysis.


( C ) TRAP of femur sections from four groups . Scale Bars , 200 μm . ( D E ) of osteoclast number per bone surface ( N . Oc / BS ) of osteoclast surface per bone surface ( Oc . S / BS ) ( n = 5 biological replicates ) . Data information : results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons , D E ) were used for statistical .

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(C) TRAP staining of femur sections from the four groups. Scale Bars, 200 μm. (D and E) Quantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 5 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test , D and E) were used for statistical analysis.


( F ) TRAP to detect osteoclastogenesis of BMMs from WT or Sigmar1 gKO mice . Scale bars , 200 μm . ( G H ) of nuclei numbers of TRAP - positive multinuclear ( n = 6 biological replicates for G n = 3 biological replicates for H ) . Data information : results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . One - way ANOVA with unpaired two - Student ' s t - ( G - H were used for statistical .

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

(F) TRAP staining to detect osteoclastogenesis of BMMs from WT or Sigmar1 gKO mice. Scale bars, 200 μm. (G and H) Quantification of the size and nuclei numbers of TRAP-positive multinuclear cells (n = 6 biological replicates for G and n = 3 biological replicates for H). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with unpaired two-tailed Student's t-test (G-H were used for statistical analysis.


( I J ) Representative of relative pit resorption of hydroxyapatite - coated plates . WT or Sigamr1 gKO BMMs were seeded on hydroxyapatite - coated plates treated with 50 ng / mL RANKL ( n = 6 biological replicates ) . Data information : results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . One - way ANOVA with unpaired two - Student ' s t - J ) were used for statistical .

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(I and J) Representative images and quantification of the relative pit resorption area of hydroxyapatite-coated plates. WT or Sigamr1 gKO BMMs were seeded on hydroxyapatite-coated plates and treated with 50 ng/mL RANKL (n = 6 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with unpaired two-tailed Student's t-test J) were used for statistical analysis.


( B ) Micro - CT of proximal femur from sham or OVX mice with different AAV injections . Scale bars , 1 mm .

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(B) Micro-CT images of the proximal femur from sham or OVX mice with different AAV injections. Scale bars, 1 mm.


( C ) of bone volume per tissue volume ( BV / TV ) , trabecular number ( Tb . N ) , trabecular separation ( Tb . Sp ) trabecular thickness ( Tb . Th ) ( n = 5 biological replicates ) . Data information : results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons ( C was used for statistical .

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

(C) Quantification of bone volume per tissue volume (BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp) and trabecular thickness (Tb. Th) (n = 5 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test (C was used for statistical analysis.


( D ) TRAP of femur sections from three groups . Scale Bars , 200 μm .

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(D) TRAP staining of femur sections from the three groups. Scale Bars, 200 μm.


( E F ) of osteoclast number per bone surface ( N . Oc / BS ) of osteoclast surface per bone surface ( Oc . S / BS ) ( n = 5 biological replicates ) . Data information : results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons E - F , was used for statistical .

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

(E and F) Quantification of osteoclast number per bone surface (N. Oc/BS) and percentage of osteoclast surface per bone surface (Oc. S/BS) (n = 5 biological replicates). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test E-F, was used for statistical analysis.


( G ) TRAP to detect osteoclastogenesis of BMMs treated with different Sigmar1 agonists ( 10μM ) or vector . Scale bars , 200 μm . ( H I ) of nuclei numbers of TRAP - positive multinuclear ( n = 6 biological replicates for H n = 3 biological replicates for I ) . Data information : results are representative data generated from at least three independent experiments . Data are presented as mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons , I , was used for statistical .

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

(G) TRAP staining to detect osteoclastogenesis of BMMs treated with different Sigmar1 agonists (10μM) or vector. Scale bars, 200 μm. (H and I) Quantification of the size and nuclei numbers of TRAP-positive multinuclear cells (n = 6 biological replicates for H and n = 3 biological replicates for I). Data information: All results are representative data generated from at least three independent experiments. Data are presented as mean ± SD. One-way ANOVA with Tukey's multiple comparisons test , I, was used for statistical analysis.


( A ) SERCA2 peptides identified through mass spectrometry are shown .

(A) SERCA2 peptides identified through mass spectrometry are shown.


( B ) HEK - 293T were transfected with indicated plasmids then subjected to immunoprecipitation followed by western blotting to detect exogenous interactions between Sigmar1 SERCA2 . Data information : results are representative data generated from at least three independent experiments .

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(B) HEK-293T cells were transfected with the indicated plasmids and then subjected to immunoprecipitation followed by western blotting to detect exogenous interactions between Sigmar1 and SERCA2. Data information: All results are representative data generated from at least three independent experiments.


( C ) lysates from mouse BMMs were incubated with either anti - Sigmar1 antibody or normal rabbit IgG , pellets were detected with indicated antibodies . Data information : results are representative data generated from at least three independent experiments .

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(C) The lysates from mouse BMMs were incubated with either anti-Sigmar1 antibody or normal rabbit IgG, and the pellets were detected with the indicated antibodies. Data information: All results are representative data generated from at least three independent experiments.


( D ) Western blots showing SERCA2 expression in HEK - 293T transfected with different amounts of Sigmar1 plasmids . were transfected with 1 μg SERCA2 plasmid Sigmar1 plasmid ( 0 . 125 μg , 0 . 25 μg , 0 . 5 μg , 1 μg ) . Data information : results are representative data generated from at least three independent experiments .

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(D) Western blots showing SERCA2 expression in HEK-293T cells transfected with different amounts of Sigmar1 plasmids. The cells were transfected with 1 μg SERCA2 plasmid and Sigmar1 plasmid (0.125 μg, 0.25 μg, 0.5 μg, 1 μg). Data information: All results are representative data generated from at least three independent experiments.


( E ) Upper : schematic representation of various Sigmar1 truncations . Bottom : mapping of Sigmar1 domains critical for SERCA2 binding . HEK - 293T were transfected with different Sigmar1 truncations , lysates were immunoprecipitated subjected to western blotting . Data information : results are representative data generated from at least three independent experiments .

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(E) Upper: schematic representation of various Sigmar1 truncations. Bottom: mapping of Sigmar1 domains critical for SERCA2 binding. HEK-293T cells were transfected with different Sigmar1 truncations, and cell lysates were immunoprecipitated and subjected to western blotting. Data information: All results are representative data generated from at least three independent experiments.


( F ) Upper : schematic representation of various SERCA2 truncations . Bottom : mapping of SERCA2 domains crucial for Sigmar1 binding . Different truncated SERCA2 plasmids were transfected into HEK - 293T . After immunoprecipitation , interaction between truncated SERCA2 Sigmar1 was detected by western blotting . Red asterisks indicate specific SERCA2 truncation bands . Data information : results are representative data generated from at least three independent experiments .

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(F) Upper: schematic representation of various SERCA2 truncations. Bottom: mapping of SERCA2 domains crucial for Sigmar1 binding. Different truncated SERCA2 plasmids were transfected into HEK-293T cells. After immunoprecipitation, the interaction between truncated SERCA2 and Sigmar1 was detected by western blotting. Red asterisks indicate specific SERCA2 truncation bands. Data information: All results are representative data generated from at least three independent experiments.


( H ) Western blots showing Q615A mutant truncated SERCA2 expression in HEK - 293T transfected with different amounts of Sigmar1 plasmids . were transfected with 1 μg Q615A mutant of truncated SERCA2 plasmid Sigmar1 plasmid ( 0 . 125 μg , 0 . 25 μg , 0 . 5 μg , 1 μg ) . Data information : results are representative data generated from at least three independent experiments .

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(H) Western blots showing Q615A mutant truncated SERCA2 expression in HEK-293T cells transfected with different amounts of Sigmar1 plasmids. The cells were transfected with 1 μg Q615A mutant of truncated SERCA2 plasmid and Sigmar1 plasmid (0.125 μg, 0.25 μg, 0.5 μg, 1 μg). Data information: All results are representative data generated from at least three independent experiments.


( I ) Interactions between Sigmar1 WT or Q615 mutants of truncated SERCA2 were detected by Co - IP assays . Data information : results are representative data generated from at least three independent experiments .

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(I) Interactions between Sigmar1 and WT or Q615 mutants of truncated SERCA2 were detected by Co-IP assays. Data information: All results are representative data generated from at least three independent experiments.


( J ) TRAP to detect osteoclastogenesis of BMMs from WT gKO mice treated with different siRNAs . Scale bars , 200 μm . Data information : results are representative data generated from at least three independent experiments .

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(J) TRAP staining to detect osteoclastogenesis of BMMs from WT and gKO mice treated with different siRNAs. Scale bars, 200 μm. Data information: All results are representative data generated from at least three independent experiments.


( A B ) Western blots showing SERCA2 expression in HEK - 293T with or without Sigmar1 cotransfection treated with eeyarestatin I or MG132 at indicated . inhibitors were applied to 8 hours prior to collection . Data information : results are representative data generated from at least three independent experiments .

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(A and B) Western blots showing SERCA2 expression in HEK-293T cells with or without Sigmar1 cotransfection treated with eeyarestatin I or MG132 at the indicated concentration. All inhibitors were applied to cells 8 hours prior to protein collection. Data information: All results are representative data generated from at least three independent experiments.