Figure 3Representative 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 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 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 . .

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

Figure 3Representative 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 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 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..


Figure 4Representative 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 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 .

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

Figure 4Representative 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 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.


Figure 5Representative 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 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 .

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

Figure 5Representative 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 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.


Figure 6Representative 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 .

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

Figure 6Representative 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.


Figure 7mtDNA 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 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 .

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

Figure 7mtDNA 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 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.


Figure 1A , 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 .

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

Figure 1A, 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.


Figure 2A - 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 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 . ) )

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

Figure 2A-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 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.))


Figure 3A , 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 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 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 .

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

Figure 3A, 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 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 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.


Figure 5A , 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 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 ) . 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 the presence ( blue ) and absence ( red ) of the HCN channel blocker ZD7288 ( 50 μM ) . 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 .

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

Figure 5A, 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 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). 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 the presence (blue) and absence (red) of the HCN channel blocker ZD7288 (50 μM).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.


Figure 6A , 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 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 .

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

Figure 6A, 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 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.


Figure 7B , 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 .

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

Figure 7B, 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.


Figure 1B ) 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 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 .

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

Figure 1B) 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 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.


Figure 2A ) 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 ) 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 animals exhibiting various aortic arch abnormalities and great vessel defects ( n = 19 ) . K ) Aortic arch and vessels in a wild - type foetus . A corresponding drawing to explain the images is shown on the right . 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 ) .

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

Figure 2A) 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) 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 animals exhibiting various aortic arch abnormalities and great vessel defects (n = 19).K) Aortic arch and vessels in a wild-type foetus. A corresponding drawing to explain the images is shown on the right.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).


Figure 3A - 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 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 .

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

Figure 3A-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 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.


Figure 4A - 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 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 .

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

Figure 4A-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 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.


Figure 9 ( 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 , 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 ) .

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

Figure 9(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, 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).


Figure 1B 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 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 .

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

Figure 1B 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 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.


Figure 2B 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 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 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 .

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

Figure 2B 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 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 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.


Figure 3A 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 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 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 .

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

Figure 3A 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 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 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.


Figure 4A 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 ) 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 ) .

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

Figure 4A 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) 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).


Figure 5A 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 - 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 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 ) .

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

Figure 5A 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-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 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).


Figure 6A 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 .

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

Figure 6A 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.


Figure 1B ) Agarose gel electrophoresis of RNA extracted from RNase treated or control K562 cells . D ) Agarose gel electrophoresis of RNA extracted from control , 4hr and 24hr ActD treated K562 cells . 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 .

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

Figure 1B) Agarose gel electrophoresis of RNA extracted from RNase treated or control K562 cells.D) Agarose gel electrophoresis of RNA extracted from control, 4hr and 24hr ActD treated K562 cells.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.


Figure 2Hi - 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 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 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 the compartmental interactions for both cis - and trans - contacts across the conditions . The bXL RNase A samples display reduced B - B type interactions .

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

Figure 2Hi-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 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 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 the compartmental interactions for both cis- and trans-contacts across the conditions. The bXL RNase A samples display reduced B-B type interactions.


Figure 3Interaction 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 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 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 .

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

Figure 3Interaction 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 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 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.


Figure 4A ) 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 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 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 the compartmental interactions for both cis - and trans - contacts across the conditions . E ) Scaling plot generated by 500kb resolution Hi - C data showing the interaction frequency as a function of genomic distance .

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

Figure 4A) 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 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 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 the compartmental interactions for both cis- and trans-contacts across the conditions.E) Scaling plot generated by 500kb resolution Hi-C data showing the interaction frequency as a function of genomic distance.


Figure 5A ) 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 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 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 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 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 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 .

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

Figure 5A) 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 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 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 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 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 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.


Figure 1A . 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 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 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 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 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 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 ) 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 ) 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 .

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

Figure 1A. 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 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 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 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 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 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) 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) 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.


Figure 2A . 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 ± SDB . 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 ± SDC , 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 ± SDE , 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 ± SDF . 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 ± SDG , 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 ± SDH . 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 ± SDI . 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 ±

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

Figure 2A. 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 ± SDB. 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 ± SDC, 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 ± SDE, 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 ± SDF. 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 ± SDG, 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 ± SDH. 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 ± SDI. 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


Figure 3A . 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 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 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 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 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 ± SDF . 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 ± SDG . 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 ±

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

Figure 3A. 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 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 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 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 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 ± SDF. 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 ± SDG. 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


Figure 4A . 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 - 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 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 - 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 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 - 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 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 - 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 assay with in - vitro translated TRIM50 and NLRP3 proteins . Data information : Data are representative of three biological replicates with similar results . 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 .

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

Figure 4A. 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-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 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-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 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-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 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-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 assay with in-vitro translated TRIM50 and NLRP3 proteins. Data information: Data are representative of three biological replicates with similar results.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.


Figure 5A . 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 - 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 . C . SDD - AGE analysis of the oligomerization of TRIM50 in 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 . D . SDD - AGE analysis of the oligomerization of NLRP3 and TRIM50 in HEK293T cells overexpressing NLRP3 together with TRIM50 or its coiled - coil domain - deleted 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 . E . Immunoblot analysis of NLRP3 in HEK293T cells transfected with NLRP3 plasmid and TRIM50 coiled - coil domain - deleted mutants , followed with the cycloheximide ( CHX ) treatment 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 ( E ) . Data are representative of three biological replicates with similar results . ( mean ± SD in E ) . F . Immunoblot analysis of ASC oligomerization in cross - linked cytosolic pellets ( insoluble ) and whole cell lysate ( soluble ) in HEK293T cells transfected with His - NLRP3 and TRIM50 or TRIM50 coiled - coil domain - deleted 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 .

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

Figure 5A. 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-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.C. SDD-AGE analysis of the oligomerization of TRIM50 in 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.D. SDD-AGE analysis of the oligomerization of NLRP3 and TRIM50 in HEK293T cells overexpressing NLRP3 together with TRIM50 or its coiled-coil domain-deleted 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.E. Immunoblot analysis of NLRP3 in HEK293T cells transfected with NLRP3 plasmid and TRIM50 coiled-coil domain-deleted mutants, followed with the cycloheximide (CHX) treatment 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(E). Data are representative of three biological replicates with similar results. (mean ± SD in E).F. Immunoblot analysis of ASC oligomerization in cross-linked cytosolic pellets (insoluble) and whole cell lysate(soluble) in HEK293T cells transfected with His-NLRP3 and TRIM50 or TRIM50 coiled-coil domain-deleted 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.