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 ) .

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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 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 ,

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

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 . 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 ,

O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O 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 O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O 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 + / + 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 ) .

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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 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 ) .

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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 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 .

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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 cells . 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 ) 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 .

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(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 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 .

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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 primary cortical neurons ( c ; 250 nM ) and SH - SY5Y cells ( 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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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

(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 ) 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 )

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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 ) 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 the indicated molar ratios was analysed by blue native PAGE ; liposome binding impaired the gel entry of proteins .

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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 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 .

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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 ) 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 .

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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 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 ) .

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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 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 )

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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 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

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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 the influence of the SPIN90 middle segment is tested on its own or in the presence of the 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 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 )

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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 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

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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 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

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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 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

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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 ) 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 ) .

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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 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 .

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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 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 ) .

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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 ) 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 .

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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 ( 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 .

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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 ( Chromatin ) centered at PionX ( left ) or HAS ( right ) in early and late mixed - sex embryos .

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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 [ 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 .

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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 [ 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 .

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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 [ Chromatin ] profiles centered at MSL2 chromatin regions called in both early and 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 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 .

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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 ) 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 .

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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 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 .

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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 embryos 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 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 ) .

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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 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 .

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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 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 .

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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 analysis of LGG ‐ 1 ‐ I and LGG ‐ 1 - II ( lipidated ) in wild type , epg ‐ 6 , and 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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 and PtdIns ( 3 , 5 ) P2 in vitro .

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


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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 puncta in each strain . At least 15 embryos were scored per strain ( 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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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(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 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 .

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(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 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 .

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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 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 .

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(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 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 .

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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 images of the 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 ) 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 .

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(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 staining of femur sections from the 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 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 .

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(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 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 .

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(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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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.


( C ) Western blots showing changes in SERCA2 expression in HEK - 293T cells in the presence of different doses of flag - tagged Hrd1 plasmids . Data information : All results are representative data generated from at least three independent experiments .

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(C) Western blots showing changes in SERCA2 expression in HEK-293T cells in the presence of different doses of flag-tagged Hrd1 plasmids. Data information: All results are representative data generated from at least three independent experiments.


( D ) Western blots showing alterations of SERCA2 expression in HEK - 293T cells pretreated with negative control siRNA ( si - NC ) or Sel1L siRNA in the presence or absence of Sigmar1 . Data information : All results are representative data generated from at least three independent experiments .

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(D) Western blots showing alterations of SERCA2 expression in HEK-293T cells pretreated with negative control siRNA (si-NC) or Sel1L siRNA in the presence or absence of Sigmar1. Data information: All results are representative data generated from at least three independent experiments.


( E ) Truncated myc - tagged SERCA2 ubiquitination levels in HEK - 293T cells transfected with empty vector or Sigmar1 were analyzed by immunoprecipitation . Cells were treated with MG132 ( 10 μM ) 8 hours before harvest . Data information : All results are representative data generated from at least three independent experiments .

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(E) Truncated myc-tagged SERCA2 ubiquitination levels in HEK-293T cells transfected with empty vector or Sigmar1 were analyzed by immunoprecipitation. Cells were treated with MG132 (10 μM) 8 hours before harvest. Data information: All results are representative data generated from at least three independent experiments.


( F ) Expression of different truncated SERCA2 lysine mutants in HEK - 293T cells transfected with different amounts of Sigmar1 plasmid . Data information : All results are representative data generated from at least three independent experiments .

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(F) Expression of different truncated SERCA2 lysine mutants in HEK-293T cells transfected with different amounts of Sigmar1 plasmid. Data information: All results are representative data generated from at least three independent experiments.


( G ) Expression of WT or 2KR ( K460A and K541A ) mutants of full - length SERCA2 in HEK - 293T cells transfected with different amounts of Sigmar1 plasmid . Data information : All results are representative data generated from at least three independent experiments .

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(G) Expression of WT or 2KR (K460A and K541A) mutants of full-length SERCA2 in HEK-293T cells transfected with different amounts of Sigmar1 plasmid. Data information: All results are representative data generated from at least three independent experiments.


( B ) Micro - CT images of calvaria from mice that received sham or LPS injection with PBS or dimemorfan treatment . Scale bars , 1 mm . ( C ) Quantification of bone volume / tissue volume ( BV / TV ) of calvaria from different groups ( 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 Tukey ' s multiple comparisons test ( C , was used for statistical analysis .

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(B) Micro-CT images of calvaria from mice that received sham or LPS injection with PBS or dimemorfan treatment. Scale bars, 1 mm. (C) Quantification of bone volume/tissue volume (BV/TV) of calvaria from different groups (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 Tukey's multiple comparisons test (C, was used for statistical analysis.


( D ) H & E and TRAP staining of calvaria from the three groups . Scale bars , 100 μm . ( 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 = 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 Tukey ' s multiple comparisons test E - F , was used for statistical analysis .

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

(D) H&E and TRAP staining of calvaria from the three groups. Scale bars, 100 μm. (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 = 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 Tukey's multiple comparisons test E-F, was used for statistical analysis.


( H ) Micro - CT images of proximal femurs from sham or ovariectomized mice with different treatments . Scale bars , 1 mm .

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(H) Micro-CT images of proximal femurs from sham or ovariectomized mice with different treatments. Scale bars, 1 mm.


( I ) Quantification of bone volume / tissue volume ( BV / TV ) , trabecular number ( Tb . N ) , trabecular separation ( Tb . Sp ) and trabecular thickness ( Tb . Th ) ( 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 Tukey ' s multiple comparisons test , I , was used for statistical analysis .

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(I) Quantification of bone volume/tissue volume (BV/TV), trabecular number (Tb. N), trabecular separation (Tb. Sp) and trabecular thickness (Tb. Th) (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 Tukey's multiple comparisons test , I, was used for statistical analysis.


( J ) TRAP staining of femurs from the three groups . Scale bars , 200 μm .

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(J) TRAP staining of femurs from the three groups. Scale bars, 200 μm.


( K and L ) 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 . One - way ANOVA with Tukey ' s multiple comparisons test was used for statistical analysis .

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

(K and L) 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. One-way ANOVA with Tukey's multiple comparisons test was used for statistical analysis.


( A ) TRAP staining of hPBMCs treated with human RANKL ( 50 ng / mL ) or dimemorfan for 12 days . Scale bars , 500 μm .

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(A) TRAP staining of hPBMCs treated with human RANKL (50 ng/mL) or dimemorfan for 12 days. Scale bars, 500 μm.


( B ) Western blot analysis of NFATc1 expression during a 7 - day induction of hPBMCs to osteoclasts .

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(B) Western blot analysis of NFATc1 expression during a 7-day induction of hPBMCs to osteoclasts.


( C ) hPBMCs were induced to differentiate into osteoclasts for 7 days , and the relative mRNA levels of marker genes were evaluated by RT - qPCR ( 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 Tukey ' s multiple comparisons test ( C ) was used for statistical analysis .

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(C) hPBMCs were induced to differentiate into osteoclasts for 7 days, and the relative mRNA levels of marker genes were evaluated by RT-qPCR (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 Tukey's multiple comparisons test (C) was used for statistical analysis.


Purified Drosophila Delta and Serrate NE3 proteins bind to liposomes ( D ) Binding is reduced in Delta ∆ β1 - 2 , but not to the Serrate equivalent using liposomes composed of PC : PS : PE - fluoroscein ( 80 : 15 : 5 ) . Notch binding to Drosophila Delta NE3 ( E ) WT and ∆ β1 - 2 ( Delta ∆ β1 - 2 ) variants both bind to Notch , unlike variant with F204A substitution in DSL domain . Comparisons were performed with a two - tailed unpaired t - test . Values are shown as scattered data points with the dark lines representing the means . ns , no significant difference , * * P < 0 . 01 ; * * * P < 0 . 0001 .

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

Purified Drosophila Delta and Serrate NE3 proteins bind to liposomes (D) Binding is reduced in Delta∆β1-2, but not to the Serrate equivalent using liposomes composed of PC:PS:PE-fluoroscein (80:15:5). Notch binding to Drosophila Delta NE3 (E) WT and ∆β1-2 (Delta∆β1-2) variants both bind to Notch, unlike variant with F204A substitution in DSL domain. Comparisons were performed with a two-tailed unpaired t-test. Values are shown as scattered data points with the dark lines representing the means. ns, no significant difference, **P < 0.01; ***P < 0.0001.


Adult wings from Dl ∆ β1 - 2 flies . No defects are detected in wings from Dl ∆ β1 - 2 / + ( B ) , Homozygous Dl ∆ β1 - 2 / Dl ∆ β1 - 2 have extra vein tissue near L5 and uneven L2 veins ( arrowheads ; C ) .

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Adult wings from Dl∆β1-2 flies. No defects are detected in wings from Dl∆β1-2/+ (B), Homozygous Dl∆β1-2/ Dl∆β1-2 have extra vein tissue near L5 and uneven L2 veins (arrowheads; C).


Microchaetae are arranged in rows on the thorax of control ( yw ) flies ; these become disordered and more dense in Dl ∆ β1 - 2 / Dl ∆ β1 - 2 . White rectangle indicates area scored for E Number of microchaetes per central area ( white rectangle in D ) in the indicated genotypes .

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Microchaetae are arranged in rows on the thorax of control (yw) flies; these become disordered and more dense in Dl∆β1-2/Dl∆β1-2. White rectangle indicates area scored for E Number of microchaetes per central area (white rectangle in D) in the indicated genotypes.


Representative images of adult females wings in combinations of Dl ∆ β1 - 2 with loss of function Delta alleles . In combinations with Dlrev10 ( A ) , Dl ∆ Exon2 ( B ) , or Df ( 3R ) DlFx3 ( C ) , vein thickening is strongly enhanced ( right panels ) compared to heterozygous mutants alone ( left panels ) . Vertical square brackets indicate the regions used for vein thickness quantification . Quantification of wing - vein thickness in females of the indicated genotypes . Representative images of adult female wings demonstrate that Dl ∆ β1 - 2 rescues the wing notching phenotype , caused by a Notch loss of function allele ( N55e11 ) . Horizontal square bracket indicates the L5 vein " delta " at the intersection with the wing margin analyzed in EV2E . Quantification of wing notching in females of the indicated genotypes , Dl ∆ β1 - 2 rescues notching in a similar manner to Dlrev10 .

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

Representative images of adult females wings in combinations of Dl∆β1-2 with loss of function Delta alleles. In combinations with Dlrev10 (A), Dl∆Exon2 (B), or Df(3R)DlFx3 (C), vein thickening is strongly enhanced (right panels) compared to heterozygous mutants alone (left panels). Vertical square brackets indicate the regions used for vein thickness quantification. Quantification of wing-vein thickness in females of the indicated genotypes. Representative images of adult female wings demonstrate that Dl∆β1-2 rescues the wing notching phenotype, caused by a Notch loss of function allele (N55e11). Horizontal square bracket indicates the L5 vein "delta" at the intersection with the wing margin analyzed in EV2E. Quantification of wing notching in females of the indicated genotypes, Dl∆β1-2 rescues notching in a similar manner to Dlrev10.


Equatorial region of eye imaginal discs where E ( spl ) mδ0 . 5 expression ( green ) becomes restricted to a single photoreceptor in each cluster ( magenta ) , as detected in control and Dl ∆ β1 - 2 / + discs ( top panels ) . In Df ( 3R ) DlFx3 / + and Dl ∆ β1 - 2 / Df ( 3R ) DlFx3 discs ( bottom panels ) E ( spl ) mδ0 . 5 expression is reduced ( Df ( 3R ) DlFx3 / + ) or absent from several clusters ( Dl ∆ β1 - 2 / Df ( 3R ) DlFx3 ) indicative of reduced Notch signalling . Scale bars correspond to 10 μm .

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

Equatorial region of eye imaginal discs where E(spl)mδ0.5 expression (green) becomes restricted to a single photoreceptor in each cluster (magenta), as detected in control and Dl∆β1-2/+ discs (top panels). In Df(3R)DlFx3 /+ and Dl∆β1-2/Df(3R)DlFx3 discs (bottom panels) E(spl)mδ0.5 expression is reduced (Df(3R)DlFx3 /+) or absent from several clusters (Dl∆β1-2/Df(3R)DlFx3) indicative of reduced Notch signalling. Scale bars correspond to 10 μm.


Proportion of photoreceptors clusters that fail to express the E ( spl ) mδ0 . 5 reporter in the indicated genotypes . ns , no significant difference , * * * P < 0 . 0001 ( one - way ANOVA ) . On the violin plot , dashed line represents the median and the dotted lines show the quartiles .

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Proportion of photoreceptors clusters that fail to express the E(spl)mδ0.5 reporter in the indicated genotypes. ns, no significant difference, *** P<0.0001 (one-way ANOVA). On the violin plot, dashed line represents the median and the dotted lines show the quartiles.


Apical view of wing imaginal disc with homozygous Dl ∆ β1 - 2 clones ( GFP negative ) stained for Dl ( red ) and Cadherin ( blue ) . ( A ' ) Z - projection of apical layers spanning a Dl ∆ β1 - 2 clone ( GFP negative ) located at the DV boundary . No change in apical localisation of Dl ( red ) and Cadherin ( blue ) is detected . Panels on the right show the Dl ( grayscale ) apical localisation on the wing imaginal disc .

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Apical view of wing imaginal disc with homozygous Dl∆β1-2 clones (GFP negative) stained for Dl (red) and Cadherin (blue). (A') Z-projection of apical layers spanning a Dl∆β1-2 clone (GFP negative) located at the DV boundary. No change in apical localisation of Dl (red) and Cadherin (blue) is detected. Panels on the right show the Dl (grayscale) apical localisation on the wing imaginal disc.


Uptake assay at t = 0 . After exposure to extracellular anti - Dl antibody , Dl protein ( red ) is detected at similar levels apical to Cadherin ( blue ) in wild - type ( GFP ) and Dl ∆ β1 - 2 mutant tissue ( GFP negative ) . ( B ' ) Cross - sectional view of B , Dl protein ( red ) is present apically relative to Cadherin ( Blue ) which marks adherens junctions . Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin ( Cad ) and Delta ( Dl ) , respectively . Uptake assay after 30 minutes , internalised anti - Dl ( red ) enters the endocytic route and in the cross sectional view ( C ' ) can similarly be detected as puncta along the cell axis in wild - type ( GFP ) and homozygous Dl ∆ β1 - 2 ( GFP negative ) tissue . Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin ( Cad ) and Delta ( Dl ) , respectively .

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

Uptake assay at t=0. After exposure to extracellular anti-Dl antibody, Dl protein (red) is detected at similar levels apical to Cadherin (blue) in wild-type (GFP) and Dl∆β1-2 mutant tissue (GFP negative). (B') Cross-sectional view of B, Dl protein (red) is present apically relative to Cadherin (Blue) which marks adherens junctions. Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin (Cad) and Delta (Dl), respectively. Uptake assay after 30 minutes, internalised anti-Dl (red) enters the endocytic route and in the cross sectional view (C') can similarly be detected as puncta along the cell axis in wild-type (GFP) and homozygous Dl∆β1-2 (GFP negative) tissue. Middle and lower panels show the crosssection in grayscale of the apical marker Cadherin (Cad) and Delta (Dl), respectively.


Average piRNA precursor abundance relative to EV in EV and ints - 11 RNAi samples , normalized to the total number of short capped RNA reads mapping to annotated WormBase TSSs ( Chen et al . , 2013 ) . Error bars represent the standard error of the mean ( 5 replicates ) .

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Average piRNA precursor abundance relative to EV in EV and ints-11 RNAi samples, normalized to the total number of short capped RNA reads mapping to annotated WormBase TSSs (Chen et al., 2013). Error bars represent the standard error of the mean (5 replicates).


Distributions of piRNA precursor length in ints - 11 RNAi - treated nematodes compared to empty vector - treated nematodes , obtained from deeply sequencing one pair of libraries ( inserts sequenced up to 75nt ) . Distributions of total sequences ( E ) and total read counts ( F ) are shown .

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Distributions of piRNA precursor length in ints-11 RNAi-treated nematodes compared to empty vector-treated nematodes, obtained from deeply sequencing one pair of libraries (inserts sequenced up to 75nt). Distributions of total sequences (E) and total read counts (F) are shown.


Co - localization between INTS - 6 : : eGFP and mCherry : : PRDE - 1 in germ cells . PRDE - 1 and INTS - 6 co - localize in early stages of meiosis ( 1 , 2 ) and up to mid - pachytene ( 3 ) , where an additional INTS - 6 focus appears ( 3 ) . When PRDE - 1 foci are about to disappear , co - localization is lost ( 4 ) , and the additional INTS - 6 focus remains present well after the loss of PRDE - 1 foci ( 5 ) .

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

Co-localization between INTS-6::eGFP and mCherry::PRDE-1 in germ cells. PRDE-1 and INTS-6 co-localize in early stages of meiosis (1,2) and up to mid-pachytene (3), where an additional INTS-6 focus appears (3). When PRDE-1 foci are about to disappear, co-localization is lost (4), and the additional INTS-6 focus remains present well after the loss of PRDE-1 foci (5).


Length distributions of nucleoplasmic and chromatin - bound piRNA precursors in N2 empty vector , N2 ints - 11 RNAi , tfiis empty vector , and tfiis ints - 11 RNAi nematodes . Length shifts in nascent RNA in tfiis mutants are indicated with dashed vertical lines and arrows . The length distributions were calculated from two merged replicates ( see Appendix Figure S2 for the length distributions in each replicate ) .

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Length distributions of nucleoplasmic and chromatin-bound piRNA precursors in N2 empty vector, N2 ints-11 RNAi, tfiis empty vector, and tfiis ints-11 RNAi nematodes. Length shifts in nascent RNA in tfiis mutants are indicated with dashed vertical lines and arrows. The length distributions were calculated from two merged replicates (see Appendix Figure S2 for the length distributions in each replicate).


Signal of 5 ′ P small RNA 5 ′ ends mapping to piRNA loci as a function of the distance to piRNA TSSs , after removal of reads corresponding to mature 21U - RNAs ( see Methods ) . The signal is normalized to counts per million of non - structural mapped reads . A peak of 5 ′ P ends centered at + 38 from piRNA TSSs is observed .

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Signal of 5′ P small RNA 5′ ends mapping to piRNA loci as a function of the distance to piRNA TSSs, after removal of reads corresponding to mature 21U-RNAs (see Methods). The signal is normalized to counts per million of non-structural mapped reads. A peak of 5′ P ends centered at +38 from piRNA TSSs is observed.


Number of loci with detected cleavage fragments in N2 empty vector nematodes across percentile bins of increasing termination signal strength ( top panel ) . Distributions of fragment read counts per locus for detected loci across percentile bins of increasing termination signal strength ( bottom panel ) . Boxplots show the interquartile range with a line at the median ; the whiskers extend to the greatest point no more than 1 . 5 times the interquartile range . The data shown here corresponds to one of two N2 EV replicates , see Appendix Figure S6 for this analysis across replicates and genotypes .

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Number of loci with detected cleavage fragments in N2 empty vector nematodes across percentile bins of increasing termination signal strength (top panel). Distributions of fragment read counts per locus for detected loci across percentile bins of increasing termination signal strength (bottom panel). Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range. The data shown here corresponds to one of two N2 EV replicates, see Appendix Figure S6 for this analysis across replicates and genotypes.