( e ) Targeting mitochondria with autophagolysosomes in absence ( − tet ) or presence ( + tet ) of wild - type CerS1 C18 - ceramide was visualized by colocalization of MTG LTR using confocal microscopy . Scale bars , 10 μm .

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(e) Targeting mitochondria with autophagolysosomes in the absence (− tet) or presence (+ tet) of wild-type CerS1 and C18-ceramide was visualized by colocalization of MTG and LTR using confocal microscopy. Scale bars, 10 μm.


( f ) Effects of wild - type mutant CerS1 ( − / + tet ) on ATP generation .

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(f) Effects of wild-type and mutant CerS1 (−/+ tet) on ATP generation.


( g ) Effects of wild - type CerS1 ( − / + tet ) on ATP generation in absence ( control shRNA ) or presence of shRNA - mediated knockdown of LC3B . Data shown are an average of at least three experiments ± s . d . ( * P 0 . 05 ) .

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(g) Effects of wild-type CerS1 (−/+ tet) on ATP generation in the absence (control shRNA) or presence of shRNA-mediated knockdown of LC3B. Data shown are an average of at least three experiments ± s.d. (*P 0.05).


( a ) Subcellular localization of ceramide , generated by wild - type CerS1 induction ( + tet ) , was visualized in mitochondria by colocalization of ceramides mitochondria with anti - ceramide anti - Tom20 , respectively , using confocal microscopy .

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(a) Subcellular localization of ceramide, generated by wild-type CerS1 induction (+ tet), was visualized in mitochondria by colocalization of ceramides and mitochondria with anti-ceramide and anti-Tom20, respectively, using confocal microscopy.


( b , c ) Mitochondrial ( Mito ) targeting of LC3B - II C18 - ceramide , generated by induction of wild - type CerS1 ( b ) , that of products of catalytically inactive mutant CerS1 ( + tet ) ( c ) was visualized by colocalization of ceramide ( Cer ; green ) , CFP - LC3B ( blue ) MTR using anti - ceramide confocal microscopy . Noninduced ( − tet ) were used as controls . Scale bars , 10 μm .

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(b,c) Mitochondrial (Mito) targeting of LC3B-II and C18-ceramide, generated by induction of wild-type CerS1 (b), and that of the products of the catalytically inactive mutant CerS1 (+ tet) (c) was visualized by colocalization of ceramide (Cer; green), CFP-LC3B (blue) and MTR using anti-ceramide and confocal microscopy. Noninduced cells (− tet) were used as controls. Scale bars, 10 μm.


( a ) Binding of Flag - tagged wild - type LC3B LC3BI35A , LC3BF52A or LC3BG120A with endogenous C18 - ceramide ( C18 - Cer ) , generated in response to wild - type CerS1 induction ( left ) or exogenous C18 - Pyr - Cer ( right ) , was measured using LC / MS / MS after pulldown using anti - Flag - conjugated beads . Data shown are an average of at least three experiments ± s . d . ( * P 0 . 05 ) .

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(a) Binding of Flag-tagged wild-type LC3B and LC3BI35A, LC3BF52A or LC3BG120A with endogenous C18-ceramide (C18-Cer), generated in response to wild-type CerS1 induction (left) or exogenous C18-Pyr-Cer (right), was measured using LC/MS/MS after pulldown using anti-Flag-conjugated beads. Data shown are an average of at least three experiments ± s.d. (*P 0.05).


( b ) Effects of CerS1 C18 - ceramide induction on lipidation of Flag - tagged wild - type , LC3BI35A , LC3BF52A LC3BG120A were examined by western blotting . Noninduced ( − tet ) were used as controls . β - actin was used as a loading control . Full blots can be found in Supplementary Figure 13 .

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(b) Effects of CerS1 and C18-ceramide induction on the lipidation of Flag-tagged wild-type, LC3BI35A, LC3BF52A and LC3BG120A proteins were examined by western blotting. Noninduced cells (− tet) were used as controls. β-actin was used as a loading control. Full blots can be found in Supplementary Figure 13.


( a , b ) Effects of G120A F52A mutations , which perturbed C18 - ceramide binding of LC3B , on targeting of mitochondria by LC3B - containing autophagosomes ( visualized by confocal microscopy using anti - Flag anti - Tom20 ) ( a ) or mitochondrial function ( measurement of OCR ) ( b ) . V , vector .

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(a,b) Effects of G120A and F52A mutations, which perturbed C18-ceramide binding of LC3B, on targeting of mitochondria by LC3B-containing autophagosomes (visualized by confocal microscopy using anti-Flag and anti-Tom20) (a) or mitochondrial function (measurement of OCR) (b). V, vector.


( c ) Effects of siRNA - mediated knockdown of p62 , NIX Drp1 on OCR in absence or presence of CerS1 C18 - ceramide induction were measured using SeaHorse . Data shown are an average of at least three experiments ± s . d . ( * P 0 . 05 ) .

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(c) Effects of siRNA-mediated knockdown of p62, NIX and Drp1 on OCR in the absence or presence of CerS1 and C18-ceramide induction were measured using the SeaHorse. Data shown are an average of at least three experiments ± s.d. (*P 0.05).


( d ) Effects of siRNA - mediated knockdown of Drp1 ( lower left right ) on localization of ceramide within mitochondrial membranes were visualized using colocalization of anti - ceramide ( red ) anti - Tom20 ( green ) under confocal microscopy , in absence ( upper lower left ) or presence ( upper lower right ) of CerS1 C18 - ceramide induction ( − / + tet ) compared to Scr siRNA - transfected controls ( upper left right ) . Scale bars , 10 μm in a d . Enlarged are shown in boxed areas in d .

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(d) Effects of siRNA-mediated knockdown of Drp1 (lower left and right) on the localization of ceramide within mitochondrial membranes were visualized using the colocalization of anti-ceramide (red) and anti-Tom20 (green) under confocal microscopy, in the absence (upper and lower left) or presence (upper and lower right) of CerS1 and C18-ceramide induction (−/+ tet) compared to Scr siRNA-transfected controls (upper left and right). Scale bars, 10 μm in a and d. Enlarged images are shown in boxed areas in d.


( a , b ) Roles of shRNA - mediated stable knockdown of endogenous LC3B expression in regulation of mitochondrial function ( a ) or UM - SCC - 22A xenograft - derived growth ( b ) in absence or presence of wild - type CerS1 C18 - ceramide induction ( − / + tet ) were determined using SeaHorse or measurement of volumes in flanks of SCID mice ( n = 6 per group ) , respectively . Data shown are an average of at least three experiments ± s . d . ( * P 0 . 05 ) .

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(a,b) Roles of shRNA-mediated stable knockdown of endogenous LC3B expression in the regulation of mitochondrial function (a) or UM-SCC-22A xenograft-derived tumor growth (b) in the absence or presence of wild-type CerS1 and C18-ceramide induction (−/+ tet) were determined using the SeaHorse or measurement of tumor volumes in the flanks of SCID mice (n = 6 per group), respectively. Data shown are an average of at least three experiments ± s.d. (*P 0.05).


Representative confocal of control ( CTL - 1 , 2 , 3 ) DOA + fibroblasts carrying indicated monoallelic mutations imaged as described in A . Scale bar = 20μm . Passage number between P12 - 15 . Mitochondrial of B . Data represent mean ± SD of two independent experiments , ( 195 - 2496 per ) , One - way ANOVA ; * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not

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Representative confocal images of control (CTL-1, 2, 3) and DOA+ patient fibroblasts carrying indicated monoallelic mutations imaged as described in A. Scale bar=20μm. Passage number between P12-15. Mitochondrial morphology quantification of B. Data represent mean ± SD of two independent experiments, (195-2496 cells per cell line), One-way ANOVA; ** p < 0.01, ****p < 0.0001, ns; not significant


Representative confocal of control ( CTL - 1 ) OPA1S545R fibroblasts treated with OPA1 , DNM1L , or non - targeting ( NT ) siRNAs for 72 hours imaged as described in A . Scale bar = 20μm . Passage number between P12 - 14 . Mitochondrial of D . Data represent mean ± SD of three independent experiments , ( 3219 - 5857 per ) , One - way ANOVA ; * * * * p < 0 . 0001 , ns ; not significant .

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Representative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with OPA1, DNM1L, or non-targeting (NT) siRNAs for 72 hours and imaged as described in A. Scale bar=20μm. Passage number between P12-14. Mitochondrial morphology quantification of D. Data represent mean ± SD of three independent experiments, (3219-5857 cells per cell line), One-way ANOVA; ****p < 0.0001, ns; not significant.


Representative confocal of control ( CTL - 1 ) OPA1S545R fibroblasts treated with 50μM cycloheximide ( CHX ) where indicated for 6 hours . Imaging as described in A . Scale bar = 20μm . Passage number between P14 - P15 . Mitochondrial of F . Data represent mean ± SD of two independent experiments , ( 879 - 4154 per ) , One - way ANOVA ; * * * * p < 0 . 0001 , ns ; not significant .

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Representative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with 50μM cycloheximide (CHX) where indicated for 6 hours. Imaging as described in A. Scale bar=20μm. Passage number between P14-P15. Mitochondrial morphology quantification of F. Data represent mean ± SD of two independent experiments, (879-4154 cells per cell line), One-way ANOVA; ****p < 0.0001, ns; not significant.


Representative confocal of control ( CTL - 1 ) OPA1S545R fibroblasts treated with OPA1 , DNM1L , PGS1 , non - targeting ( NT ) siRNAs or indicated combinations for 72 hours . Mitochondria ( anti - TOM40 , green ) nuclei ( DAPI , blue ) . Scale bar = 20μm . Passages number between P10 - 15 . Mitochondrial of ( A ) using control fibroblasts with fragmented ( OPA1 siRNA ) , normal ( non - targeting NT siRNA ) , hypertubulated ( DNM1L siRNA ) mitochondria . Data represent mean ± SD of three independent experiments , One - way ANOVA ( 905 - 3695 per ) , ( % fragmented ) ; ; * * * * p < 0 . 0001 , ns ; not significant . .

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Representative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with OPA1, DNM1L, PGS1, and non-targeting (NT) siRNAs or indicated combinations for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=20μm. Passages number between P10-15. Mitochondrial morphology quantification of (A) using control fibroblasts with fragmented (OPA1 siRNA), normal (non-targeting NT siRNA), and hypertubulated (DNM1L siRNA) mitochondria. Data represent mean ± SD of three independent experiments, One-way ANOVA (905-3695 cells per cell line), (% fragmented); ; ****p < 0.0001, ns; not significant..


Representative confocal of wild type ( WT ) Opa1Crispr MEFs treated with NT or Pgs1 siRNA for 72 hours . Live imaging of mitochondria ( mitoYFP , green ) nuclei ( NucBlue , blue ) . Scale bar = 10μm . Mitochondrial of ( C ) using WT MEFs treated with Opa1 siRNA ( fragmented ) , NT siRNA ( normal ) , or Dnm1l siRNA ( hypertubulated ) ground truth training sets . Data represent mean ± SD of three independent experiments , One - way ANOVA ( 6613 - 8758 per ) , ( % fragmented ) ; * * * * p < 0 . 0001 , ns ; not significant .

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Representative confocal images of wild type (WT) and Opa1Crispr MEFs treated with NT or Pgs1 siRNA for 72 hours. Live imaging of mitochondria (mitoYFP, green) and nuclei (NucBlue, blue). Scale bar=10μm. Mitochondrial morphology quantification of (C) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubulated) ground truth training sets. Data represent mean ± SD of three independent experiments, One-way ANOVA (6613-8758 cells per cell line), (% fragmented) ; ****p < 0.0001, ns; not significant.


Representative confocal of WT , Opa1Crispr MEFs complemented with pLenti - Opa1 , Opa1CrisprPgs1Crispr MEFs Pgs1Crispr MEFs complemented with pLenti - Pgs1 by lentiviral delivery . Live imaging of mitochondria ( mitoYFP , green ) nuclei ( NucBlue , blue ) . Scale bar = 10μm . Supervised ML mitochondrial of ( E ) using WT MEFs treated with Opa1 siRNA ( fragmented ) , NT siRNA ( normal ) , or Dnm1l siRNA ( hypertubulated ) training sets . Data represent mean ± SD of three independent experiments , One - way ANOVA ( 691 - 3990 per ) , ( % fragmented ) ; * * * * p < 0 . 0001 , ns ; not significant . .

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Representative confocal images of WT, Opa1Crispr MEFs complemented with pLenti-Opa1, Opa1CrisprPgs1Crispr MEFs and Pgs1Crispr MEFs complemented with pLenti-Pgs1 by lentiviral delivery. Live imaging of mitochondria (mitoYFP, green) and nuclei (NucBlue, blue). Scale bar=10μm. Supervised ML mitochondrial morphology quantification of (E) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubulated) training sets. Data represent mean ± SD of three independent experiments, One-way ANOVA (691-3990 cells per cell line), (% fragmented) ; ****p < 0.0001, ns; not significant..


Representative confocal of live imaging of MEFs of indicated genotypes subjected fragmentation with 5μM carbonyl cyanide m - chlorophenyl hydrazine ( CCCP ) for indicated time points . were captured every hour for 18 hours . Scale bar = 10μm . Supervised ML mitochondrial using WT MEFs treated with 5μM CCCP for 18h ( fragmented ) , untreated ( normal ) , or treated with 10μM CHX for 9h ( hypertubular ) training sets . Data represent mean ± SD of three independent experiments , ( 131 - 426 per ) , One - way ANOVA ; * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not significant .

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Representative confocal images of live cell imaging of MEFs of the indicated genotypes subjected fragmentation with 5μM carbonyl cyanide m-chlorophenyl hydrazine (CCCP) for the indicated time points. Images were captured every hour for 18 hours. Scale bar=10μm. Supervised ML mitochondrial morphology quantification using WT MEFs treated with 5μM CCCP for 18h (fragmented), untreated (normal), or treated with 10μM CHX for 9h (hypertubular) training sets. Data represent mean ± SD of three independent experiments, (131-426 cells per cell line), One-way ANOVA; *p < 0.05, ** p < 0.01, ****p < 0.0001, ns; not significant.


Representative confocal of live imaging of MEFs of indicated genotypes subjected hyperfusion ( SiMH ) with 10μM cycloheximide ( CHX ) for indicated time points . were captured every hour for 9 hours . Mitochondrial of using WT MEFs treated with 5μM CCCP for 18h ( fragmented ) , untreated ( normal ) , or treated with 10μM CHX for 9h ( hypertubular ) training sets . Data represent mean ± SD of four independent experiments , ( 155 - 745 per ) , One - way ANOVA .

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Representative confocal images of live cell imaging of MEFs of the indicated genotypes subjected hyperfusion (SiMH) with 10μM cycloheximide (CHX) for the indicated time points. Images were captured every hour for 9 hours. Mitochondrial morphology quantification of using WT MEFs treated with 5μM CCCP for 18h (fragmented), untreated (normal), or treated with 10μM CHX for 9h (hypertubular) training sets. Data represent mean ± SD of four independent experiments, (155-745 cells per cell line), One-way ANOVA.


Representative confocal of MEFs WT Opa1Crispr MEFs treated with indicated siRNAs for 72 hours . Mitochondria ( anti - TOM40 , green ) nuclei ( DAPI , blue ) . Scale bar = 10μm . Supervised ML mitochondrial of ( B ) using WT MEFs with fragmented ( Opa1 siRNA ) , normal ( non - targetting NT siRNA ) , hypertubular ( Dnm1l siRNA ) mitochondria . Data represent mean ± SD of three independent experiments , One - way ANOVA ( 726 - 4236 per ) , ( % fragmented ) ; * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns ; not significant .

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Representative confocal micrographs of MEFs WT and Opa1Crispr MEFs treated with indicated siRNAs for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=10μm. Supervised ML mitochondrial morphology quantification of (B) using WT MEFs with fragmented (Opa1 siRNA), normal (non-targetting NT siRNA), and hypertubular (Dnm1l siRNA) mitochondria. Data represent mean ± SD of three independent experiments, One-way ANOVA (726-4236 cells per cell line), (% fragmented); *** p < 0.001, ****p < 0.0001, ns; not significant.


Representative confocal of MEFs WT , Pgs1Crispr Dnm1lCrispr MEFs treated with indicated siRNAs for 72 hours . Mitochondria ( anti - TOM40 , green ) nuclei ( DAPI , blue ) . Scale bar = 10μm . Supervised ML mitochondrial of ( G ) using WT MEFs with fragmented ( Opa1 siRNA ) , normal ( non - targetting NT siRNA ) , hypertubulated ( Dnm1l siRNA ) mitochondria . Data represent mean ± SD of > 3 independent experiments , ( 3096 - 7238 per ) , One - way ANOVA ( % fragmented ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not significant .

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Representative confocal micrographs of MEFs WT, Pgs1Crispr and Dnm1lCrispr MEFs treated with indicated siRNAs for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=10μm. Supervised ML mitochondrial morphology quantification of (G) using WT MEFs with fragmented (Opa1 siRNA), normal (non-targetting NT siRNA), and hypertubulated (Dnm1l siRNA) mitochondria. Data represent mean ± SD of >3 independent experiments, (3096-7238 cells per cell line), One-way ANOVA (% fragmented) ; *p < 0.05, ** p < 0.01, ****p < 0.0001, ns; not significant.


Representative transmission electron of MEFs of indicated genotypes showing loss of lamellar cristae in Opa1Crispr Opa1CrisprPgs1Crispr MEFs . Scale bar = 200nm . of ( C ) of mitochondrial ultrastructure ; outer / inner ration ( IMM / OMM ) cristae number per mitochondrion . Violin plot of > 50 mitochondria per , One - way ANOVA ; * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not significant .

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Representative transmission electron micrographs of MEFs of the indicated genotypes showing loss of lamellar cristae in Opa1Crispr and Opa1CrisprPgs1Crispr MEFs. Scale bar=200nm. Quantification of (C) of mitochondrial ultrastructure; outer membrane/inner membrane ration (IMM/OMM) and cristae number per mitochondrion. Violin plot of >50 mitochondria per cell line, One-way ANOVA; *p < 0.05, ** p < 0.01, ****p < 0.0001, ns; not significant.


mtDNA content in MEFs from ( F ) was quantified by amplification of MTTL1 , 16S MT - ND1 genes relative to GAPDH gene in MEFs . Data represent mean ± SD of three independent experiments , One - way ANOVA ; * * * * p < 0 . 0001 , ns ; not significant . mtDNA content in WT mutant MEFs treated with indicated siRNAs for 72 hours was quantified by amplification of MTTL1 , 16S ND1 genes relative to GAPDH gene in MEFs . Data represent mean ± SD of three independent experiments , One - way ANOVA ; * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not significant .

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mtDNA content in MEFs from (F) was quantified by amplification of MTTL1, 16S and MT-ND1 genes relative to the GAPDH nuclear gene in MEFs. Data represent mean ± SD of three independent experiments, One-way ANOVA; ****p < 0.0001, ns; not significant. mtDNA content in WT and mutant MEFs treated with indicated siRNAs for 72 hours was quantified by amplification of MTTL1, 16S and ND1 genes relative to the GAPDH nuclear gene in MEFs. Data represent mean ± SD of three independent experiments, One-way ANOVA; **p < 0.01, ****p < 0.0001, ns; not significant.


( G ) Equal amounts of extracted from WT mutant MEFs were separated by SDS - PAGE ( horizontal denotes separate membranes ) , immunoblotted with indicated antibodies quantified by densitometry ( H ) . Data represent mean ± SD of three independent experiments , One - way ANOVA ; * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns ; not significant .

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(G) Equal amounts of protein extracted from WT and mutant MEFs were separated by SDS-PAGE (horizontal line denotes separate membranes), immunoblotted with indicated antibodies and quantified by densitometry (H). Data represent mean ± SD of three independent experiments, One-way ANOVA; **p < 0.01, ***p < 0.001, ****p < 0.0001, ns; not significant.


A , Schematic representation of signaling events . Left : time course of changes in cAMP ( dotted ) cGMP ( solid ) ; right : time course of changes in Vm ( green ) , pHi ( red ) ; [ Na + ] i ( orange ) , [ Ca2 + ] i ( pink ) . Vertical dotted lines indicate time of stimulation .

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A, Schematic representation of signaling events. Left: time course of changes in cAMP (dotted line) and cGMP (solid line); right: time course of changes in Vm (green), pHi (red); [Na+]i (orange), and [Ca2+]i (pink). Vertical dotted lines indicate time of stimulation.


A - D , Longitudinal ( A C , proximal view on left ) cross - sectional ( B D , proximal - to - distal view ) tomographic slices of representative intact flagella from wild - type Chlamydomonas reinhardtii ( C . r . ) flagella ( A B ) sea urchin Strongylocentrotus purpuratus ( S . p . ) sperm ( C D ) . Note that in Chlamydomonas , flagellar ( m ) is surrounded by a glycocalyx ( g ) . Other labels : central pair ( c ) doublet microtubule ( d ) . Scale bar , 100 nm .

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A-D, Longitudinal (A and C, proximal view on left) and cross-sectional (B and D, proximal-to-distal view) tomographic slices of representative intact flagella from wild-type Chlamydomonas reinhardtii (C.r.) flagella (A and B) and sea urchin Strongylocentrotus purpuratus (S.p.) sperm (C and D). Note that in Chlamydomonas, the flagellar membrane (m) is surrounded by a glycocalyx (g). Other labels: central pair complex (c) and doublet microtubule (d). Scale bar, 100 nm.


E , Diameter of intact flagella from Chlamydomonas sea urchin sperm measured on cross - sectional views of cryo - tomographic reconstructions . Numbers indicate average diameters ± s . d . ( n = 84 for sea urchin ( S . p . ) n = 18 for Chlamydomonas ( C . r . ) )

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E, Diameter of intact flagella from Chlamydomonas and sea urchin sperm measured on cross-sectional views of cryo-tomographic reconstructions. Numbers indicate average diameters ± s.d. (n = 84 for sea urchin (S.p.) and n = 18 for Chlamydomonas (C.r.))


A , Time course of change in voltage Vm evoked by a 2 - ms flash in sperm that was loaded with caged cGMP ( 15 μM ) recorded in 100KASW . Inset : extended timescale showing rise of Vm . Fit of data ( black ) using two exponentials with time constants τrise = 17 . 4 ms τrelax = 354 ms . flash in panels is depicted as dashed magenta .

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A, Time course of the change in voltage Vm evoked by a 2-ms light flash in sperm that was loaded with caged cGMP (15 μM) and recorded in 100KASW. Inset: extended timescale showing the rise of Vm. Fit of data (black) using two exponentials with time constants τrise = 17.4 ms and τrelax = 354 ms. Light flash in all panels is depicted as dashed magenta line.


B , Time course of changes in Vm evoked by 100 - ms flash in 100KASW using caged cGMP ( blue ) caged 8 - Br - cGMP ( black ) . flashes released 260 nM cGMP or 2 , 600 nM 8 - Br - cGMP .

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B, Time course of the changes in Vm evoked by 100-ms flash in 100KASW using caged cGMP (blue) and caged 8-Br-cGMP (black). The flashes released 260 nM cGMP or 2,600 nM 8-Br-cGMP.


C , Time course of changes in Vm in 100KASW evoked by 2 - s flashes at different energy that correspond to 440 , 220 , 75 , 8 nM s - 1 of cGMP . Data was fitted using a heuristic model described by equation 3 ( Methods section ) ( black lines ) . D , Amplitude ( red ) decay time τrelax ( black ) for different energies release rates for experiments as shown in panel C . τrelax was determined using a single exponential fit . linear fit of Vm amplitude was restricted to low ; at higher , Vm response saturates ( see panel C ) . Points indicate mean ± s . d . from 5 experiments .

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C, Time course of changes in Vm in 100KASW evoked by 2-s flashes at different light energy that correspond to 440, 220, 75, and 8 nM s-1 of cGMP. Data was fitted using a heuristic model described by equation 3 (Methods section) (black lines). D, Amplitude (red) and decay time τrelax (black) for different light energies and release rates for experiments as shown in panel C. τrelax was determined using a single exponential fit. The linear fit of the Vm amplitude was restricted to low light levels; at higher levels, the Vm response saturates (see panel C). Points indicate mean ± s.d. from 5 experiments.


A , Time course of cGMP - evoked changes in Vm in 100KASW ( red , 500 nM cGMP ; pink , 9 nM cGMP ) normal ASW ( blue , 500 nM cGMP ; navy , 9 nM cGMP ) . time constants of recovery after cGMP release ( 9 nM ) were 289 ms ( depolarization ) 303 ms ( hyperpolarization ) . Signals in panels were detected using potentiometric probe FluoVolt ; dashed magenta lines indicate timing of photolysis flash .

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A, Time course of cGMP-evoked changes in Vm in 100KASW (red, 500 nM cGMP; pink, 9 nM cGMP) and normal ASW (blue, 500 nM cGMP; navy, 9 nM cGMP ). The time constants of recovery after cGMP release (9 nM) were 289 ms (depolarization) and 303 ms (hyperpolarization). Signals in panels were detected using the potentiometric probe FluoVolt; dashed magenta lines indicate the timing of the photolysis flash.


, Representative measurement of dependence of recovery - time constant on amount of released cGMP . Inset : decay time was constant for low stimulation equivalent to ≲ 30 cGMP molecules or ≤ 3 resact molecules .

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, Representative measurement of the dependence of recovery-time constant on the amount of released cGMP. Inset: decay time was constant for low stimulation levels equivalent to ≲ 30 cGMP molecules or ≤ 3 resact molecules.


, Voltage response evoked by photolysis of DEACM - caged cGMP ( blue ) or 8 - Br - cGMP ( red ) . energy was adjusted such that Vm response amplitudes became similar ; for 8 - Br - cGMP , about 5 - times more energy was required . 10 ms .

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, Voltage response evoked by photolysis of DEACM-caged cGMP (blue) or 8-Br-cGMP (red). The light energy was adjusted such that Vm response amplitudes became similar; for 8-Br-cGMP, about 5-times more light energy was required. Light pulse 10 ms.


, Vm responses evoked by cGMP in presence ( blue ) absence ( red ) of HCN channel blocker ZD7288 ( 50 μM ) .

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, Vm responses evoked by cGMP in the presence (blue) and absence (red) of the HCN channel blocker ZD7288 (50 μM).


E , cGMP - evoked Vm response in ASW ( black ) , in presence of IBMX ( 1 mM ) ( red ) , in Ca2 + - free ASW ( [ Ca2 + ] ≤ 500 nM ) in presence of IBMX ( 1 mM ) ( blue ) . Signals were detected using potentiometric probe Di - 8 - ANEPPS in mode . F , Normalized responses from panel E .

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E, cGMP-evoked Vm response in ASW (black), in the presence of IBMX (1 mM) (red), and in Ca2+-free ASW ([Ca2+] ≤ 500 nM) in the presence of IBMX (1 mM) (blue). Signals were detected using the potentiometric probe Di-8-ANEPPS in the ratio mode. F, Normalized responses from panel E.


A , B , Ca2 + signals elicited by cGMP release recorded with FluoForte . UV flash released 28 ( A ) 215 B ) cGMP molecules . flashes are indicated by dashed magenta lines .

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A, B, Ca2+ signals elicited by cGMP release and recorded with FluoForte. The UV flash released 28 (A) and 215 B) cGMP molecules. Light flashes are indicated by dashed magenta lines.


C , pHi signals , elicited by release of approximately 1 , 400 cGMP molecules , were recorded with pHrodo Red . An increase of pHi is indicated by a decrease of ∆ F / F . Exemplary recording from a single . D , Average of pHi signals recorded from n = 25 . Error bars represent s . d .

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C, pHi signals, elicited by the release of approximately 1,400 cGMP molecules, were recorded with pHrodo Red. An increase of pHi is indicated by a decrease of ∆F/F. Exemplary recording from a single cell. D, Average of pHi signals recorded from n = 25 cells. Error bars represent s.d.


B , Relationship between cGMP synthesis rate free cGMP . We assume that a single GC molecule can synthesize 10 cGMP molecules during its lifetime of 150 ms Inset : expanded scale .

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B, Relationship between cGMP synthesis rate and free cGMP concentration. We assume that a single GC molecule can synthesize 10 cGMP molecules during its lifetime of 150 ms Inset: expanded scale.


B ) weights of E18 . 5 wild - type , TKO QKO foetuses . Data for are presented as circles with mean ± . Data were analysed by one - way ANOVA followed by multiple comparison with Benjamini Hochberg correction .

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B) Body weights of E18.5 wild-type, TKO and QKO foetuses. Data for individual animals are presented as circles with mean ± SEM. Data were analysed by one-way ANOVA followed by multiple comparison with Benjamini and Hochberg correction.


C ) Percentages of E18 . 5 QKO foetuses exhibiting indicated externally visible developmental defects ( numbers above bars represent number of examined ) . - matched TKO are included for comparison . An example of a QKO E18 . 5 pup presenting with an omphalocele ( asterisk ) , exencephaly ( arrow ) , spina bifida ( arrowhead ) is shown alongside an - matched WT control . Scale bar = 5 mm .

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C) Percentages of E18.5 QKO foetuses exhibiting the indicated externally visible developmental defects (numbers above bars represent the number of animals examined). Age-matched TKO animals are included for comparison. An example of a QKO E18.5 pup presenting with an omphalocele (asterisk), exencephaly (arrow), and spina bifida (arrowhead) is shown alongside an age-matched WT control. Scale bar = 5 mm.


A ) Percentages of E18 . 5 QKO foetuses exhibiting indicated defects when examined under a dissection microscope . - matched TKO are included for comparison . Numbers above bars represent number of examined .

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A) Percentages of E18.5 QKO foetuses exhibiting the indicated defects when examined under a dissection microscope. Age-matched TKO animals are included for comparison. Numbers above bars represent number of animals examined.


B - D ) depicting hindlimbs ( HL ) , tail ( T ) , forelimbs ( FL ) , ventral view of palate ( P ) in a wild - type foetus at E18 . 5 of development . E - G ) Corresponding of a representative - matched QKO foetus . Panel G shows a cleft palate ( indicated by arrowheads ) . H , I ) Magnified of panels E F . Arrows indicate excess tissue at anterior posterior edges of hindlimb forelimb , respectively , of QKO foetuses .

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B-D) Images depicting the hindlimbs (HL), tail (T), forelimbs (FL), and ventral view of the palate (P) in a wild-type foetus at E18.5 of development. E-G) Corresponding images of a representative age-matched QKO foetus. Panel G shows a cleft palate (indicated by arrowheads). H, I) Magnified images of panels E and F. Arrows indicate excess tissue at the anterior and posterior edges of the hindlimb and forelimb, respectively, of QKO foetuses.


J ) Percentages of QKO exhibiting various aortic arch abnormalities great vessel defects ( n = 19 ) .

J) Percentages of QKO animals exhibiting various aortic arch abnormalities and great vessel defects (n = 19).


K ) Aortic arch vessels in a wild - type foetus . A corresponding drawing to explain is shown on right .

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K) Aortic arch and vessels in a wild-type foetus. A corresponding drawing to explain the images is shown on the right.


L , M ) of QKO foetuses accompanying illustration of vessel defects . Absence of right subclavian artery ( L ) persistent truncus arteriosus ( pTA ) with a right curving pulmonary trunk / ductus arteriosus ( PT / rcDA ; M ) .

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L, M) Images of QKO foetuses and accompanying illustration of vessel defects. Absence of the right subclavian artery (L) and persistent truncus arteriosus (pTA) with a right curving pulmonary trunk / ductus arteriosus (PT/rcDA; M).


A - C ) Heads of E18 . 5 foetuses were serially sectioned in coronal orientation stained with H & E . Representative of rostro - occipital level - matched sections from - matched wild - type ( n = 2 ) , Bok - / - Bak - / - Bax - / - triple knockout ( TKO ; n = 4 ) QKO foetuses ( n = 4 ) depicting forebrain , nasal cavity , tongue are shown . BP , bony palate ; Cx , cerebral cortex ; ES , epidural space ; In , incisor ; LV ; lateral ventricle ; Ma , mandible ; MC , mouth cavity ; NC , nasal cavity ; NS , nasal septum ; St , striatum ; SVZ , subventricular zone ; To , tongue ; WP , whisker follicles . Scale bar = 850 µm ( A ) , 1200 µm ( B , C ) .

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A-C) Heads of E18.5 foetuses were serially sectioned in coronal orientation and stained with H&E. Representative images of rostro-occipital level-matched sections from age-matched wild-type (n = 2), Bok-/-Bak-/-Bax-/- triple knockout (TKO; n = 4) and QKO foetuses (n = 4) depicting the forebrain, nasal cavity, and tongue are shown. BP, bony palate; Cx, cerebral cortex; ES, epidural space; In, incisor; LV; lateral ventricle; Ma, mandible; MC, mouth cavity; NC, nasal cavity; NS, nasal septum; St, striatum; SVZ, subventricular zone; To, tongue; WP, whisker follicles. Scale bar = 850 µm (A), 1200 µm (B, C).


D ) Morphometric assessment of indicated tissue areas . Data of are displayed as circles with means standard error of mean . Data were analysed by unpaired , two - Student ' s t - . Related data normalised to mean bodyweight are displayed in Figure EV1 .

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D) Morphometric assessment of indicated tissue areas. Data of individual animals are displayed as circles with means and standard error of the mean. Data were analysed by unpaired, two-tailed Student's t-test. Related data normalised to mean bodyweight are displayed in Figure EV1.


A - C ) Representative H & E - stained coronal serial sections of eye from E18 . 5 wild - type ( n = 2 ) , Bok - / - Bak - / - Bax - / - triple knockout ( n = 3 ) , Bok - / - Bak - / - Bax - / - Bid - / - quadruple knockout ( n = 3 ) foetuses .

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A-C) Representative H&E-stained coronal serial sections of the eye from E18.5 wild-type (n = 2), Bok-/-Bak-/-Bax-/- triple knockout (n = 3), and Bok-/-Bak-/-Bax-/-Bid-/- quadruple knockout (n = 3) foetuses.


D ) Densitometry of hyaloid cavity tissue section as an assessment of blood vessel endothelial present . Data of are displayed as circles with means standard error of mean were analysed by unpaired , two - Student ' s t - .

D) Densitometry of hyaloid cavity tissue section as an assessment of blood vessel endothelial cells present. Data of individual animals are displayed as circles with means and standard error of the mean and were analysed by unpaired, two-tailed Student's t-test.


( A ) Estimation of BID in TKO MEFs . DKO MEFs re - expressing human BAK , TKO MEFs , two human leukaemia lines RS4 ; 11 MV4 ; 11 , were lysed analysed by Western blot for BID , using a rat monoclonal antibody raised against human BID ( anti - hBID clone 2D1 ) that also recognises mouse BID . Also present are samples of human FL - BID added at equivalent concentrations used in ( B ) ( C ) . On right gel , duplicate samples of DKO TKO permeabilised DKO TKO ( from B C ) showing that endogenous mouse BID is in these . Data shown are representative of 2 independent experiments .

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(A) Estimation of BID protein levels in TKO MEFs. DKO MEFs re-expressing human BAK, TKO MEFs, and two human leukaemia cell lines RS4;11 and MV4;11, were lysed and analysed by Western blot for BID levels, using a rat monoclonal antibody raised against human BID (anti-hBID clone 2D1) that also recognises mouse BID. Also present are samples of the human FL-BID protein added at the equivalent concentrations used in (B) and (C). On the right gel, duplicate samples of DKO and TKO cells and permeabilised DKO and TKO cells (from B and C) showing that endogenous mouse BID is cytosolic in these cells. Data shown are representative of 2 independent experiments.


( B , C ) Partial cytochrome c release by FL - BID from mitochondria of TKO MEFs when combined with BIM BH3 peptide or cBID . DKO MEFs re - expressing human BAK or TKO MEFs were permeabilised , then incubated with indicated concentrations of Bim BH3 peptide , cBID or FL - BID , alone or in indicated combinations , at 30oC for 30 min . were then co - stained for cytochrome c TOM20 analysed by flow cytometry . Left panels show median data from replicate experiments ( squares denote experiment 1 , circles denote experiment 2 ) . Right panels show histograms of cytochrome c TOM20 from experiment 2 ( treatments are as listed in left - panels ) .

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(B, C) Partial cytochrome c release by FL-BID from mitochondria of TKO MEFs when combined with BIM BH3 peptide or cBID. DKO MEFs re-expressing human BAK or TKO MEFs were permeabilised, and then the cells incubated with the indicated concentrations of Bim BH3 peptide, cBID or FL-BID, alone or in the indicated combinations, at 30oC for 30 min. Cells were then co-stained for cytochrome c and TOM20 and analysed by flow cytometry. Left hand panels show median fluorescence data from replicate experiments (squares denote experiment 1, circles denote experiment 2). Right hand panels show histograms of cytochrome c staining and TOM20 staining from experiment 2 (treatments are as listed in the left-hand panels).


B RT - PCR of from mid - temporal autopsy brain sections obtained from NCI , MCI or AD subjects . Representative gel of results is shown . C Quantitation of RT - PCR products of samples analyzed from post - mortem brain tissues ( mean s . e . m . ) . An omnibus significant effect was obtained using one - way of variance ( one - way ANOVA ) with Tukey post - to determine p - value . Error bars are s . e . m .

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B RT-PCR analysis of RNA from mid-temporal autopsy brain sections obtained from NCI, MCI or AD subjects. Representative gel of results is shown.C Quantitation of RT-PCR products of all samples analyzed from post-mortem brain tissues (mean s.e.m.). An omnibus significant effect was obtained using the one-way analysis of variance (one-way ANOVA) with Tukey post-test to determine the p-value. Error bars are s.e.m.


D RT - PCR of from hippocampus of 1 - 2 3 - 7 month old non - transgenic ( WT ) or TgCRND8 ( AD ) mice . E of relative of exon 19 inclusion for TgCRND8 ( AD ) WT mice ( mean s . e . m . ; one - way ANOVA with Tukey post - ) . number of samples analyzed ( n ) is indicated .

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D RT-PCR analysis of RNA from the hippocampus of 1-2 and 3-7 month old non-transgenic (WT) or TgCRND8 (AD) mice.E Quantification of the relative ratio of exon 19 inclusion for TgCRND8 (AD) and WT mice (mean s.e.m.; one-way ANOVA with Tukey post-test). The number of samples analyzed (n) is indicated.


B Top , diagram of ASOs tested below , mapped to their position of complementarity on ApoER2 . Bottom , RT - PCR of isolated from HeLa transfected with indicated ASO at a final of 80 nM . spliced forms are labeled . of inclusion is shown below image calculated as : Inclusion ( % ) = [ exon 19 included / ( exon 19 included + exon 19 skipped ) ] x 100 .

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B Top, diagram of ASOs tested below, mapped to their position of complementarity on ApoER2. Bottom, RT-PCR of RNA isolated from HeLa cells transfected with the indicated ASO at a final concentration of 80 nM. RNA spliced forms are labeled. Quantification of the percentage inclusion is shown below the image and calculated as: Inclusion (%)=[exon 19 included/(exon 19 included+exon 19 skipped)] x 100.


D Top , RT - PCR of ApoER2 exon 19 splicing in HeLa depleted of SRSF1 using an SRSF1 - specific siRNA . Control is a non - specific siRNA . Bottom , Immunoblot of SRSF1 from HeLa lysates depleted of SRSF1 . β - actin is included as a control . E of exon 19 splicing in ( D ) . Error bars show s . e . m . P - value was determined using Student ' s two - t - .

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D Top, RT-PCR analysis of ApoER2 exon 19 splicing in HeLa cells depleted of SRSF1 using an SRSF1-specific siRNA. Control is a non-specific siRNA. Bottom, Immunoblot analysis of SRSF1 from HeLa lysates depleted of SRSF1. β-actin is included as a control.E Quantification of exon 19 splicing in (D). Error bars show s.e.m. P-value was determined using Student's two-tailed t-test.


F Top , RT - PCR of exon 19 splicing in a mouse kidney depleted of SRSF1 by RNAi . Control is a non - specific siRNA . Bottom , Immunoblot of SRSF1 from HeLa lysates depleted of SRSF1 . β - actin is included as a control . G of exon 19 splicing in E . Error bars show s . e . m . P - value was calculated using Student ' s two - t - .

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F Top, RT-PCR analysis of exon 19 splicing in a primary mouse kidney cell line depleted of SRSF1 by RNAi. Control is a non-specific siRNA. Bottom, Immunoblot analysis of SRSF1 from HeLa lysates depleted of SRSF1. β-actin is included as a control.G Quantification of exon 19 splicing in E. Error bars show s.e.m. P-value was calculated using Student's two-tailed t-test.


A Top , ASOs mapped onto mouse ApoER2 exon 19 gene region . Bottom , RT - PCR of isolated from mouse transfected with different ASOs . of exon 19 inclusion is shown below gel image .

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A Top, ASOs mapped onto the mouse ApoER2 exon 19 gene region. Bottom, RT-PCR of RNA isolated from mouse cells transfected with different ASOs. Quantification of percent exon 19 inclusion is shown below the gel image.


B ASO improves exon 19 inclusion in a dose - responsive manner . RT - PCR of from mouse transfected with increasing doses of ASO - 15 . inclusion of exon 19 is shown below gel image . half - maximal effective ( EC50 ) was calculated using GraphPad Prism version 6 . 0 ( GraphPad Software , San Diego , CA ) after fitting data using nonlinear regression with normalized response variable slope .

B ASO improves exon 19 inclusion in a dose-responsive manner. RT-PCR analysis of RNA from mouse cells transfected with increasing doses of ASO-15. The percent inclusion of exon 19 is shown below the gel image. The half-maximal effective concentration (EC50) was calculated using GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA) after fitting the data using nonlinear regression with normalized response and variable slope.


D RT - PCR of isolated from hippocampus of mice treated as adults by ICV injection of indicated ASOs . C refers to a non - specific control ASO . E Quantitation of ApoER2 exon 19 inclusion from D ( mean s . e . m . , one - way ANOVA with Bonferroni multiple comparison .

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D RT-PCR analysis of RNA isolated from the hippocampus of mice treated as adults by ICV injection of indicated ASOs. C refers to a non-specific control ASO.E Quantitation of ApoER2 exon 19 inclusion from D (mean s.e.m., one-way ANOVA with Bonferroni multiple comparison test.


A RT - PCR of ApoER2 exon 19 splicing in isolated from hippocampus of 4 month old AD WT mice treated with ASO - 21 or ASO - C at P1 - 2 . B Quantitation of exon 19 splicing . ( mean s . e . m . ; Kruskal - Wallis with Dunn ' s multiple comparison .

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A RT-PCR analysis of ApoER2 exon 19 splicing in RNA isolated from hippocampus of 4 month old AD and WT mice treated with ASO-21 or ASO-C at P1-2.B Quantitation of exon 19 splicing. (mean s.e.m.; Kruskal-Wallis test with Dunn's multiple comparison test.


C Immunoblot for exon 19 containing ApoER2 ( top ) total ApoER2 ( bottom ) . β - actin was used as loading control . D of ApoER2 isoforms shown in C ( mean s . e . m . , student ' s t - ) .

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C Immunoblot for exon 19 containing ApoER2 (top) and total ApoER2 (bottom). β-actin was used as loading control.D Quantification of ApoER2 protein isoforms shown in C (mean s.e.m., student's t-test).


A Input - output curves show hippocampal CA1 field excitatory postsynaptic potential ( fEPSP ) response amplitudes as a function of stimulus delivered to Schaffer collateral afferent . Two - way repeated - measures ANOVA revealed a significant effect of treatment ( F ( 2 , 119 ) = 31 . 62 , p < 0 . 0001 ) with Bonferroni multiple comparisons showing significant differences between ASO - C treated AD - matched ASO - C treated WT ( * p < 0 . 05 ) . Responses from ASO - 21 treated AD mice were not significantly different from ASO - C treated WT or AD mice .

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A Input-output curves show hippocampal CA1 field excitatory postsynaptic potential (fEPSP) response amplitudes as a function of stimulus intensity delivered to Schaffer collateral afferent fibers. Two-way repeated-measures ANOVA revealed a significant effect of treatment (F(2,119) = 31.62, p < 0.0001) with the Bonferroni multiple comparisons test showing significant differences between ASO-C treated AD and age-matched ASO-C treated WT (* p < 0.05). Responses from ASO-21 treated AD mice were not significantly different from ASO-C treated WT or AD mice.


B Representative paired - facilitation ( PPF ) responses from ASO - C treated WT ( top ) , ASO - C treated AD ( middle ) ASO - 21 treated AD ( bottom ) mice are shown to left . Scale bars = 0 . 2 mV by 10 msec apply to traces . Bar graph ( right ) summarizes paired - ratios . PPF was not significantly different in AD compared to WT samples , nor was it affected by ASO - 21 treatment .

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B Representative paired-pulse facilitation (PPF) responses from ASO-C treated WT (top), ASO-C treated AD (middle) and ASO-21 treated AD (bottom) mice are shown to the left. Scale bars = 0.2 mV by 10 msec apply to all traces. Bar graph (right) summarizes the paired-pulse ratios. PPF was not significantly different in AD compared to WT samples, nor was it affected by ASO-21 treatment.


C LTP was unaffected after ASO - 21 treatment similar in AD compared to WT mice . To combine data from multiple slices , individually measured fEPSP rise slopes were normalized to average baseline value taken 0 - 10 minutes before tetanic stimulation ( arrows ) . normalized values are plotted as a function of time for ASO - C treated WT ( gray , left ) , ASO - C treated AD ( red , middle ) ASO - 21 treated AD mice ( blue , right ) . Representative traces taken before 50 - 60 minutes after tetanic stimulation are shown in insets ( scale bars = 0 . 2 mV by 10 msec for traces ) . D Graph summarizes LTP results as of mean fEPSP rise slope measured 50 - 60 minutes after tetanic stimulation divided by that taken 0 - 10 minutes before tetanic stimulation . There was no significant difference among groups . data are shown as mean ± s . e . m are from same hippocampal brain slices ( n = 6 slices from 4 , 3 , 4 WT / ASO - C , AD / ASO - C AD / ASO - 21 , per group , respectively ) .

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C LTP was unaffected after ASO-21 treatment and similar in AD compared to WT mice. To combine data from multiple slices, individually measured fEPSP rise slopes were normalized to the average baseline value taken 0-10 minutes before tetanic stimulation (arrows). The normalized values are plotted as a function of time for ASO-C treated WT (gray, left), ASO-C treated AD (red, middle) and ASO-21 treated AD mice (blue, right). Representative traces taken before and 50-60 minutes after tetanic stimulation are shown in the insets (scale bars = 0.2 mV by 10 msec for all traces).D Graph summarizes LTP results as the ratio of mean fEPSP rise slope measured 50-60 minutes after tetanic stimulation divided by that taken 0-10 minutes before tetanic stimulation. There was no significant difference among groups. All data are shown as mean ± s.e.m and are from the same hippocampal brain slices (n=6 slices from 4, 3, and 4 WT/ASO-C, AD/ASO-C and AD/ASO-21, animals per group, respectively).


A Representative water - maze pathway traces of last four trials of day 3 . Trial 1 is shown in red , trial 2 in green , trial 3 in blue trial 4 in black . B Morris water maze ( MWM ) of acquisition performance of ( left ) ( right ) mice represented as mean ( cm ) that mice traveled to find submerged platform plotted per day ( mean s . e . m . ) Symbols represent statistically significant difference within same genotype ( * AD ASO - C vs AD ASO - 21 # WT ASO - C vs WT ASO - 21 ) or between groups with same treatment ( ΔAD ASO - C vs WT ASO - C ^ AD ASO - 21 vs WT ASO - 21 ) on corresponding day ( two - way repeated - measures ANOVA with Tukey multiple comparisons ; * * p < 0 . 01 , ^ p < 0 . 05 , ΔΔΔΔp < 0 . 0001 ) . See source data for statistical results . C Morris water maze integrated ( Zhang et al , 2014 ) ( under curve , AUC ) for ( left ) ( right ) WT AD mice treated with control ASO ( ASO - C ) or ASO - 21 . ( mean s . e . m . ; one - way ANOVA , Tukey post - . D , E Correlation of performance on day 3 of MWM with exon 19 skipping among groups of mice ( D ) or between AD mice treated with ASO - C or ASO - 21 ( E ) . mice are indicated . Pearson correlation coefficient ( r ) , covariance ( σ ) two - p - value are shown .

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A Representative water-maze pathway traces of the last four trials of day 3. Trial 1 is shown in red, trial 2 in green, trial 3 in blue and trial 4 in black.B Morris water maze (MWM) analysis of acquisition performance of male (left) and female (right) mice represented as the mean distance (cm) that mice traveled to find the submerged platform plotted per day (mean s.e.m.) Symbols represent statistically significant difference within the same genotype (*AD ASO-C vs AD ASO-21 and #WT ASO-C vs WT ASO-21) or between groups with same treatment (ΔAD ASO-C vs WT ASO-C and ^AD ASO-21 vs WT ASO-21) on the corresponding day (two-way repeated-measures ANOVA with Tukey multiple comparisons test; **p<0.01, ^p<0.05, ΔΔΔΔp<0.0001). See source data for statistical results.C Morris water maze analysis integrated distance (Zhang et al, 2014) (area under the curve, AUC) for male (left) and female (right) WT and AD mice treated with control ASO (ASO-C) or ASO-21. (mean s.e.m.; one-way ANOVA, Tukey post-test.D,E Correlation analysis of performance on day 3 of the MWM with exon 19 skipping among all groups of mice (D) or between AD mice treated with ASO-C or ASO-21 (E). Male and female mice are indicated. Pearson correlation coefficient (r), covariance (σ) and two-tailed p-value are shown.


B ) Agarose gel electrophoresis of extracted from RNase treated or control K562 .

B) Agarose gel electrophoresis of RNA extracted from RNase treated or control K562 cells.


D ) Agarose gel electrophoresis of extracted from control , 4hr 24hr ActD treated K562 .

D) Agarose gel electrophoresis of RNA extracted from control, 4hr and 24hr ActD treated K562 cells.


E ) Barplots showing qRT - PCR gene expression ( mean + / - S . D . ) of PTEN , FNDC3B , STAM genes in control , 4hr 24hr actinomycin D treated K562 . p - value : two - t - , ( * * * p < 0 . 05 ) . results represent data from 3 technical replicates from 3 independent biological preparations .

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E) Barplots showing qRT-PCR gene expression levels (mean +/- S.D.) of PTEN, FNDC3B, and STAM genes in control, 4hr and 24hr actinomycin D treated K562 cells. p-value: two-tailed t-test, (*** p < 0.05). The results represent data from 3 technical replicates from 3 independent biological preparations.


Hi - C interaction heatmaps at 500kb ( left ) resolution showing of chromosome 11 , at 100kb resolution ( right ) showing chr11 : 79 - 115Mb for aXL control RNase A treated . arrows represent similar patterning of long - range interactions in samples treated with RNase after crosslinking . Hi - C interaction heatmaps at 500kb ( left ) resolution showing of chromosome 11 , at 100kb resolution ( right ) showing chr11 : 79 - 115Mb for bXL control RNase A treated . Black arrow indicates reduction in long - range interactions in bXL RNase A dataset . arrows represent perturbed patterning of long - range interactions in samples treated with RNase before crosslinking .

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Hi-C interaction heatmaps at 500kb (left) resolution showing all of chromosome 11, and at 100kb resolution (right) showing chr11: 79-115Mb for aXL control and RNase A treated cells. The arrows represent similar patterning of long-range interactions in samples treated with RNase after crosslinking.   Hi-C interaction heatmaps at 500kb (left) resolution showing all of chromosome 11, and at 100kb resolution (right) showing chr11: 79-115Mb for bXL control and RNase A treated cells. Black arrow indicates the reduction in long-range interactions in the bXL RNase A dataset. The arrows represent perturbed patterning of long-range interactions in samples treated with RNase before crosslinking.


C - D ) Pearson correlation heatmaps showing genomic interactions at 500kb resolution for chromosome 4 , with 1st principal components ( 1st PC ) below for aXL ( C ) bXL ( D ) samples . bXL RNase A sample shows reduced Pearson correlations across genome .

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C-D) Pearson correlation heatmaps showing the genomic interactions at 500kb resolution for chromosome 4, with the 1st principal components (1st PC) below for aXL (C) and bXL (D) samples. The bXL RNase A sample shows reduced Pearson correlations across the genome.


E ) Violin plot showing first eigenvalues ( calculated by trans - chromosomal data ) in control RNase treated before after crosslinking . bXL RNase A sample show reduction of negative eigenvalues , indicative of perturbation of B - type compartments . p - value : Wilcoxon rank - sum . In violin plots , horizontal bands represent median , error bars represent + / - 1 . 5 x IQR values , outer shapes represent density of data points . figures represent data generated from pooled Hi - C datasets with 2 biological replicates .

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E) Violin plot showing the first eigenvalues (calculated by the trans-chromosomal data) in control and RNase treated cells before and after crosslinking. The bXL RNase A sample show reduction of the negative eigenvalues, indicative of perturbation of B-type compartments. p-value: Wilcoxon rank-sum test. In the violin plots, the horizontal bands represent the median, the error bars represent +/- 1.5 x IQR values, and the outer shapes represent the density of the data points. The figures represent data generated from the pooled Hi-C datasets with 2 biological replicates.


F ) Saddle plots showing compartmental interactions for both cis - trans - contacts across conditions . bXL RNase A samples display reduced B - B type interactions .

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F) Saddle plots showing the compartmental interactions for both cis- and trans-contacts across the conditions. The bXL RNase A samples display reduced B-B type interactions.


Interaction heatmaps at 40kb resolution zooming in chr21 : 19 . 5 - 33Mb showing TAD structures in aXL bXL datasets . aXL bXL RNase A / control log2 ratios are shown below heatmaps .

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Interaction heatmaps at 40kb resolution zooming in chr21:19.5-33Mb and showing the TAD structures in aXL and bXL datasets. The aXL and bXL RNase A / control log2 ratios are shown below the heatmaps.


Boxplot showing TAD boundary scores for aXL bXL datasets . p - value : Wilcoxon rank - sum . ( * * * p < 0 . 05 ) . horizontal bands in boxplots represent median , error bars represent + / - 1 . 5 x IQR values , outer shapes represent density of data points . figures represent data generated from pooled Hi - C datasets with 2 biological replicates .

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Boxplot showing the TAD boundary scores for the aXL and bXL datasets. p-value: Wilcoxon rank-sum test. (*** p < 0.05). The horizontal bands in the boxplots represent the median, the error bars represent +/- 1.5 x IQR values, and the outer shapes represent the density of the data points. The figures represent data generated from the pooled Hi-C datasets with 2 biological replicates.


C - D ) Mean interactions frequencies + / - 1Mb centered on TAD boundaries for each dataset at 40kb resolution , as well as log2 ratios of aXL ( left panel ) or bXL ( right panel ) samples . TAD structures are not altered upon RNase A treatment in samples treated with RNase after crosslinking .

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C-D) Mean interactions frequencies +/- 1Mb centered on all TAD boundaries for each dataset at 40kb resolution, as well as the log2 ratios of aXL (left panel) or bXL (right panel) samples. The TAD structures are not altered upon RNase A treatment in samples treated with RNase after crosslinking.


A ) Hi - C interaction heatmaps at 500kb ( left ) resolution showing of chromosome 11 , at 100kb resolution ( right ) showing chr11 : 79 - 115Mb for control 24hr ActD treated . arrows point out to perturbed long - range interactions at shorter scales .

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A) Hi-C interaction heatmaps at 500kb (left) resolution showing all of chromosome 11, and at 100kb resolution (right) showing chr11: 79-115Mb for control and 24hr ActD treated cells. The arrows point out to perturbed long-range interactions at shorter scales.


B ) Pearson correlation heatmaps showing genomic interactions at 500kb resolution for chromosome 4 , with 1st principal component ( 1st PC ) below for control 24hr ActD treated .

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B) Pearson correlation heatmaps showing the genomic interactions at 500kb resolution for chromosome 4, with the 1st principal component (1st PC) below for control and 24hr ActD treated cells.


C ) Violin plot showing first eigenvalues ( calculated by trans - chromosomal data ) in control ActD treated . p - value : Wilcoxon rank - sum . In violin plots , horizontal bands represent median , error bars represent + / - 1 . 5 x IQR values , outer shapes represent density of data points . figures represent data generated from pooled Hi - C datasets with 2 biological replicates .

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C) Violin plot showing the first eigenvalues (calculated by the trans-chromosomal data) in control and ActD treated cells. p-value: Wilcoxon rank-sum test. In the violin plots, the horizontal bands represent the median, the error bars represent +/- 1.5 x IQR values, and the outer shapes represent the density of the data points. The figures represent data generated from the pooled Hi-C datasets with 2 biological replicates.


D ) Saddle plots showing compartmental interactions for both cis - trans - contacts across conditions .

D) Saddle plots showing the compartmental interactions for both cis- and trans-contacts across the conditions.


E ) Scaling plot generated by 500kb resolution Hi - C data showing interaction frequency as a function of genomic .

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E) Scaling plot generated by 500kb resolution Hi-C data showing the interaction frequency as a function of genomic distance.


A ) Interaction heatmaps at 40kb resolution zooming in chr9 : 104 - 114Mb showing TAD structures , as well as log2 ratios , in control transcriptionally inhibited datasets . Black arrow indicates increase in inter - TAD interactions .

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A) Interaction heatmaps at 40kb resolution zooming in chr9:104-114Mb and showing the TAD structures, as well as the log2 ratios, in control and transcriptionally inhibited datasets. Black arrow indicates the increase in inter-TAD interactions.


B ) Boxplot showing TAD boundary scores in control 24hr ActD treated . p - value : Wilcoxon rank - sum . horizontal bands in boxplots represent median , error bars represent + / - 1 . 5 x IQR values , outer shapes represent density of data points . figures represent data generated from pooled Hi - C datasets with 2 biological replicates .

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B) Boxplot showing the TAD boundary scores in control and 24hr ActD treated cells. p-value: Wilcoxon rank-sum test. The horizontal bands in the boxplots represent the median, the error bars represent +/- 1.5 x IQR values, and the outer shapes represent the density of the data points. The figures represent data generated from the pooled Hi-C datasets with 2 biological replicates.


C ) Mean interaction frequencies centered on TAD boundaries + / - 1Mb for each dataset at 40kb resolution , as well as log2 ratios . inter - TAD interactions are increased upon transcriptional inhibition .

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C) Mean interaction frequencies centered on all TAD boundaries +/- 1Mb for each dataset at 40kb resolution, as well as the log2 ratios. The inter-TAD interactions are increased upon transcriptional inhibition.


D ) Aggregate Peak on K562 loops coordinates heatmaps are centered on loop positions + / - 250kb using 25kb resolution Hi - C data . There is a ~ 50 % reduction of loop as assessed by z - scores log2 fold change .

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D) Aggregate Peak Analysis based on the K562 loops coordinates The heatmaps are centered on the loop positions +/- 250kb using 25kb resolution Hi-C data. There is a ~50% reduction of loop intensity as assessed by z-scores and log2 fold change.


E ) Boxplot showing TAD boundary scores in control 24hr ActD treated for boundaries that are bound with differing numbers of CTCF binding sites . reduction of TAD boundary scores upon ActD treatment is independent of CTCF binding . p - value : Wilcoxon rank - sum . horizontal bands in boxplots represent median , error bars represent + / - 1 . 5 x IQR values , outer shapes represent density of data points . figures represent data generated from pooled Hi - C datasets with 2 biological replicates .

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E) Boxplot showing the TAD boundary scores in control and 24hr ActD treated cells for boundaries that are bound with differing numbers of CTCF binding sites. The reduction of TAD boundary scores upon ActD treatment is independent of CTCF binding. p-value: Wilcoxon rank-sum test. The horizontal bands in the boxplots represent the median, the error bars represent +/- 1.5 x IQR values, and the outer shapes represent the density of the data points. The figures represent data generated from the pooled Hi-C datasets with 2 biological replicates.


F ) Scaling plot generated by 40kb resolution Hi - C data showing interaction frequency as a function of genomic , with upper limit of 3Mb . 24hr ActD treated samples shows a slower rate of decay at shorter distances , correlating with reduction of insulation scores .

F) Scaling plot generated by 40kb resolution Hi-C data showing the interaction frequency as a function of genomic distance, with upper distance limit of 3Mb. The 24hr ActD treated samples shows a slower rate of decay at shorter distances, correlating with the reduction of insulation scores.


A . Exogenous immunoprecipitation of interaction between TRIM50 NLRP3 . Data information : Data are representative of three biological replicates with similar results .

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A. Exogenous immunoprecipitation assay of the interaction between TRIM50 and NLRP3. Data information: Data are representative of three biological replicates with similar results.


B . Endogenous immunoprecipitation of interaction between TRIM50 NLRP3 in mouse peritoneal macrophages . Data information : Data are representative of three biological replicates with similar results .

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B. Endogenous immunoprecipitation assay of the interaction between TRIM50 and NLRP3 in mouse peritoneal macrophages. Data information: Data are representative of three biological replicates with similar results.


C . TRIM50 NLRP3 were expressed obtained from in vitro translation system their interaction were further analyzed . Data information : Data are representative of three biological replicates with similar results .

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C. TRIM50 and NLRP3 proteins were expressed and obtained from the in vitro translation system and their interaction were further analyzed. Data information: Data are representative of three biological replicates with similar results.


D . Co - IP of interaction between NLRP3 TRIM50 after stimulation with nigericin for 0 , 15 , 30 60 min . Data information : Data are representative of three biological replicates with similar results .

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D. Co-IP assay of the interaction between NLRP3 and TRIM50 after stimulation with nigericin for 0, 15, 30 and 60 min. Data information: Data are representative of three biological replicates with similar results.


E . Co - IP of interaction between NLRP3 TRIM50 in mouse peritoneal macrophages primed with LPS for 6h treated with ATP for 30min . Data information : Data are representative of three biological replicates with similar results .

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E. Co-IP assay of the interaction between NLRP3 and TRIM50 in mouse peritoneal macrophages primed with LPS for 6h and treated with ATP for 30min. Data information: Data are representative of three biological replicates with similar results.


F . Confocal microscopy of interaction between TRIM50 NLRP3 in THP1 with stimulation by LPS ATP . Scale bars : 10μm . Data information : Data are representative of three biological replicates with similar results .

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F. Confocal microscopy assay of the interaction between TRIM50 and NLRP3 in THP1 cells with stimulation by LPS and ATP. Scale bars: 10μm. Data information: Data are representative of three biological replicates with similar results.


G . Schematic diagram of human TRIM50 ( WT ) its truncation mutants was presented ( top ) , interaction of TRIM50 ( TRIM50 truncation mutants ) NLRP3 was detected by IP . Data information : Data are representative of three biological replicates with similar results .

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G. Schematic diagram of human TRIM50 (WT) and its truncation mutants was presented(top), and the interaction of TRIM50 (TRIM50 truncation mutants) and NLRP3 was detected by IP assay. Data information: Data are representative of three biological replicates with similar results.


H , I . Schematic diagram of human NLRP3 ( WT ) its truncation mutants ( top ) , immunoprecipitation of interaction of NLRP3 ( NLRP3 truncation mutants ) with TRIM50 in HEK293T . Data information : Data are representative of three biological replicates with similar results .

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H, I. Schematic diagram of human NLRP3 (WT) and its truncation mutants (top), and immunoprecipitation assay of the interaction of NLRP3 (NLRP3 truncation mutants) with TRIM50 in HEK293T cells. Data information: Data are representative of three biological replicates with similar results.


A . Immunoblot of NLRP3 level in mouse peritoneal macrophages transfected with Si - Trim50 . Data information : Band densities were quantitated by ' Image J ' software normalized to GAPDH . P - values are shown , two - Student ' s t - ( A , Data are representative of three biological replicates with similar results . ( mean ±

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A. Immunoblot analysis of the NLRP3 protein level in the mouse peritoneal macrophages transfected with Si-Trim50. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-tailed Student's t-test (A, Data are representative of three biological replicates with similar results. (mean ± SD


B . qRT - PCR of Nlrp3 IL - 1β mRNA in LPS - primed Si - Trim50 transfected mouse peritoneal macrophages . Data information : P - values are shown , two - way of variance ( ANOVA ) ( B , Data are representative of three biological replicates with similar results . ( mean ±

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B. qRT-PCR analysis of Nlrp3 and IL-1β mRNA levels in LPS-primed Si-Trim50 transfected mouse peritoneal macrophages. Data information: P-values are shown, two-way analysis of variance (ANOVA) (B, Data are representative of three biological replicates with similar results. (mean ± SD


C , D . Immunoblot of Caspase1 - P20 ( C ) Gasdermin D ( D ) of WT or Trim50 - / - mouse peritoneal macrophages after LPS stimulation for 6h ATP treatment for 30min . Data information : Band densities were quantitated by ' Image J ' software normalized to GAPDH . P - values are shown , two - Student ' s t - Data are representative of three biological replicates with similar results . ( mean ±

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C, D. Immunoblot analysis of Caspase1-P20 (C) and Gasdermin D (D) of WT or Trim50-/- mouse peritoneal macrophages after LPS stimulation for 6h and ATP treatment for 30min. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SD


E , Immunoblot of Caspase1 - P20 ( E ) of WT or Trim50 - / - BMDM after LPS stimulation for 6h ATP treatment for 30min . Data information : Band densities were quantitated by ' Image J ' software normalized to GAPDH . P - values are shown , two - Student ' s t - Data are representative of three biological replicates with similar results . ( mean ±

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E, Immunoblot analysis of Caspase1-P20 (E) of WT or Trim50-/- BMDM cells after LPS stimulation for 6h and ATP treatment for 30min. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SD


F . Immunoblot of Gasdermin D ( F ) of WT or Trim50 - / - BMDM after LPS stimulation for 6h ATP treatment for 30min . Data information : Band densities were quantitated by ' Image J ' software normalized to GAPDH . P - values are shown , two - Student ' s t - Data are representative of three biological replicates with similar results . ( mean ±

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F. Immunoblot analysis of Gasdermin D (F) of WT or Trim50-/- BMDM cells after LPS stimulation for 6h and ATP treatment for 30min. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SD


G , ELISA of IL - 1β , TNF - α IL - 6 in supernatant of WT or Trim50 - / - peritoneal macrophages with activation of NLRP3 inflammasome . n = 3 Data information : P - values are shown , two - way of variance ( ANOVA ) G , Data are representative of three biological replicates with similar results . ( mean ±

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G, ELISA analysis of IL-1β, TNF-α and IL-6 in the supernatant of WT or Trim50-/- peritoneal macrophages with the activation of NLRP3 inflammasome. n=3 Data information: P-values are shown, two-way analysis of variance (ANOVA) G, Data are representative of three biological replicates with similar results. (mean ± SD


H . ELISA of IL - 1β , TNF - α IL - 6 in supernatant of WT or Trim50 - / - BMDM with activation of NLRP3 inflammasome . n = 3 Data information : P - values are shown , two - way of variance ( ANOVA ) Data are representative of three biological replicates with similar results . ( mean ±

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H. ELISA analysis of IL-1β, TNF-α and IL-6 in the supernatant of WT or Trim50-/- BMDM cells with the activation of NLRP3 inflammasome. n=3 Data information: P-values are shown, two-way analysis of variance (ANOVA) Data are representative of three biological replicates with similar results. (mean ± SD


I . LDH of WT or Trim50 - / - mouse peritoneal macrophages with activation of NLRP3 inflammasome . n = 3 Data information : P - values are shown , two - way of variance ( ANOVA ) I ) . Data are representative of three biological replicates with similar results . ( mean ±

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I. LDH analysis of WT or Trim50-/- mouse peritoneal macrophages with the activation of NLRP3 inflammasome. n=3 Data information: P-values are shown, two-way analysis of variance (ANOVA) I). Data are representative of three biological replicates with similar results. (mean ± SD


A . Immunoprecipitation of NLRP3 ubiquitination in HEK293T transfected with HA - ubiquitin , His - NLRP3 Flag - TRIM50 plasmids . Data information : Band densities were quantitated by ' Image J ' software normalized to GAPDH . Data are representative of three biological replicates with similar results .

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A. Immunoprecipitation analysis of the NLRP3 ubiquitination in HEK293T cells transfected with HA-ubiquitin, His-NLRP3 and Flag-TRIM50 plasmids. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.


B . Immunoprecipitation of endogenous NLRP3 ubiquitination in WT or Trim50 - / - BMDM . Data information : Band densities were quantitated by ' Image J ' software normalized to GAPDH . Data are representative of three biological replicates with similar results .

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B. Immunoprecipitation analysis of the endogenous NLRP3 ubiquitination in WT or Trim50-/- BMDM cells. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.


C . Immunoprecipitation of NLRP3 ubiquitination in HEK293T transfected with NLRP3 , HA - Ubiquitin , TRIM50 or TRIM50 ( ΔRING ) mutant plasmids . Data information : Band densities were quantitated by ' Image J ' software normalized to GAPDH . Data are representative of three biological replicates with similar results .

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C. Immunoprecipitation analysis of NLRP3 ubiquitination in HEK293T cells transfected with NLRP3, HA-Ubiquitin, TRIM50 or TRIM50 (ΔRING) mutant plasmids. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.


D . Immunoblot of ASC oligomerization in cross - linked pellets ( insoluble ) whole lysate ( soluble ) in HEK293T with nigericin treatment for 30 min . Data information : Band densities were quantitated by ' Image J ' software normalized to GAPDH . Data are representative of three biological replicates with similar results .

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D. Immunoblot analysis of the ASC oligomerization in cross-linked cytosolic pellets (insoluble) and the whole cell lysate (soluble) in HEK293T cells with nigericin treatment for 30 min. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.


E . Immunoblot of NLRP3 in cycloheximide ( CHX ) treated HEK293T . Data information : Band densities were quantitated by ' Image J ' software normalized to GAPDH . P - values are shown , two - way ANOVA Data are representative of three biological replicates with similar results . ( mean ±

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E. Immunoblot analysis of NLRP3 protein levels in cycloheximide (CHX) treated HEK293T cells. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-way ANOVA Data are representative of three biological replicates with similar results. (mean ± SD


F . Immunoblot of NLRP3 in HEK293T transfected with NLRP3 plasmid TRIM50 or TRIM50 ( ΔRING ) mutant , followed with further treatment with CHX for 0h , 4h or 8h . Data information : Band densities were quantitated by ' Image J ' software normalized to GAPDH . P - values are shown , two - way ANOVA Data are representative of three biological replicates with similar results . ( mean ±

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F. Immunoblot analysis of NLRP3 in HEK293T cells transfected with NLRP3 plasmid and TRIM50 or TRIM50 (ΔRING) mutant, followed with the further treatment with CHX for 0h, 4h or 8h. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-way ANOVA Data are representative of three biological replicates with similar results. (mean ± SD


G . Immunoblot of NLRP3 in mouse peritoneal macrophages , followed with further treatment with CHX for 0h , 4h or 8h . Data information : Band densities were quantitated by ' Image J ' software normalized to GAPDH . P - values are shown , two - way ANOVA Data are representative of three biological replicates with similar results . ( mean ±

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G. Immunoblot analysis of NLRP3 in mouse peritoneal macrophages, followed with the further treatment with CHX for 0h, 4h or 8h. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-way ANOVA Data are representative of three biological replicates with similar results. (mean ± SD


A . SDD - of NLRP3 oligomerization in HEK293T transfected with NLRP3 TRIM50 plasmids . Data information : Data are representative of three biological replicates with similar results .

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A. SDD-AGE analysis of NLRP3 oligomerization in HEK293T cells transfected with NLRP3 and TRIM50 plasmids. Data information: Data are representative of three biological replicates with similar results.


B . SDD - of NLRP3 oligomerization in NLRP3 TRIM50 transfected HEK293T followed with nigericin treatment for 30min . Data information : Data are representative of three biological replicates with similar results .

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B. SDD-AGE analysis of NLRP3 oligomerization in NLRP3 and TRIM50 transfected HEK293T cells followed with nigericin treatment for 30min. Data information: Data are representative of three biological replicates with similar results.


C . Immunoblot of ASC oligomerization in cross - linked pellets ( insoluble ) whole lysate ( soluble ) in HEK293T transfected with NLRP3 , ASC TRIM50 plasmids followed with nigericin treatment for 30 min . Data information : Data are representative of three biological replicates with similar results .

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C. Immunoblot analysis of ASC oligomerization in cross-linked cytosolic pellets (insoluble) and whole cell lysate (soluble) in HEK293T cells transfected with NLRP3, ASC and TRIM50 plasmids followed with nigericin treatment for 30 min. Data information: Data are representative of three biological replicates with similar results.


D . SDD - of NLRP3 oligomerization in WT or Trim50 - / - peritoneal macrophages with LPS treatment for 6h ATP treatment for 30 min . Data information : Data are representative of three biological replicates with similar results .

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D. SDD-AGE analysis of NLRP3 oligomerization in WT or Trim50-/- peritoneal macrophages with LPS treatment for 6h and ATP treatment for 30 min. Data information: Data are representative of three biological replicates with similar results.


E . Immunoblot of ASC oligomerization in cross - linked pellets ( insoluble ) whole lysate ( soluble ) in WT or Trim50 - / - peritoneal macrophages by LPS stimulation for 6h ATP treatment for 30min . Data information : Data are representative of three biological replicates with similar results .

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E. Immunoblot analysis of ASC oligomerization in cross-linked cytosolic pellets (insoluble) and whole cell lysate (soluble) in WT or Trim50-/- peritoneal macrophages by LPS stimulation for 6h and ATP treatment for 30min. Data information: Data are representative of three biological replicates with similar results.


F . SDD - of NLRP3 oligomerization in WT or Trim50 - / - BMDM with LPS treatment for 6h ATP treatment for 30 min . Data information : Data are representative of three biological replicates with similar results .

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F. SDD-AGE analysis of NLRP3 oligomerization in WT or Trim50-/- BMDM cells with LPS treatment for 6h and ATP treatment for 30 min. Data information: Data are representative of three biological replicates with similar results.


G . Immunoblot of ASC oligomerization in cross - linked pellets ( insoluble ) whole lysate ( soluble ) in WT or Trim50 - / - BMDM by LPS stimulation for 6h ATP treatment for 30min . Data information : Data are representative of three biological replicates with similar results .

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G. Immunoblot analysis of ASC oligomerization in cross-linked cytosolic pellets (insoluble) and whole cell lysate (soluble) in WT or Trim50-/- BMDM cells by LPS stimulation for 6h and ATP treatment for 30min. Data information: Data are representative of three biological replicates with similar results.


H . SDD - of oligomerization of NLRP3 in HEK293T transfected with His - NLRP3 TRIM50 or TRIM50 ( ΔRING ) mutant . Data information : Data are representative of three biological replicates with similar results .

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H. SDD-AGE analysis of the oligomerization of NLRP3 in HEK293T cells transfected with His-NLRP3 and TRIM50 or TRIM50(ΔRING) mutant. Data information: Data are representative of three biological replicates with similar results.


I . In vitro NLRP3 oligomerization with in - vitro translated TRIM50 NLRP3 . Data information : Data are representative of three biological replicates with similar results .

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I. In vitro NLRP3 oligomerization assay with in-vitro translated TRIM50 and NLRP3 proteins. Data information: Data are representative of three biological replicates with similar results.


J . Immunoprecipitation of NLRP3 ubiquitination in HA - ubiquitin , His - NLRP3 Myc - TRIM50 transfected HEK293T with further treatment with tranilast or MCC950 for 24h . Data information : Data are representative of three biological replicates with similar results .

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J. Immunoprecipitation analysis of NLRP3 ubiquitination in HA-ubiquitin, His-NLRP3 and Myc-TRIM50 transfected HEK293T cells with further treatment with tranilast or MCC950 for 24h. Data information: Data are representative of three biological replicates with similar results.


A . Immunoprecipitation of NLRP3 ubiquitination in HEK293T transfected with NLRP3 , HA - Ubiquitin , TRIM50 or its truncation mutants . Data information : Band densities were quantitated by ' Image J ' software normalized to GAPDH . Data are representative of three biological replicates with similar results .

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A. Immunoprecipitation analysis of NLRP3 ubiquitination in HEK293T cells transfected with NLRP3, HA-Ubiquitin, TRIM50 or its truncation mutants. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.


B . SDD - of NLRP3 oligomerization in NLRP3 together with TRIM50 or its coiled - coil domain - deleted mutants ( ΔC - C1 ΔC - C2 ) transfected HEK293T . Data information : Band densities were quantitated by ' Image J ' software normalized to GAPDH . Data are representative of three biological replicates with similar results .

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B. SDD-AGE analysis of NLRP3 oligomerization in NLRP3 together with TRIM50 or its coiled-coil domain-deleted mutants (ΔC-C1 and ΔC-C2) transfected HEK293T cells. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.