( D ) HeLa were transfected with VAMP3 - HA for 20 hr stained for EEA1 or RAB11 . Histogram shows colocalization between VAMP3 - HA EEA1 or VAMP3 - HA RAB11 ( Manders ' coefficient ) . Error bar , .

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(D) HeLa cells were transfected with VAMP3-HA for 20 hr and stained for EEA1 or RAB11. Histogram shows colocalization between VAMP3-HA and EEA1 or VAMP3-HA and RAB11 (Manders' coefficient). Error bar, SEM.


( E ) HeLa were transfected for 20 hr with VAMP3 - HA , incubated for 1 hr at 37 ° C or 18 ° C , fixed , labeled for HA mATG9 . Histogram shows of colocalization between VAMP3 with mATG9 ( Manders ' coefficient ) . Error bar , . NS , not significant .

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(E) HeLa cells were transfected for 20 hr with VAMP3-HA, incubated for 1 hr at 37°C or 18°C, fixed, and labeled for HA and mATG9. Histogram shows the percentage of colocalization between VAMP3 with mATG9 (Manders' coefficient). Error bar, SEM. NS, not significant.


( A - C ) HeLa transfected with control , VAMP3 siRNA for 4 days were transfected during last 20 hr with ATG9L1 - pEGFP labeled with anti - EEA1 ( for early endosomes ) or anti - RAB11 ( for recycling endosomes ) . histogram in ( C ) shows of colocalization of mATG9 in EEA1 or RAB11 ( Manders ' coefficient ) . Error bars , . ∗ ∗ ∗ p < 0 . 001 .

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(A-C) HeLa cells transfected with control, VAMP3 siRNA for 4 days were transfected during the last 20 hr with ATG9L1-pEGFP and labeled with anti-EEA1 (for early endosomes) or anti-RAB11 (for recycling endosomes). The histogram in (C) shows the quantification of the colocalization of mATG9 in EEA1 or RAB11 (Manders' coefficient). Error bars, SEM. ∗∗∗p < 0.001.


( D - F ) HeLa were treated as in ( A ) - ( C ) transfected during last 20 hr with pEGFP - ATG16L1 labeled with anti - EEA1 ( early endosomes ) or anti - RAB11 ( recycling endosomes ) . Histogram in ( D ) shows of colocalization of ATG16L1 in EEA1 or RAB11 ( Manders ' coefficient ) . Error bars , . ∗ ∗ ∗ p < 0 . 001 ; NS , not significant .

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(D-F) HeLa cells were treated as in (A)-(C) and transfected during the last 20 hr with pEGFP-ATG16L1 and labeled with anti-EEA1 (early endosomes) or anti-RAB11 (recycling endosomes). Histogram in (D) shows quantification of the colocalization of ATG16L1 in EEA1 or RAB11 (Manders' coefficient). Error bars, SEM. ∗∗∗p < 0.001; NS, not significant.


( A ) HeLa transfected with control , VAMP3 siRNA for 4 days were transfected during last 20 hr with mStrawberry - ATG16L1 labeled for mATG9 . correlation between mATG9 ATG16L1 was quantified ( Pearson ' s coefficient ) . Error bar , . ∗ ∗ ∗ p < 0 . 001 .

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(A) HeLa cells transfected with control, VAMP3 siRNA for 4 days were transfected during the last 20 hr with mStrawberry-ATG16L1 and labeled for mATG9. The correlation between mATG9 and ATG16L1 was quantified (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.


( B ) HeLa were transfected with control or VAMP3 siRNA then with mStrawberry - ATG16 , 5 min movies were recorded . Homotypic fusion events between mStrawberry - ATG16 vesicles were assessed in control VAMP3 knockdown . Error bar , . NS , not significant .

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(B) HeLa cells were transfected with control or VAMP3 siRNA and then with mStrawberry-ATG16, and 5 min movies were recorded. Homotypic fusion events between mStrawberry-ATG16 vesicles were assessed in control and VAMP3 knockdown cells. Error bar, SEM. NS, not significant.


( C ) In vitro fusion of ATG16L1 mATG9 vesicles . HeLa were transfected with control or VAMP3 siRNA for 4 days separately , on fourth day , either transfected with mStrawberry - ATG16L1 or ATG9L1 - pEGFP . PNS of expressing two constructs were mixed for 1 hr at 37C ° observed by confocal microscopy . Histogram shows correlation of vesicles carrying ATG16L1 that fuse with vesicles carrying mATG9 ( Pearson ' s coefficient ) . Error bar , . ∗ ∗ ∗ p < 0 . 001 . total number of structures per field ( ATG16L1 ATG9L1 ) is not significantly different in control KD samples ( control , 37 ± 3 ; VAMP3 KD , 45 ± 3 ; ± ; p = 0 . 106 ) .

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(C) In vitro fusion assay of ATG16L1 and mATG9 vesicles. HeLa cells were transfected with control or VAMP3 siRNA for 4 days separately and, on the fourth day, either transfected with mStrawberry-ATG16L1 or ATG9L1-pEGFP. PNS of cells expressing the two constructs were mixed for 1 hr at 37C° and observed by confocal microscopy. Histogram shows the correlation of vesicles carrying ATG16L1 that fuse with vesicles carrying mATG9 (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001. The total number of structures per field (ATG16L1 and ATG9L1) is not significantly different in the control and KD samples (control, 37 ± 3; VAMP3 KD, 45 ± 3; ± SEM; p = 0.106).


( A ) THP - 1 were subjected to knockdowns as indicated treated with 100 ng / mL LPS overnight , followed by 0 . 25 mM LLOMe for 3 h , of IL - 1β were determined in supernatants .

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(A) THP-1 cells were subjected to knockdowns as indicated and treated with 100 ng/mL LPS overnight, followed by 0.25 mM LLOMe for 3 h, and the levels of IL-1β were determined in supernatants.


( B ) THP - 1 were subjected to GABARAP knockdown processed as in A .

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(B) THP-1 cells were subjected to GABARAP knockdown and processed as in A.


( C ) IL - 1β in supernatants of THP - 1 subjected to TRIM knockdowns treated with LPS then with LLOMe .

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(C) IL-1β levels in supernatants of THP-1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe.


( D ) Confocal microscopy of THP - 1 treated sequentially with LPS LLOMe , stained for LC3B TRIM16 . tracings correspond to arrows . Arrowheads indicate colocalization . Scale bars , 5 μm .

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(D) Confocal microscopy of THP-1 cells treated sequentially with LPS and LLOMe, and stained for LC3B and TRIM16. Line tracings correspond to arrows. Arrowheads indicate colocalization. Scale bars, 5 μm.


( E ) IL - 1β in supernatants of THP - 1 subjected to TRIM knockdowns treated with LPS then with LLOMe .

(E) IL-1β levels in supernatants of THP-1 cells subjected to TRIM knockdowns and treated with LPS and then with LLOMe.


( F ) of IL - 1β were determine in supernatants from THP - 1 subjected to knockdown as indicated , treated with 100 ng / mL LPS for overnight then with 0 . 25mM of Silica , Alum , or monosodium urate ( MSU ) .

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(F) Levels of IL-1β were determine in supernatants from THP-1 cells subjected to knockdown as indicated, treated with 100 ng/mL LPS for overnight and then with 0.25mM of Silica, Alum, or monosodium urate (MSU).


( G ) IL - 1β in supernatants of human macrophages ( MDM ) subjected to knockdowns sequentially treated with LPS LLOMe . Data , means ± ; n ≥ 5 ( except for B where a representative data set from 3 repeats ) . * P < 0 . 05 ( t or ANOVA ) .

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(G) IL-1β in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe. Data, means ± SEM; n≥5 (except for B where a representative data set from 3 repeats). *P < 0.05 (t test or ANOVA).


( A B ) of IL - 1β in supernatants from THP - 1 that were subjected to knockdown as indicated were treated with LPS , then ( A ) treated with LLOMe or ( B ) starved in EBSS .

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(A and B) Levels of IL-1β in supernatants from THP-1 cells that were subjected to knockdown as indicated and were treated with LPS, and then (A) treated with LLOMe or (B) starved in EBSS.


( C ) IL - 1β in supernatants of human macrophages ( MDM ) subjected to knockdowns sequentially treated with LPS LLOMe .

(C) IL-1β in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe.


( D ) Co - immunoprecipitation ( co - IP ) of flag - galectin - 8 ( Gal - 8 ) GFP - TRIM16 ( T16 ) in HEK293T .

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(D) Co-immunoprecipitation (co-IP) of flag-galectin-8 (Gal-8) and GFP-TRIM16 (T16) in HEK293T cells.


( E ) Co - IP of endogenous galectin - 8 TRIM16 in complexes from LLOMe - treated THP - 1 .

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(E) Co-IP analysis of endogenous galectin-8 and TRIM16 in protein complexes from LLOMe-treated THP-1 cells.


( F ) Confocal microscopy of THP - 1 treated with LPS LLOMe , stained for TRIM16 galectin - 8 . tracings correspond to arrowed dashed lines . Pearson correlation coefficient was calculated using SlideBook as described previously . Scale bars , 5 μm .

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(F) Confocal microscopy of THP-1 cells treated with LPS and LLOMe, and stained for TRIM16 and galectin-8. Line tracings correspond to arrowed dashed lines. Pearson correlation coefficient was calculated using SlideBook as described previously. Scale bars, 5 μm.


( G ) In vitro translated radiolabeled [ 35S ] myc - HA - TRIM16 was incubated with GST - galectin - 8 in presence ( + ) or absence ( - ) of flag - ULK1 cold ATP , GST pulldowns were performed , [ 35S ] radiolabeled Myc - HA - TRIM16 in pulled - down material detected by PAGE autoradiography . Amounts of GST fusion are shown in coommassie brilliant blue ( CBB ) - stained gels . Data , means  ; n ≥ 5 , except for immunoblot quantifications where n ≥ 3 . * P < 0 . 05 , † P ≥ 0 . 05 ( t or ANOVA ) .

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(G) In vitro translated and radiolabeled [35S] myc-HA-TRIM16 was incubated with GST-galectin-8 in the presence (+) or absence (-) of flag-ULK1 and cold ATP, GST pulldowns were performed, and [35S] radiolabeled Myc-HA-TRIM16 in pulled-down material detected by PAGE and autoradiography. Amounts of GST fusion proteins are shown in coommassie brilliant blue (CBB)-stained gels. Data, means  SEM; n≥5, except for immunoblot quantifications where n≥3. *P < 0.05, †P ≥ 0.05 (t test or ANOVA).


( A B ) Immunoblot analyses of indicated fractions prepared by differential centrifugation . HeLa reconstituted with flag - pro - IL - 1β myc - pro - Caspase - 1 were treated with 1mM LLOMe for 1h , lysates were subjected to differential centrifugations at 3 , 000xg ( 3k ) , 25 , 000xg ( 25k ) 100 , 000xg ( 100k ) as depicted in cartoon . Arrows , two ways membranes were analyzed - pellets from velocity sedimentation were analyzed in panels B - D , or by further sucrose OptiPrep density separation of 25k pellets in panels F G .

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(A and B) Immunoblot analyses of indicated membrane fractions prepared by differential centrifugation. HeLa cells reconstituted with flag-pro-IL-1β and myc-pro-Caspase-1 were treated with 1mM LLOMe for 1h, and lysates were subjected to differential centrifugations at 3,000xg (3k), 25,000xg (25k) and 100,000xg (100k) as depicted in the cartoon. Arrows, two ways the membranes were analyzed - pellets from velocity sedimentation were analyzed in panels B-D, or by further sucrose and OptiPrep density separation of 25k pellets in panels F and G.


( C ) IL - 1β in 25k fractions ( pellets ) from HeLa their CRISPR knockout derivatives ( TRIM16KO ) that were reconstituted for IL - 1β secretion treated with 1mM LLOMe for 1 h .

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(C) IL-1β levels in 25k membrane fractions (pellets) from HeLa cells and their CRISPR knockout derivatives (TRIM16KO) that were reconstituted for IL-1β secretion and treated with 1mM LLOMe for 1 h.


( D ) of analyses in ( B ) . RI , relative .

(D) Quantification of analyses in (B). RI, relative intensity.


( E ) Whole lysates ( immunoblots ) analyzed in F G .

(E) Whole cell lysates (immunoblots) analyzed in F and G.


( F , G ) Sucrose density gradient of 25k membranes . Note that mature IL - 1β ( mIL - 1β ) is recruited to co - fractionates with TRIM16 + , Sec22b + , LC3 - II + membranes in TRIM16wt HeLa reconstituted with pro - IL - 1β pro - caspase 1 , but not in TRIM16KO mutant .

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(F,G) Sucrose density gradient analysis of 25k membranes. Note that mature IL-1β (mIL-1β) is recruited to and co-fractionates with TRIM16+, Sec22b+, and LC3-II+ membranes in TRIM16wt HeLa cells reconstituted with pro-IL-1β and pro-caspase 1, but not in TRIM16KO mutant cells.


( H ) Confocal of LC3 in HeLa expressing GFP - Sec22b treated with LLOMe or untreated ( control ) . Arrowheads , colocalization . Scale bars , 5 μm . ( I ) GFP - Sec22b LC3 overlapping obtained by high content microscopy .

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(H) Confocal images of LC3 in HeLa cells expressing GFP-Sec22b treated with LLOMe or untreated (control). Arrowheads, colocalization. Scale bars, 5 μm. (I) GFP-Sec22b and LC3 overlapping area obtained by high content microscopy.


( J ) Co - IP between flag - TRIM16 with GFP - Sec22b or GFP - syntaxin - 17 co - expressed in HEK293T .

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(J) Co-IP analysis between flag-TRIM16 with GFP-Sec22b or GFP-syntaxin-17 co-expressed in HEK293T cells.


( K ) Co - IP of endogenous , TRIM16 Sec22b , in lysates from THP - 1 treated with LLOMe .

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(K) Co-IP analysis of endogenous proteins, TRIM16 and Sec22b, in lysates from THP-1 cells treated with LLOMe.


( L ) Co - IP of interactions between deletion variants of flag - TRIM16 with GFP - Sec22b in HEK293T .

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(L) Co-IP analysis of interactions between deletion variants of flag-TRIM16 with GFP-Sec22b in HEK293T cells.


( M N ) IL - 1β secretion complementation experiments of TRIM16KO with TRIM16 Sec22b - interacting its Sec22b - nonbinding mutant shown .

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(M and N) IL-1β secretion complementation experiments of TRIM16KO cells with TRIM16 Sec22b-interacting and its Sec22b-nonbinding mutant shown.


( O ) Two color super - resolution image showing co - clustering of flag - TRIM16 ( red ) GFP - Sec22b ( green ) ; Scale bar , 200 nm . ( P ) Cross - correlation of flag - TRIM16 GFP - Sec22b super - resolution data showing a characteristic separation of ~ 70 nm .

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(O) Two color super-resolution image showing co-clustering of flag-TRIM16 (red) and GFP-Sec22b (green); Scale bar, 200 nm. (P) Cross-correlation analysis of flag-TRIM16 and GFP-Sec22b super-resolution data showing a characteristic separation of ~ 70 nm.


( A B ) IL - 1β in supernatants of THP - 1 subjected to knockdowns , treated with LPS , then ( A ) with LLOMe or ( B ) starved in EBSS .

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(A and B) IL-1β levels in supernatants of THP-1 cells subjected to knockdowns, treated with LPS, and then (A) with LLOMe or (B) starved in EBSS.


( C ) IL - 1β in supernatants of human macrophages ( MDM ) subjected to knockdowns sequentially treated with LPS LLOMe .

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(C) IL-1β levels in supernatants of primary human macrophages (MDM) subjected to knockdowns and sequentially treated with LPS and LLOMe.


( D ) Immunoblot analyses of p62 in THP - 1 subjected to knockdowns as indicated starved for 3 h .

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(D) Immunoblot analyses of p62 levels in THP-1 cells subjected to knockdowns as indicated and starved for 3 h.


( E ) mRFP - GFP overlapping areas of tandem HeLa reconstituted with flag - pro - IL - 1β myc - Caspase 1 starved for 3 h . Mask overlay , iDev software - defined objects ( objects , outlines ; internal secondary objects , GFP ( top ) , mRFP ( middle ) , or their overlapping ( bottom ) ) . Bars , 10 μm .

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(E) mRFP-GFP overlapping areas of tandem HeLa cells reconstituted with flag-pro-IL-1β and myc-Caspase 1 and starved for 3 h. Mask overlay, iDev software-defined objects (primary objects, cell outlines; internal secondary objects, GFP (top), mRFP (middle), or their overlapping area (bottom)). Bars, 10 μm.


( F ) IL - 1β in supernatants of starved tandem HeLa reconstituted with flag - pro - IL - 1β myc - Caspase 1 as in E .

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(F) IL-1β levels in supernatants of starved tandem HeLa cells reconstituted with flag-pro-IL-1β and myc-Caspase 1 as in E.


( A - C ) IL - 1β in supernatants of THP - 1 subjected to knockdowns , treated with LPS , then with LLOMe ( A C ) or starved in EBSS ( B ) .

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(A-C) IL-1β levels in supernatants of THP-1 cells subjected to knockdowns, treated with LPS, and then with LLOMe (A and C) or starved in EBSS (B).


( A - C ) IL - 1β in supernatants of THP - 1 subjected to knockdowns , treated with LPS , then with LLOMe ( A C ) .

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(A-C) IL-1β levels in supernatants of THP-1 cells subjected to knockdowns, treated with LPS, and then with LLOMe (A and C).


( C ) Ferritin in supernatants of THP - 1 subjected to knockdowns as indicated sequentially treated with LPS LLOMe .

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(C) Ferritin levels in supernatants of THP-1 cells subjected to knockdowns as indicated and sequentially treated with LPS and LLOMe.


( D ) Ferritin in supernatants of THP - 1 subjected to knockdowns as indicated infected with M . tuberculosis as indicated .

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(D) Ferritin levels in supernatants of THP-1 cells subjected to knockdowns as indicated and infected with M. tuberculosis as indicated.


( E ) of ferritin were determine in supernatants from THP - 1 that were subjected to knockdown as indicated were sequentially treated with LPS LLOMe .

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(E) Levels of ferritin were determine in supernatants from THP-1 cells that were subjected to knockdown as indicated and were sequentially treated with LPS and LLOMe.


( F ) Confocal microscopy of HeLa expressing GFP - Sec22b were treated with LLOMe , stained for FTH1 . tracings correspond to arrows . Scale bars , 5 μm ;

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(F) Confocal microscopy of HeLa cells expressing GFP-Sec22b were treated with LLOMe, and stained for FTH1. Line tracings correspond to arrows. Scale bars, 5 μm;


( F ) Malate dehydrogenase ( MDH ; 0 . 2 µM ) was incubated at 40 ° C in absence ( black ) or presence of 0 . 5 µM WT HSP27 ( red ) or 0 . 5 µM P182L HSP27 ( blue ) . ( G ) Insulin ( 40 µM ) was incubated at 40 ° C in absence ( black ) or presence of 20 µM WT HSP27 ( red ) or 20 µM P182L HSP27 ( blue ) . y - axes depict absorbance at 340 nm due to formation of large . solid lines represent average of three replicates with filled reflecting ± one standard deviation .

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(F) Malate dehydrogenase (MDH; 0.2 µM) was incubated at 40 °C in the absence (black) or presence of 0.5 µM WT HSP27 (red) or 0.5 µM P182L HSP27 (blue). (G) Insulin (40 µM) was incubated at 40 °C in the absence (black) or presence of 20 µM WT HSP27 (red) or 20 µM P182L HSP27 (blue). The y-axes depict the absorbance at 340 nm due to the formation of large aggregates. The solid lines represent the average of three replicates with the filled area reflecting ± one standard deviation.


( A ) SEC - MALS data from WT HSP27 P182L variant , with cHSP27 included for comparison . samples were injected at 40 µM ( monomer ) . respective average molecular masses are 14 MDa , 470 kDa , 18 kDa for P182L , WT HSP27 , cHSP27 . normalized UV signals are plotted on left y - axis as solid lines molecular masses are plotted on right y - axis as circles .

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(A) SEC-MALS data from WT HSP27 and the P182L variant, with cHSP27 included for comparison. All samples were injected at 40 µM concentration (monomer). The respective average molecular masses are 14 MDa, 470 kDa, and 18 kDa for P182L, WT HSP27, and cHSP27. The normalized UV signals are plotted on the left y-axis as solid lines and the molecular masses are plotted on the right y-axis as circles.


( B ) Fractionation of HeLa overexpressing either HSP27 or P182L mutant by a 10 % to 80 % sucrose gradient . Western blot against anti - V5 is shown .

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(B) Fractionation of HeLa cells overexpressing either HSP27 or the P182L mutant by a 10% to 80% sucrose gradient. Western blot against anti-V5 is shown.


( C ) Expression of V5 - epitope - tagged HSP27 ( WT or P182L mutant ) in HSP27 knock - out HeLa immunostaining of HSP27 . scale bars correspond to non - expanded dimensions , derived by dividing by sample expansion factor of 4 . 3 . ( D ) P182L mutation decreases density of HSP27 spots upon detection of local maxima of number of detected spots in expanded samples ( n = 30 ) . error bar is standard deviation for different regions . ( E ) Distribution of of spots ( n = 3342 ) after Gaussian fitting of in neighborhood of detected spots .

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(C) Expression of V5-epitope-tagged HSP27 (WT or P182L mutant) in HSP27 knock-out HeLa cells and immunostaining of HSP27. The scale bars correspond to the non-expanded dimensions, derived by dividing by the sample expansion factor of 4.3. (D) The P182L mutation decreases the density of HSP27 spots upon detection of local fluorescence intensity maxima and quantification of the number of detected spots in expanded samples (n = 30). The error bar is the standard deviation for the different cytoplasmic regions. (E) Distribution of the intensity of individual spots (n = 3342) after Gaussian fitting of fluorescence intensity in the neighborhood of detected spots.


( F ) Immunostaining of HSP27 ( ) in motor neurons differentiated from CMT - derived iPSCs that harbor heterozygous P182L mutation reveals large , ( center right panels ) . control motor neurons that contain WT HSP27 do not show evidence of any . nucleus is stained with Hoechst ( blue ) . Scale bar , 10 µm .

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(F) Immunostaining of HSP27 (white) in motor neurons differentiated from CMT patient-derived iPSCs that harbor the heterozygous P182L mutation reveals large, cytoplasmic aggregates (center and right panels). The control motor neurons that contain WT HSP27 do not show evidence of any aggregates. The nucleus is stained with Hoechst (blue). Scale bar, 10 µm.


( A ) sequences of WT peptide ( red ) P182L peptide ( blue ) . IxI / V motif is indicated with text . Below : regions of 2D 1H - 15N HSQC spectra of 15N - labeled cHSP27 ( black ) in presence of increasing amounts of WT ( red ) or P182L peptide ( blue ) . Resonances in β4 β8 strands broaden disappear upon peptide binding , indicative of intermediate exchange . ( B ) Zoomed - in regions of resonances during peptide titrations . color bars indicate amount of added WT or P182L peptide . ( C ) combined 1HN 15N chemical shift changes , denoted as chemical shift perturbation ( CSP ) , are plotted as a function of added peptide . ( D ) CSPs shown as a function of residue number for WT ( red ) P182L peptide ( blue ) . ( E ) X - ray structure of cHSP27 bound to WT peptide ( PDB : 4mjh ) , shown in sticks , with results from ( D ) plotted onto structure .

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(A) Primary sequences of the WT peptide (red) and P182L peptide (blue). The IxI/V motif is indicated with white text. Below: regions of 2D 1H-15N HSQC spectra of 15N-labeled cHSP27 (black) in the presence of increasing amounts of WT (red) or P182L peptide (blue). Resonances in the β4 and β8 strands broaden and disappear upon peptide binding, indicative of intermediate exchange. (B) Zoomed-in regions of resonances during the peptide titrations. The color bars indicate the amount of added WT or P182L peptide. (C) The combined 1HN and 15N chemical shift changes, denoted as chemical shift perturbation (CSP), are plotted as a function of added peptide concentration. (D) CSPs shown as a function of residue number for the WT (red) and P182L peptide (blue). (E) X-ray structure of cHSP27 bound to the WT peptide (PDB: 4mjh), shown in white sticks, with the results from (D) plotted onto the structure.


( A , B ) i . 15N CPMG RD data for residues in β4 strand ( T113 ) β8 strand ( S154 ) in absence of added peptide . CPMG data are shown for same residues in presence of ii . ca . 2 % cHSP27 - WT peptide or iii . ca . 2 % of cHSP27 - P182L peptide . Uncertainties in R2 , eff values are derived from standard deviations of fitted peak intensities obtained from duplicated νCPMG values . ( C ) i . association ( kon ) dissociation ( koff ) rates for WT ( red ) P182L ( blue ) IxI / V peptide binding to ACD , calculated directly from CPMG RD data . Residues that are impacted by presence of a small amount of peptide are indicated by red ( ii . , WT ) blue ( iii . , P182L ) spheres . In both cases , residues in similar ACD regions show enhanced Rex , indicative of chemical exchange at similar sites .

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(A, B) i. 15N CPMG RD data for residues in the β4 strand (T113) and the β8 strand (S154) in the absence of added peptide. CPMG data are shown for the same residues in the presence of ii. ca. 2% cHSP27-WT peptide complex or iii. ca. 2% of the cHSP27-P182L peptide complex. Uncertainties in R2,eff values are derived from the standard deviations of fitted peak intensities obtained from duplicated νCPMG values. (C) i. The association (kon) and dissociation (koff) rates for WT (red) and P182L (blue) IxI/V peptide binding to the ACD, calculated directly from the CPMG RD data. Residues that are impacted by the presence of a small amount of peptide are indicated by red (ii., WT) and blue (iii., P182L) spheres. In both cases, residues in similar ACD regions show enhanced Rex, indicative of chemical exchange at similar sites.


( B ) Volcano plot representing interactors that are enriched for P182L mutant versus GFP as a negative control , obtained with affinity - enrichment mass spectrometry . that co - immunoprecipitated significantly more ( p < 0 . 05 ) with P182L variant are shown in upper right quadrant . Interactors with one or more [ I / V ] - x - [ I / V ] motifs are displayed in red while other significantly enriched interactors are displayed in black .

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(B) Volcano plot representing the interactors that are enriched for the P182L mutant versus GFP as a negative control, obtained with affinity-enrichment mass spectrometry. Proteins that co-immunoprecipitated significantly more (p<0.05) with the P182L variant are shown in the upper right quadrant. Interactors with one or more [I/V]-x-[I/V] motifs are displayed in red while other significantly enriched interactors are displayed in black.


( C - D ) Co - immunoprecipitation from HeLa stably overexpressing V5 - epitope - tagged HSP27 ( WT or P182L mutant ) using anti - V5 beads . Co - immunoprecipitation was quantified presented as a relative to P182L variant ( n = 3 ) . mean ± one standard deviation ( n = 3 ) is shown . Right : location of Pro182 ( red ) in crystal structure of peptide - bound cHSP27 ( PDB : 4mjh ) in silico creation of P182L mutation ( blue ) . peptide is shown in sticks . P182L specifically weakens binding of HSP27 IxI / V motif .

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(C-D) Co-immunoprecipitation from HeLa cells stably overexpressing V5-epitope-tagged HSP27 (WT or P182L mutant) using anti-V5 beads. Co-immunoprecipitation was quantified and presented as a percentage relative to the P182L variant (n=3). The mean ± one standard deviation (n=3) is shown. Right: the location of Pro182 (red) in the crystal structure of peptide-bound cHSP27 (PDB: 4mjh) and in silico creation of the P182L mutation (blue). The peptide is shown in white sticks. P182L specifically weakens binding of the HSP27 IxI/V motif.


( E ) Co - immunoprecipitation from HeLa stably overexpressing V5 - epitope - tagged HSP27 ( WT or P182L mutant ) transiently transfected with BAG3 - eGFP ( WT or IPV - mutant ) using anti - GFP beads . Western blots for anti - GFP ( BAG3 ) , V5 ( HSPB1 ) , α - tubulin . NS indicates a non - specific band . Note that detection of BAG3 ( anti - GFP ) HSP27 ( anti - V5 ) occurs with different antibodies . locations of IPV - to - GPG mutations in BAG3 are depicted below .

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(E) Co-immunoprecipitation from HeLa cells stably overexpressing V5-epitope-tagged HSP27 (WT or P182L mutant) and transiently transfected with BAG3-eGFP (WT or IPV-mutant) using anti-GFP beads. Western blots for anti-GFP (BAG3), V5 (HSPB1), and α-tubulin. NS indicates a non-specific band. Note that detection of BAG3 (anti-GFP) and HSP27 (anti-V5) occurs with different antibodies. The locations of the IPV-to-GPG mutations in BAG3 are depicted below.


( F ) Co - immunoprecipitation from HeLa transiently transfected with V5 - epitope - tagged HSP27 ( P182L or S155Q / P182L mutant ) using anti - V5 beads ( n = 2 ) . Right : location of Ser155 ( red ) in crystal structure of peptide - bound cHSP27 ( PDB : 4mjh ) in silico creation of S155Q mutation ( purple ) showing a clash with bound peptide . peptide is shown in sticks . S155Q disrupts binding of IxI / V motifs .

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(F) Co-immunoprecipitation from HeLa cells transiently transfected with V5-epitope-tagged HSP27 (P182L or S155Q/P182L mutant) using anti-V5 beads (n=2). Right: the location of Ser155 (red) in the crystal structure of peptide-bound cHSP27 (PDB: 4mjh) and in silico creation of the S155Q mutation (purple) showing a clash with the bound peptide. The peptide is shown in white sticks.S155Q disrupts binding of all IxI/V motifs.


B ) Western blot assays for expression level of TFAM in ten HCC lines , one normal liver ( LO2 ) two non - liver tissues . level was quantified as ratios between TFAM GAPDH ( n = 3 independent experiments ) . Data information : Graphs show mean ± s . e . m . , two - unpaired t - . * * P < 0 . 01 .

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B) Western blot assays for the expression level of TFAM in ten HCC cell lines, one normal liver cell line (LO2) and two non-tumor liver tissues. The protein level was quantified as ratios between TFAM and GAPDH (n = 3 independent experiments). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.


( C ) Western blot quantitative for expression of TFAM in HCC with TFAM stable knockdown control ( n = 3 independent experiments ) . shCtrl , control shRNA ; shTFAM , shRNA against TFAM ; EV , empty vector ; TFAM , expression vector encoding TFAM . Data information : Graphs show mean ± s . e . m . , two - unpaired t - . * * P < 0 . 01 .

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(C) Western blot and quantitative analysis for the expression of TFAM in HCC cells with TFAM stable knockdown and control cells (n = 3 independent experiments). shCtrl, control shRNA; shTFAM, shRNA against TFAM; EV, empty vector; TFAM, expression vector encoding TFAM. Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.


( D , E ) Wound - healing migration ( D ) Transwell migration invasion assays ( E ) for HCC with treatment as indicated ( n = 3 independent experiments ) . Scale bars , 100μm . Data information : Graphs show mean ± s . e . m . , two - unpaired t - . * * P < 0 . 01 .

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(D, E) Wound-healing migration assay (D) and Transwell migration and invasion assays (E) for HCC cells with treatment as indicated (n = 3 independent experiments). Scale bars, 100μm. Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.


( G ) Representative H & E of lung metastasis in mice used in ( F ) . Scale bars , 100μm . ( H , Number of metastatic nodules per lung ( H ) in mice used in ( F ) . Data information : Graphs show mean ± s . e . m . , two - unpaired t - . * * P < 0 . 01 .

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(G) Representative H&E images of lung metastasis in mice used in (F). Scale bars, 100μm. (H, Number of metastatic nodules per lung (H) in mice used in (F). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.


( C ) Immunofluorescence of TFAM Phalloidin in human HCC from paired clinical samples ( n = 100 ) . 3D reconstruction of z - stacks from region is showed in right side . Red : LifeAct , Blue : DAPI , Green : TFAM . Scale bars , 20μm for microscopy , 40μm for 3D reconstruction .

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(C) Immunofluorescence analysis of TFAM and Phalloidin staining in human HCC cells from paired clinical samples (n=100). The 3D reconstruction of z-stacks from nuclear region is showed in the right side. Red: LifeAct, Blue: DAPI, Green: TFAM. Scale bars, 20μm for microscopy images, 40μm for 3D reconstruction.


( D ) Statistical for of F - actin in ( A ) , ( B ) ( C ) . are quantified from at least 30 for each group in ( A ) ( B ) . One - way ANOVA . * P < 0 . 05 ; * * P < 0 . 01 .

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(D) Statistical analysis for fluorescent intensity of nuclear F-actin in (A), (B) and (C). Fluorescence intensity are quantified from at least 30 cells for each group in (A) and (B). One-way ANOVA. *P < 0.05; **P < 0.01.


( E ) Representative H & E of lung metastasis in mice used in ( D ) . Scale bars , 100μm . ( F G ) Number of metastatic nodules per lung in mice used in ( F ) incidence of lung metastasis ( G ) in mice used in ( D ) . Data information : Graphs show mean ± s . e . m . , two - unpaired t - . * * P < 0 . 01 .

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(E) Representative H&E images of lung metastasis in mice used in (D). Scale bars, 100μm. (F and G) Number of metastatic nodules per lung in mice used in (F) and the incidence of lung metastasis (G) in mice used in (D). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. **P < 0.01.


F ) Western blot assays for expression of metastasis related gene in HCC ( n = 3 independent experiments ) tissues from mice ( n = 4 per group ) with treatments as indicated . Data information : Graphs show mean ± s . e . m . , two - unpaired t - . * P < 0 . 05 ; * * P < 0 . 01 .

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F) Western blot assays for protein expression levels of metastasis related gene in HCC cells (n = 3 independent experiments) and tumor tissues from mice (n =4 per group) with treatments as indicated. Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. *P < 0.05; **P < 0.01.


( H Western blot assays of FN1 , ITGB3 in HCC with treatment as indicated ( n = 3 independent experiments ) . Data information : Graphs show mean ± s . e . m . , two - unpaired t - . * P < 0 . 05 ; * * P < 0 . 01 .

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(H Western blot assays levels of FN1, ITGB3 in HCC cells with treatment as indicated (n = 3 independent experiments). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test. *P < 0.05; **P < 0.01.


( A ) Western blot analyses for level of key regulators of actin polymerization in cytoplasm nucleus of HCC with treatment as indicated . Tublin Lamin B1 were used as loading controls for cytoplasm nucleus , respectively ( n = 3 independent experiments ) .

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(A) Western blot analyses for the protein level of key regulators of actin polymerization in the cytoplasm and nucleus of HCC cells with treatment as indicated. Tublin and Lamin B1 were used as loading controls for the cytoplasm and nucleus, respectively (n = 3 independent experiments).


( B ) Western blot analyses for level in tissues of mouse liver induced by Akt / β - catenin ( n = 3 independent experiments ) .

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(B) Western blot analyses for the protein level in tissues of mouse liver tumor induced by Akt/β-catenin (n = 3 independent experiments).


Representative of mDia2 translocation in human ( E ) HCC tissues . NT , non - liver tissues , n = 100 ; MT , metastatic tissues , n = 58 ; NMT , non - metastatic tissues n = 42 . Scale bars , 25μm . ( F ) Positive rates of mDia2 in nucleus cytoplasm was calculated on in ( E ) . Graphs show mean ± s . e . m . One - way ANOVA . * * P < 0 . 01 .

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Representative IHC images of mDia2 nuclear translocation in human (E) HCC tissues. NT, non-tumor liver tissues, n=100; MT, metastatic tumor tissues, n=58; NMT, non-metastatic tumor tissues n=42. Scale bars, 25μm. (F) Positive rates of mDia2 in nucleus and cytoplasm was calculated based on the IHC assay in (E). Graphs show mean ± s.e.m. One-way ANOVA. **P < 0.01.


( H I ) Spearman correlation between positive rates of mDia2 TFAM expression level ( or positive rates of F - actin ) on in ( E ) .

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(H and I) Spearman correlation analysis between the positive rates of nuclear mDia2 and the TFAM expression level (or positive rates of nuclear F-actin) based on the IHC assay in (E).


( D ) Western blot of anti - mDia2 IPs whole - lysates ( WCL ) derived from HCC mouse tissues ( n = 3 independent experiments ) . NT , non - liver tissues ; MT , metastatic tissues ; NMT , non - metastatic tissues .

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(D) Western blot analysis of anti-mDia2 IPs and whole-cell lysates (WCL) derived from HCC cells and mouse tumor tissues (n = 3 independent experiments). NT, non-tumor liver tissues; MT, metastatic tumor tissues; NMT, non-metastatic tumor tissues.


( E ) level of malonylated mDia2 . Equal amount of immunoprecipitated mDia2 either from TFAM knockdown or control was loaded in Western blot .

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(E) The level of malonylated mDia2. Equal amount of immunoprecipitated mDia2 either from TFAM knockdown or control cells was loaded in Western blot assay.


( F ) Western blot for distribution of mDia2 in SNU - 368 incubated with malonyl - CoA ( 5 μM ) or ND - 630 ( 10 μM ) ( n = 3 independent experiments ) .

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(F) Western blot analysis for the distribution of mDia2 in SNU-368 cells incubated with malonyl-CoA (5 μM) or ND-630 (10 μM) (n = 3 independent experiments).


( H ) Representative H & E of lung metastasis in mice used in ( G ) . Scale bars , 100μm . ( I ) Number of metastatic nodules per lung in mice used in ( G ) . Data information : Graphs show mean ± s . e . m . , two - unpaired t - for , I ) . * P < 0 . 05 ; * * P < 0 . 01 .

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(H) Representative H&E images of lung metastasis in mice used in (G). Scale bars, 100μm. (I) Number of metastatic nodules per lung in mice used in (G). Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test for , I). *P < 0.05; **P < 0.01.


( A ) scores of TFAM in 429 paired tissues from HCC . relative expression of to non - was log2 - transformed . T , tissues ; NT , non - liver tissues .

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(A) IHC scores of TFAM in 429 paired tissues from HCC patients. The relative expression ratio of tumor to non-tumor was log2-transformed. T, tumor tissues; NT, non-tumor liver tissues.


( B ) mRNA level of TFAM in 50 HCC ' paired tissues from TCGA database . relative expression of to non - was log2 - transformed .

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(B) mRNA level of TFAM in 50 HCC patients' paired tissues from TCGA database. The relative expression ratio of tumor to non-tumor was log2-transformed.


( C ) Representative of of TFAM ( top panel ) scores ( bottom panel ) in HCC tissues . One - way ANOVA . * P < 0 . 05 ; * * P < 0 . 01 . MT , metastatic tissues ; NMT , non - metastatic tissues . Scale bars , 25μm . Boxes : first quartile to third quartile ; Whiskers : minimum value to maximum value ; Central band : median .

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(C) Representative images of IHC staining of TFAM (top panel) and scores (bottom panel) in HCC tumor tissues. One-way ANOVA. *P < 0.05; **P < 0.01. MT, metastatic tumor tissues; NMT, non-metastatic tumor tissues. Scale bars, 25μm. Boxes: first quartile to third quartile; Whiskers: minimum value to maximum value; Central band: median.


( D Kaplan - Meier curves for overall survival of HCC stratified by TFAM expression . ( F Kaplan - Meier curves for overall survival of HCC stratified by F - actin positive rates .

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(D Kaplan-Meier curves for overall survival of HCC patients stratified by TFAM expression. (F Kaplan-Meier curves for overall survival of HCC patients stratified by nuclear F-actin positive rates.


E ) recurrence of HCC stratified by TFAM expression . G ) recurrence of HCC stratified by F - actin positive rates .

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E) the recurrence of HCC patients stratified by TFAM expression. G) the recurrence of HCC patients stratified by nuclear F-actin positive rates.


( H ) Representative of of TFAM , FN1 ITGB3 from two HCC . Scale bars , 25μm . ( I ) Spearman correlation between of TFAM FN1 , IL - 6 , TGFB1 or ITGB3 .

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(H) Representative images of IHC staining of TFAM, FN1 and ITGB3 from two HCC patients. Scale bars, 25μm. (I) Spearman correlation analysis between the levels of TFAM and FN1, IL-6, TGFB1 or ITGB3.


b , Representative confocal of NeuN ( green ) Npas4 ( red ) immunostaining performed 1h after last injection of cocaine or saline ( scale bar : 50 µm ) .

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b, Representative confocal images of NeuN (green) and Npas4 (red) immunostaining performed 1h after the last injection of cocaine or saline (scale bar: 50 µm).


c , Quantifications of Npas4 - positive ( fold change normalized to 30min saline control group ) in NAc dStr . Mean + ; n = 4 mice per group . 10 ( 5 per hemisphere ) per mouse for NAc 10 ( 5 per hemisphere ) for dStr . p * * < 0 . 01 , unpaired Student ' s t - .

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c, Quantifications of Npas4-positive cells (fold change normalized to the 30min saline control group) in the NAc and dStr. Mean + SEM; n = 4 mice per group. 10 images (5 per hemisphere) per mouse for the NAc and 10 images (5 per hemisphere) for the dStr. p** < 0.01, unpaired Student's t-test.


c , MSN cultures at DIV11 were treated for 2h with either 10 µM of D1 - agonist SKF - 38393 ( SKF ) alone or with SKF 10 µM of selective D1 - antagonist SCH - 23390 ( SCH ) . qPCR was performed to determine relative mRNA of immediate early genes cFos Arc . Mean + ; one - way ANOVA with Holm - Sidak post - hoc correction ; p * * < 0 . 01 , p * * * * < 0 . 0001 ; n = 3 independent cultures as biological replicates .

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c, MSN cultures at DIV11 were treated for 2h with either 10 µM of the D1-agonist SKF-38393 (SKF) alone or with SKF and 10 µM of the selective D1-antagonist SCH-23390 (SCH). qPCR was performed to determine the relative mRNA levels of the immediate early genes cFos and Arc. Mean + SEM; one-way ANOVA with Holm-Sidak post-hoc correction; p** < 0.01, p**** < 0.0001; n = 3 independent cultures as biological replicates.


d , MSNs were depolarized with potassium chloride ( HiK , 55 mM ) or treated with D1 - agonist SKF - 38393 ( 10 µM ) for 1h , 2h or 4h . Subsequently , qPCR was performed to assess immediate early gene mRNA . Mean + ; two - way ANOVA with Bonferroni post - hoc ; indicated p - values for induction over control for each treatment ; p * < 0 . 05 , p * * < 0 . 01 , p * * * < 0 . 001 ; n = 4 independent cultures as biological replicates .

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d, MSNs were depolarized with potassium chloride (HiK, 55 mM) or treated with the D1-agonist SKF-38393 (10 µM) for 1h, 2h or 4h. Subsequently, qPCR was performed to assess immediate early gene mRNA levels. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test; indicated p-values for induction over control for each treatment; p* < 0.05, p** < 0.01, p***<0.001; n = 4 independent cultures as biological replicates.


g , Differential induction of Npas4 through HiK not forskolin in hiPSC - derived forebrain organoids . Organoids were treated with HiK ( 55 mM ) or PKA activator forskolin plus TTX ( Fsk , 10 µM ; TTX 1 µM ) for 1h before qPCR was performed to assess mRNA of Npas4 Arc . Mean + ; n = 7 organoids in control condition , n = 5 organoids for HiK , n = 5 organoids for Fsk / TTX . One - way ANOVA with Tukey ' s post - hoc , ns = not significant , p * < 0 . 05 , p * * * < 0 . 001 .

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g, Differential induction of Npas4 through HiK and not forskolin in hiPSC-derived forebrain organoids. Organoids were treated with HiK (55 mM) or the PKA activator forskolin plus TTX (Fsk, 10 µM; TTX 1 µM) for 1h before qPCR analysis was performed to assess mRNA levels of Npas4 and Arc. Mean + SEM; n = 7 organoids in control condition, n = 5 organoids for HiK, n = 5 organoids for Fsk/TTX. One-way ANOVA with Tukey's post-hoc test, ns = not significant, p* < 0.05, p***<0.001.


MSN cultures were stimulated with HiK ( 55 mM ) for 1h in presence of various pharmacologicals to dissect synapse - to - nucleus cascades mediating transcription of Npas4 , Fosb Arc . Values were normalized to induced condition to compare for differences in induction between treatments . Mean + one - way ANOVA with Bonferroni post - hoc correction were used in experiments . p * < 0 . 05 , p * * < 0 . 01 , p * * * < 0 . 001 , p * * * * < 0 . 0001 . n - number indicated in each panel . a , co - incubation with NMDA receptor blocker APV ( 100 mM , applied 5min before ) reveals that IEG inductions through HiK in MSNs depend on NDMA receptor activity . Mean + ; n = 4 independent cultures as biological replicates . b , blockade of voltage - dependent calcium channels through verapamil ( 30 µM , 5min before ) TTX ( 1 µM , 5min , to prevent verapamil - induced bursting ) leads to significant reduction in gene - inductions for Npas4 Fosb . Mean + ; n = 4 independent cultures as biological replicates . c , inhibition of ERK1 / 2 through UO126 ( 10 µM , 20min before ) leads to significant blockade of mRNA induction for Arc Fosb , but not Npas4 . Mean + ; n = 3 independent cultures as biological replicates . d , inhibition of p38alpha p38beta through SB203580 or SB202190 respectively ( both 10 µM 20min before ) leads to significant reductions in mRNA induction for Arc Fosb , but not Npas4 . Mean + ; n = 3 independent cultures as biological replicates . e , blockade of PKA activity through H - 89 ( 10 µM , 20min before ) leads to reduced induction of Arc , unaffected induction of Fosb a superinduction of Npas4 . Mean + ; n = 3 independent cultures as biological replicates . f , inhibition of calcineurin through co - application of Cyclosporin FK506 ( 1 µM each , 20min before ) leads to unaffected induction of mRNAs for Arc Fosb , but significantly reduced of Npas4 . Mean + ; n = 3 independent cultures as biological replicates .

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MSN cultures were stimulated with HiK (55 mM) for 1h in the presence of various pharmacologicals to dissect the synapse-to-nucleus cascades mediating transcription of Npas4, Fosb and Arc. Values were normalized to the induced condition to compare for differences in induction between treatments. Mean + SEM and one-way ANOVA with Bonferroni post-hoc correction were used in all experiments. p* < 0.05, p** < 0.01, p***<0.001, p****<0.0001. n-number indicated in each panel. a, co-incubation with the NMDA receptor blocker APV (100 mM, applied 5min before) reveals that all IEG inductions through HiK in MSNs depend on NDMA receptor activity. Mean + SEM; n = 4 independent cultures as biological replicates. b, blockade of voltage-dependent calcium channels through verapamil (30 µM, 5min before) and TTX (1 µM, 5min, to prevent verapamil-induced bursting) leads to significant reduction in gene-inductions for Npas4 and Fosb. Mean + SEM; n =4 independent cultures as biological replicates. c, inhibition of ERK1/2 through UO126 (10 µM, 20min before) leads to significant blockade of mRNA induction for Arc and Fosb, but not Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. d, inhibition of p38alpha and p38beta through SB203580 or SB202190 respectively (both 10 µM and 20min before) leads to significant reductions in mRNA induction for Arc and Fosb, but not Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. e, blockade of PKA activity through H-89 (10 µM, 20min before) leads to reduced induction of Arc, unaffected induction of Fosb and a superinduction of Npas4. Mean + SEM; n = 3 independent cultures as biological replicates. f, inhibition of calcineurin through co-application of Cyclosporin and FK506 (1 µM each, 20min before) leads to unaffected induction of mRNAs for Arc and Fosb, but significantly reduced levels of Npas4. Mean + SEM; n = 3 independent cultures as biological replicates.


MSN cultures were stimulated with HiK ( 55 mM ) for 1h in presence of various pharmacologicals to dissect synapse - to - nucleus cascades mediating transcription of Npas4 , Fosb Arc . Values were normalized to induced condition to compare for differences in induction between treatments . Mean + one - way ANOVA with Bonferroni post - hoc correction were used in experiments . p * < 0 . 05 , p * * < 0 . 01 , p * * * < 0 . 001 , p * * * * < 0 . 0001 . n - number indicated in each panel . g , blockade of CaMKII / IV through application of KN - 93 ( 1 µM , 20min before ) leads to significantly reduced mRNA inductions of Arc , Fosb Npas4 . Mean + ; n = 4 independent cultures as biological replicates . h , co - application of HiK SKF reveals synergistic effects on mRNA induction of Fosb but not Arc or Npas4 , Mean + ; n = 6 independent cultures as biological replicates .

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MSN cultures were stimulated with HiK (55 mM) for 1h in the presence of various pharmacologicals to dissect the synapse-to-nucleus cascades mediating transcription of Npas4, Fosb and Arc. Values were normalized to the induced condition to compare for differences in induction between treatments. Mean + SEM and one-way ANOVA with Bonferroni post-hoc correction were used in all experiments. p* < 0.05, p** < 0.01, p***<0.001, p****<0.0001. n-number indicated in each panel. g, blockade of CaMKII/IV through application of KN-93 (1 µM, 20min before) leads to significantly reduced mRNA inductions of Arc, Fosb and Npas4. Mean + SEM; n = 4 independent cultures as biological replicates. h, co-application of HiK and SKF reveals synergistic effects on mRNA induction of Fosb but not Arc or Npas4, Mean + SEM; n = 6 independent cultures as biological replicates.


investigating role of calcium signaling in mRNA induction of Arc , Fosb Npas4 . k , immunohistochemical of MSNs infected with mCherry - NLS as control or competitive Ca2 + / CaM antagonist CaMBP4 - mCherry . Infection on DIV4 , fixation on DIV11 . D32 = anti - Darpp - 32 . scale bar = 10 µm . l , MSNs expressing rAAV - mCherry - control or CaMBP4 ( BP4 ) were treated with 55mM HiK for 1h qPCR was performed for indicated IEGs . Mean + ; two - way ANOVA with Bonferroni post - hoc , ns = not significant , p * * * * < 0 . 0001 . n = 5 independent cultures as biological replicates . m , immunohistochemical of MSNs infected with mCherry - NLS as control or Ca2 + - buffer Parvalbumin - NLS - mCherry . Infection on DIV4 , fixation on DIV11 . D32 = anti - Darpp - 32 . n , MSNs expressing rAAV - mCherry - control or PV - NLS were treated with 55mM HiK for 1h qPCR was performed for indicated IEGs . Mean + ; two - way ANOVA with Bonferroni post - hoc , p * < 0 . 05 , p * * < 0 . 01 , p * * * * < 0 . 0001 . n = 3 independent cultures as biological replicates .

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investigating the role of nuclear calcium signaling in mRNA induction of Arc, Fosb and Npas4. k, immunohistochemical analysis of MSNs infected with mCherry-NLS as control or the competitive nuclear Ca2+/CaM antagonist CaMBP4-mCherry. Infection on DIV4, fixation on DIV11. D32 = anti-Darpp-32. scale bar = 10 µm. l, MSNs expressing rAAV-mCherry-control or CaMBP4 (BP4) were treated with 55mM HiK for 1h and qPCR analysis was performed for the indicated IEGs. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test, ns = not significant, p****<0.0001. n = 5 independent cultures as biological replicates. m, immunohistochemical analysis of MSNs infected with mCherry-NLS as control or the nuclear Ca2+-buffer Parvalbumin-NLS-mCherry. Infection on DIV4, fixation on DIV11. D32 = anti-Darpp-32. n, MSNs expressing rAAV-mCherry-control or PV-NLS were treated with 55mM HiK for 1h and qPCR analysis was performed for the indicated IEGs. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test, p* < 0.05, p** < 0.01, p****<0.0001. n = 3 independent cultures as biological replicates.


e , Npas4 controls activity - dependent upregulation of genes for glutamatergic synapse formation Ca2 + channels . MSNs were infected with rAAVs expressing shRNA - scrambled ( control ) or shNpas4 ( Npas4 knockdown ) on DIV5 . On DIV 12 , cultures were stimulated with HiK ( 55 mM ) for 1h or 6h mRNA were analysed by qPCR . mRNA level = fold change in mRNA compared to shscrambled unstimulated control . Mean + ; two - way ANOVA with Bonferroni post - hoc , p * < 0 . 05 , p * * * < 0 . 001 . n = 5 independent cultures as biological replicates .

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e, Npas4 controls activity-dependent upregulation of genes for glutamatergic synapse formation and Ca2+ channels. MSNs were infected with rAAVs expressing shRNA-scrambled (control) or shNpas4 (Npas4 knockdown) on DIV5. On DIV 12, cultures were stimulated with HiK (55 mM) for 1h or 6h and mRNA levels were analysed by qPCR. mRNA level = fold change in mRNA compared to shscrambled unstimulated control. Mean + SEM; two-way ANOVA with Bonferroni post-hoc test, p* < 0.05, p***<0.001. n = 5 independent cultures as biological replicates.


e , Confocal of tdTomato - labelled D1 - MSNs expressing shscr - GFP or shNpas4 - GFP ( left panels ; scale bar : 20 µm ) . 3D volume - renderings of MSN dendrites from NAc shell ( right panels ; scale bar : 2 µm ) .

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e, Confocal images of tdTomato-labelled D1-MSNs expressing shscr-GFP or shNpas4-GFP (left panels; scale bar: 20 µm). 3D volume-renderings of MSN dendrites from the NAc shell (right panels; scale bar: 2 µm).


g , Confocal of tdTomato - labelled D2 - MSNs expressing shscr - GFP or shNpas4 - GFP ( left panels ; scale bar : 20 µm ) . 3D volume - renderings of MSN dendrites from NAc shell ( right panels ; scale bar : 2 µm ) .

g, Confocal images of tdTomato-labelled D2-MSNs expressing shscr-GFP or shNpas4-GFP (left panels; scale bar: 20 µm). 3D volume-renderings of MSN dendrites from the NAc shell (right panels; scale bar: 2 µm).


a - d , Scatter plot of passive active properties of MSNs in ex vivo slice preparations from shRNA - AAV - infected mice , plotted for MSNs as D1 - positive ( D1 + ) , D1 - negative ( D1 - ) those two populations pooled ( ) . a , rheobase ; mean ± . b , firing rate estimated at twice value of rheobase ; mean ± . c , input / output gain values recorded in shscr - or shNpas4 - injected mice , mean ± . increased rheobase lower input - output gain indicate a lower excitability for MSNs in Npas4 - knockdown group . d , facilitation at corticostriatal synapses examined in MSNs with paired - stimulation at 20 4 Hz . Representative current traces scatter plots of paired - experiments illustrate a potent facilitation in MSNs in mice injected with scr - shRNA a significant decrease of facilitation in mice injected with shNpas4 , with a stronger effect in D1 - negative MSNs . Mean ± . Data information : For n - numbers , please see Table EV1 . Mann - Whitney - , p * < 0 . 05 , p * * < 0 . 01 .

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a-d, Scatter plot of passive and active membrane properties of MSNs in ex vivo slice preparations from shRNA-AAV-infected mice, plotted for MSNs as D1-positive (D1+), D1-negative (D1-) and those two populations pooled (all). a, rheobase; mean ± SEM. b, firing rate estimated at twice the value of the rheobase; mean ± SEM. c, input/output gain values recorded in shscr- or shNpas4-injected mice, mean ± SEM. The increased rheobase and lower input-output gain indicate a lower excitability for MSNs in the Npas4-knockdown group. d, facilitation at corticostriatal synapses examined in MSNs with paired-pulse stimulation at 20 and 4 Hz. Representative current traces and scatter plots of paired-pulse experiments illustrate a potent facilitation in MSNs in mice injected with scr-shRNA and a significant decrease of the facilitation in mice injected with shNpas4, with a stronger effect in D1-negative MSNs. Mean ± SEM. Data information: For n-numbers, please see Table EV1. Mann-Whitney-Test, p*<0.05, p**<0.01.


b , cocaine - induced hyperlocomotion is attenuated by Npas4 - knockdown while locomotor behaviour after saline injection is unaffected on days . Mean + ; two - way ANOVA followed by Bonferroni post hoc . p * < 0 . 05 , p * * < 0 . 01 , ns = not significant . n = 12 - 16 per condition .

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b, cocaine-induced hyperlocomotion is attenuated by Npas4-knockdown while locomotor behaviour after saline injection is unaffected on all days. Mean + SEM; two-way ANOVA followed by Bonferroni post hoc test. p* < 0.05, p** < 0.01, ns = not significant. n = 12 - 16 animals per condition.


B GFP + EBs derived from CT RA cultures at day 10 ; scale bar : 100 μm .

B GFP+ EBs derived from CT and RA cultures at day 10; scale bar: 100 μm.


C Representative FC plots depicting of GFP + obtained at day 15 , from CT RA cultures in a typical experiment .

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C Representative FC plots depicting percentage of GFP+ cells obtained at day 15, from CT and RA cultures in a typical experiment.


D Heat map demonstrating enrichment of cardiac genes in GFP + fractions ( CT + , RA + ) compared to GFP − fractions ( CT − , RA − ) at day 31 .

D Heat map demonstrating enrichment of cardiac genes in GFP+ fractions (CT+, RA+) compared to GFP− fractions (CT−, RA−) at day 31.


E , F Heat map of a select list of genes ( E ) upregulated ( F ) downregulated in RA + compared to CT + at day 31 . Fold change > 2 .

E, F Heat map of a select list of genes (E) upregulated and (F) downregulated in RA+ compared to CT+ at day 31. Fold change > 2.


A - C qPCR of selected transcripts at day 31 to validate ( A ) enrichment of cardiac markers in GFP + fractions against GFP − fractions , ( B ) upregulation of atrial ( C ) downregulation of ventricular genes in RA + compared to CT + ( n = 3 ) . Data information : Data are presented as mean ± . In ( A - C ) , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by unpaired t - . In ( A ) , for TNNT2 , P = 0 . 0001 for CT − against CT + P = 0 . 0002 for RA − against RA + ; for NKX2 . 5 , P = 0 . 00005 for CT − against CT + P = 0 . 00007 for RA − against RA + . In ( B ) , P = 0 . 0006 for NPPA P = 0 . 0002 for PITX2 . In ( C ) , P = 0 . 02 for HEY2 P = 0 . 007 for IRX4 .

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A-C qPCR of selected transcripts at day 31 to validate (A) enrichment of cardiac markers in GFP+ fractions against GFP− fractions, (B) upregulation of atrial and (C) downregulation of ventricular genes in RA+ compared to CT+ (n = 3).Data information: Data are presented as mean ± SEM. In (A-C), *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired t-test. In (A), for TNNT2, P = 0.0001 for CT− against CT+ and P = 0.0002 for RA− against RA+; for NKX2.5, P = 0.00005 for CT− against CT+ and P = 0.00007 for RA− against RA+. In (B), P = 0.0006 for NPPA and P = 0.0002 for PITX2. In (C), P = 0.02 for HEY2 and P = 0.007 for IRX4.


B Representative APs of day 31 CMs from control ( CT ) RA - treated ( RA ) groups at 1 Hz .

B Representative APs of day 31 CMs from control (CT) and RA-treated (RA) groups at 1 Hz.


C - E RMP , APAmax APAplat ( C ) , dV / dtmax ( D ) APD20 , APD50 APD90 of CT RA CMs ( E ) . F Plot showing measured APAplat values of CT RA CMs .

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C-E RMP, APAmax and APAplat (C), dV/dtmax (D) and APD20, APD50 and APD90 of CT and RA CMs (E).F Plot showing all measured APAplat values of CT and RA CMs.


G Representative APs of CT RA CMs at 0 . 5 - 4 Hz . H Average APAplat at 0 . 5 - 4 Hz . Please note that AP differences in are present at measured frequencies .

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G Representative APs of CT and RA CMs at 0.5-4 Hz.H Average APAplat at 0.5-4 Hz. Please note that the AP differences in morphology are present at all measured frequencies.


A , B plot illustrating relative mRNA of ( A ) COUP - TFI ( B ) COUP - TFII in VM AM differentiations from day 5 through day 9 ( left ) in GFP + CMs at day 31 ( right ) ; n = 3 . Data information : Data are presented as mean ± . In ( A , B ) , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by unpaired t - . In ( A ) , left panel , P = 0 . 03 , 0 . 02 , 0 . 03 , 0 . 005 0 . 0004 for comparison of COUP - TFI expression between AM VM at days 5 , 6 , 7 , 8 9 of differentiation . In ( A ) , right panel , P = 0 . 0002 for comparison of COUP - TFI expression at day 31 between AM VM . In ( B ) , left panel , P = 0 . 02 , 0 . 03 , 0 . 006 , 0 . 004 0 . 003 for comparison of COUP - TFII expression between AM VM at days 5 , 6 , 7 , 8 9 of differentiation . In ( B ) , right panel , P = 0 . 0001 for comparison of COUP - TFII expression at day 31 between AM VM . CT = control differentiation ; hESC - atrial ( AM ) ; hESC - ventricular ( VM ) .

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

A, B Line plot illustrating relative mRNA levels of (A) COUP-TFI and (B) COUP-TFII in VM and AM differentiations from day 5 through day 9 (left) and in GFP+CMs at day 31 (right); n = 3.Data information: Data are presented as mean ± SEM. In (A, B), *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired t-test. In (A), left panel, P = 0.03, 0.02, 0.03, 0.005 and 0.0004 for comparison of COUP-TFI expression between AM and VM at days 5, 6, 7, 8 and 9 of differentiation. In (A), right panel, P = 0.0002 for comparison of COUP-TFI expression at day 31 between AM and VM. In (B), left panel, P = 0.02, 0.03, 0.006, 0.004 and 0.003 for comparison of COUP-TFII expression between AM and VM at days 5, 6, 7, 8 and 9 of differentiation. In (B), right panel, P = 0.0001 for comparison of COUP-TFII expression at day 31 between AM and VM. CT = control differentiation; hESC-atrial (AM); hESC-ventricular (VM).


C , D COUP - TFI ( C ) COUP - TFII ( D ) immunofluorescence at day 31 in AM ( top ) VM ( bottom ) . Scale bars : 40 μm .

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C, D COUP-TFI (C) and COUP-TFII (D) immunofluorescence at day 31 in AM (top) and VM (bottom). Scale bars: 40 μm.


A , B mRNA expression of COUP - TFI COUP - TFII following shRNA - mediated knockdown of ( A ) COUP - TFI or ( B ) COUP - TFII in hESC - atrial cardiomyocytes ( AM ) at day 30 .

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

A, B mRNA expression of COUP-TFI and COUP-TFII following shRNA-mediated knockdown of (A) COUP-TFI or (B) COUP-TFII in hESC-atrial cardiomyocytes (AM) at day 30.


C , D mRNA expression of ion channel genes KCNA5 , KCNJ3 KCNJ5 after knockdown of ( C ) COUP - TFI or ( D ) COUP - TFII in AM at day 30 .

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

C, D mRNA expression of ion channel genes KCNA5, KCNJ3 and KCNJ5 after knockdown of (C) COUP-TFI or (D) COUP-TFII in AM at day 30.


E , F Schematic of NR2F binding sites in ( E ) KCNA5 ( F ) KCNJ3 promoters . G , H ChIP - qPCR at day 30 shows enriched binding of COUP - TFI COUP - TFII to promoter region of ( G ) KCNA5 ( H ) KCNJ3 , compared to IgG in AM .

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

E, F Schematic of NR2F binding sites in (E) KCNA5 and (F) KCNJ3 promoters.G, H ChIP-qPCR analysis at day 30 shows enriched binding of COUP-TFI and COUP-TFII to the promoter region of (G) KCNA5 and (H) KCNJ3, compared to IgG in AM.


A Expression of KCNA5 ( left ) KCNJ3 ( right ) in GFP + pools of VM AM CMs at day 31 , as well as in ventricles atria of human heart .

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A Expression of KCNA5 (left) and KCNJ3 (right) in GFP+ pools of VM and AM CMs at day 31, as well as in ventricles and atria of human heart.


B , C Typical examples ( left ) current - voltage relationships ( right ) of ( B ) IKur ( C ) IK , ACh in VM AMCMs . Data information : Data are presented as mean ± . In ( A ) , * P < 0 . 05 by unpaired t - . In ( B , C ) , * P < 0 . 05 by two - way repeated measures ANOVA followed by pairwise comparison using Student - Newman - Keuls for ( B ) Mann - Whitney rank - sum for ( C ) . In ( B ) , P = 0 . 778 , 0 . 350 , 0 . 03 , 0 . 02 , 0 . 002 , 0 . 001 , < 0 . 001 < 0 . 001 , respectively , for comparison between VM AM within potentials of − 20 , − 10 , 0 , 10 , 20 , 30 , 40 50 mV . In ( C ) , P = 0 . 01 , 0 . 01 , 0 . 01 , 0 . 01 , 0 . 03 , 0 . 397 , 0 . 397 , 0 . 397 , 0 . 671 , 0 . 207 , 0 . 207 , 0 . 09 , 0 . 01 , 0 . 039 0 . 015 , respectively , for comparison between VM AM within potentials of − 120 , − 110 , − 100 , − 90 , − 80 , − 70 , − 60 , − 50 , − 40 , − 30 , − 20 , − 10 , 0 , 10 20 mV . CMs = cardiomyocytes ; hESC - atrial ( AM ) hESC - ventricular ( VM ) CMs ; IK , ACh = acetylcholine - activated potassium current ; IKur = potassium ultra - rapid delayed rectifier current . 4 - AP = 4 - aminopyridine ; CCh = carbachol .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C Typical examples (left) and current-voltage relationships (right) of (B) IKur and (C) IK,ACh in VM and AMCMs.Data information: Data are presented as mean ± SEM. In (A), *P < 0.05 by unpaired t-test. In (B, C), *P < 0.05 by two-way repeated measures ANOVA followed by pairwise comparison using the Student-Newman-Keuls test for (B) and Mann-Whitney rank-sum test for (C). In (B), P = 0.778, 0.350, 0.03, 0.02, 0.002, 0.001, < 0.001 and < 0.001, respectively, for comparison between VM and AM within membrane potentials of −20, −10, 0, 10, 20, 30, 40 and 50 mV. In (C), P = 0.01, 0.01, 0.01, 0.01, 0.03, 0.397, 0.397, 0.397, 0.671, 0.207, 0.207, 0.09, 0.01, 0.039 and 0.015, respectively, for comparison between VM and AM within membrane potentials of −120, −110, −100, −90, −80, −70, −60, −50, −40, −30, −20, −10, 0, 10 and 20 mV. CMs = cardiomyocytes; hESC-atrial (AM) and hESC-ventricular (VM) CMs; IK,ACh = acetylcholine-activated potassium current; IKur = potassium ultra-rapid delayed rectifier current. 4-AP = 4-aminopyridine; CCh = carbachol.


D , E Representative APs of VM AM at 1 Hz in response to ( D ) IKur block by 4 - AP ( E ) IK , ACh activation by CCh . AP parameters are shown in Supplementary Table S5 .

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

D, E Representative APs of VM and AM at 1 Hz in response to (D) IKur block by 4-AP and (E) IK,ACh activation by CCh. AP parameters are shown in Supplementary Table S5.


A Representative APs at 1 Hz of VM AM CMs in response to vernakalant . Inset shows dV / dtmax . B , C Average APAplat ( B ) dV / dtmax ( C ) in absence presence of vernakalant at 1 - 4 Hz . AP parameters are shown in Supplementary Table S6 . Abbreviations as in Figs3 6 . Data information : Data are presented as mean ± . * P < 0 . 05 by Mann - Whitney rank - sum for ( B ) . P = 0 . 01 , 0 . 008 , 0 . 008 0 . 007 , respectively , for comparison of APAplat between VM AM groups at frequencies of 1 . 0 , 2 . 0 , 3 . 0 4 . 0 Hz . Two - way repeated measures ANOVA followed by pairwise comparison using Student - Newman - Keuls for ( C ) . P = 0 . 686 between VM AM groups hence not statistically significant . For VM , P = 0 . 06 for 1 versus 2 Hz ; P < 0 . 001 for 1 versus 3 Hz ; P < 0 . 001 for 1 versus 4 Hz ; P = 0 . 001 for 2 versus 3 Hz ; P = 0 . 002 for 2 versus 4 Hz ; P = 0 . 857 for 3 versus 4 Hz . For AM , P = 0 . 03 for 1 versus 2 Hz ; P < 0 . 001 for 1 versus 3 Hz ; P < 0 . 001 for 1 versus 4 Hz ; P = 0 . 02 for 2 versus 3 Hz ; P = 0 . 02 for 2 versus 4 Hz ; P = 0 . 621 for 3 versus 4 Hz .

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

A Representative APs at 1 Hz of VM and AM CMs in response to vernakalant. Inset shows dV/dtmax.B, C Average APAplat (B) and dV/dtmax (C)in the absence and presence of vernakalant at 1-4 Hz. AP parameters are shown in Supplementary Table S6. Abbreviations as in Figs3 and 6.Data information: Data are presented as mean ± SEM. *P < 0.05 by Mann-Whitney rank-sum test for (B). P = 0.01, 0.008, 0.008 and 0.007, respectively, for comparison of APAplat between VM and AM groups at frequencies of 1.0, 2.0, 3.0 and 4.0 Hz. Two-way repeated measures ANOVA followed by pairwise comparison using the Student-Newman-Keuls test for (C). P = 0.686 between VM and AM groups and hence not statistically significant. For VM, P = 0.06 for 1 versus 2 Hz; P < 0.001 for 1 versus 3 Hz; P < 0.001 for 1 versus 4 Hz; P = 0.001 for 2 versus 3 Hz; P = 0.002 for 2 versus 4 Hz; and P = 0.857 for 3 versus 4 Hz. For AM, P = 0.03 for 1 versus 2 Hz; P < 0.001 for 1 versus 3 Hz; P < 0.001 for 1 versus 4 Hz; P = 0.02 for 2 versus 3 Hz; P = 0.02 for 2 versus 4 Hz; and P = 0.621 for 3 versus 4 Hz.


A Representative APs of VM AM CMs in absence , presence following washout of 3 μmol / l XEN - D0101 . AP parameters are shown in Supplementary Table S7 .

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

A Representative APs of VM and AM CMs in the absence, presence and following washout of 3 μmol/l XEN-D0101. AP parameters are shown in Supplementary Table S7.


B Representative APs ( 1 Hz ) of VM AM in CCh , to activate IK , ACh subsequent addition of XEN - R0703 . AP parameters are shown in Supplementary Table S9 .

O O O O O O O O O O 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 Representative APs (1 Hz) of VM and AM in the CCh, to activate IK,ACh and subsequent addition of XEN-R0703. AP parameters are shown in Supplementary Table S9.


C , D Experiments performed in RAP conscious dogs in presence of vehicle or following 1 , 3 10 mg / kg XEN - R0703 show ( C ) mean right AERP values ( left ) , mean Van de Water ' s QTc ( right ) ( D ) AF inducibility plotted as a function of dose . Data information : For RAP dog experiments , n = 5 ; statistical significance tested with paired t - . Data are presented as mean ± . AERP = atrial effective refractory period ; AF = atrial fibrillation ; RAP = rapid atrial pacing ; N . S . = not significant . Other abbreviations as in Figs3 , 6 7 .

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

C, D Experiments performed in RAP conscious dogs in the presence of vehicle or following 1, 3 and 10 mg/kg XEN-R0703 show (C) mean right AERP values (left), mean Van de Water's QTc (right) and (D) AF inducibility plotted as a function of dose.Data information: For RAP dog experiments, n = 5; statistical significance tested with paired t-test. Data are presented as mean ± SEM. AERP = atrial effective refractory period; AF = atrial fibrillation; RAP = rapid atrial pacing; N.S. = not significant. Other abbreviations as in Figs3, 6 and 7.


( B ) Disintegration reaction ( 1 hour ) performed by RAG1 aa 216 - 1008 R848M / E649V , RAG2 aa 1 - 361 full human HMGB1 with Mg2 + or Mn2 + at indicated temperatures . Denaturing gel displays fluorophore - labeled strand from RAG STC before ( 16 nt band , lane 2 ) after ( 37 nt ) disintegration reaction . Lane 1 , fluorophore - labeled 37 nt marker . Representative of 2 independent experiments .

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

(B) Disintegration reaction (1 hour) performed by RAG1 aa 216-1008 R848M/E649V, RAG2 aa 1-361 and full length human HMGB1 with Mg2+ or Mn2+ at the indicated temperatures. Denaturing gel displays the fluorophore-labeled DNA strand from the RAG STC before (16 nt band, lane 2) and after (37 nt) the disintegration reaction. Lane 1, fluorophore-labeled 37 nt DNA marker. Representative of 2 independent experiments.


( D ) Results of in vivo plasmid - to - plasmid transposition using WT or R848M RAG1 indicated forms of RAG2 containing or lacking ( ∆ L ) four at tip of 333 - 342 loop , performed as described in ( Zhang et al . , 2019 ) ; ( * * * * , p < 0 . 0001 ) . Three biological replicates for each condition except in last two columns where data from 11 biological replicates are presented .

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

(D) Results of in vivo plasmid-to-plasmid transposition assay using WT or R848M RAG1 and the indicated forms of RAG2 containing or lacking (∆L) the four amino acids at the tip of the 333-342 loop, performed as described in (Zhang et al., 2019); (****, p<0.0001). Three biological replicates for each condition except in the last two columns where data from 11 biological replicates are presented.


( E ) Results of in vivo plasmid - to - genome transposition using WT or R848M RAG1 different forms of RAG2 as indicated . As previously demonstrated ( Zhang et al . , 2019 ) , R848 ( present in WT RAG1 ) RAG2 acidic hinge residues 362 - 383 each potently suppress transposition . Substantial transposition is observed with combination of RAG1 R848M RAG2 1 - 361 , which is further increased by ∆ L ; ( * * * , p = 0 . 001 ) . Four biological replicates for each condition .

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

(E) Results of in vivo plasmid-to-genome transposition assay using WT or R848M RAG1 and different forms of RAG2 as indicated. As previously demonstrated (Zhang et al., 2019), R848 (present in WT RAG1) and RAG2 acidic hinge residues 362-383 each potently suppress transposition. Substantial transposition is observed with the combination of RAG1 R848M and RAG2 1-361, which is further increased by ∆L; (***, p=0.001). Four biological replicates for each condition.


( F ) In vivo recombination activity in HEK293T of mouse wild - type RAG1 or RAG1 R848M with RAG2 1 - 361 or 1 - 361 ∆ L . ( p = 0 . 42 for wild - type RAG1 group , p = 0 . 41 for RAG1 R848M group ) . Three biological replicates for each condition .

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

(F) In vivo recombination activity in HEK293T cells of mouse wild-type RAG1 or RAG1 R848M with RAG2 1-361 or 1-361 ∆L. (p=0.42 for wild-type RAG1 group, p=0.41 for RAG1 R848M group). Three biological replicates for each condition.


NoV in vaccinated mice hindlimb muscle tissue at 2 , 3 or 4 days post infection ( dpi ) with WT NoV were determined by Western blotting to detect viral coat ( CP ) B2 of β - actin was used as a loading control ( B ) .

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NoV levels in the vaccinated mice hindlimb muscle tissue at 2, 3 or 4 days post infection (dpi) with WT NoV were determined by Western blotting to detect the viral coat protein (CP) and B2 Staining of β-actin was used as a loading control (B).


D , Western blotting of HSP70 , tsg 101 , CD81 , CD63 , CD9 in EVs , whole blood , blood clot derived from suckling mice infected with DMEM or NoVΔB2 . of β - actin was used as a loading control .

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

D, Western blotting of HSP70, tsg 101, CD81, CD63, and CD9 in EVs, whole blood, and blood clot derived from suckling mice infected with DMEM or NoVΔB2. Staining of β-actin was used as a loading control.


J , Total were extracted from hindlimb muscle tissue of NoVWT - infected BALB / c suckling mice to measure viral coat ( CP ) B2 by Western blotting . of β - actin was used as a loading control .

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

J, Total proteins were extracted from the hindlimb muscle tissue of NoVWT-infected BALB/c suckling mice to measure the viral coat protein (CP) and B2 protein by Western blotting. Staining of β-actin was used as a loading control.


( A ) Nissl - stained sagittal brain sections reveal no change in hippocampal between Clcn3unc / unc Clcn3 + / + mice at P45 or after 20 months . In contrast , hippocampus was absent ( indicated by asterisk ) in 3 months - old Clcn3 - / - mice ( scale bar : 200 µm ) .

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(A) Nissl-stained sagittal brain sections reveal no change in hippocampal morphology between Clcn3unc/unc and Clcn3+/+ mice at P45 or after 20 months. In contrast, the hippocampus was absent (indicated by asterisk) in 3 months-old Clcn3-/- mice (scale bar: 200 µm).


( B ) Nissl - stained paraffin sections show intact retinal layers in 20 months old Clcn3unc / un mice . Neurodegeneration in Clcn3 - / - mice however , results in a loss of retinal structure already at 11 of ( scale bar : 100 µm ) . RPE , retinal pigment epithelium ; OS , photoreceptor outer segments ; IS , photoreceptor inner segments ; ONL , outer layer ; OPL , outer plexiform layer ; INL , inner layer ; IPL , inner plexiform layer ; GCL , ganglion layer .

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(B) Nissl-stained paraffin sections show intact retinal layers in 20 months old Clcn3unc/un mice. Neurodegeneration in Clcn3-/- mice however, results in a loss of retinal structure already at 11 weeks of age (scale bar: 100 µm). RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.


( C ) Immunoblots for ClC - 3 , - 4 - 5 of fractions of WT ( + / + ) , Clcn3unc / unc ( unc / unc ) , Clcn3unc / + ( unc / + ) Clcn3 - / - ( - / - ) mice . β - actin , loading control .

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(C) Immunoblots for ClC-3, -4 and -5 of membrane fractions of WT (+/+), Clcn3unc/unc (unc/unc), Clcn3unc/+ (unc/+) and Clcn3-/- (-/-) mice. β-actin, loading control.


( D ) Representative Western blot for ClC - 4 of fractions from brain kidney of WT ( + / + ) , Clcn3 + / - ( + / - ) Clcn3 - / - ( - / - ) mice . β - actin , loading control . ( E ) of ClC - 4 immunoblots including those shown in ( C D ) ( normalized to actin ) . Mean values ± s . e . m . Average from ≥ 5 per genotype at ≥ 2 immunoblots per . * * * p < 0 . 0005 , * * p < 0 . 005 , * p < 0 . 05 ( two - unpaired t ) .

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

(D) Representative Western blot for ClC-4 of membrane fractions from brain and kidney of WT (+/+), Clcn3+/- (+/-) and Clcn3-/- (-/-) mice. β-actin, loading control. (E) Quantification of ClC-4 immunoblots including those shown in (C and D) (normalized to actin). Mean values ± s.e.m. Average from ≥5 animals per genotype and at ≥2 immunoblots per animal. *** p < 0.0005 , ** p < 0.005, * p < 0.05 (two-tailed unpaired t test).


( A ) Co - immunoprecipitation reveals a ClC - 3 - ClC - 4 . 10 % of solubilized brain membranes of WT Clcn3unc / unc mice were directly loaded on gel ( input , or first immunoprecipitated ( IP ) with antibodies against ClC - 3 or ClC - 4 . Western blots were probed for ClC - 3 ClC - 4 . Equivalent amounts of lysates precipitates were loaded . * unspecific band / contamination .

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

(A) Co-immunoprecipitation reveals a ClC-3-ClC-4 complex. 10% of solubilized brain membranes of WT and Clcn3unc/unc mice were directly loaded on the gel (input, or first immunoprecipitated (IP) with antibodies against ClC-3 or ClC-4. Western blots were probed for ClC-3 and ClC-4. Equivalent amounts of lysates and precipitates were loaded. * unspecific band / contamination.


( B ) FRET experiments show homo - heteromerization of ClC - 3 ClC - 4 constructs ( fused to yellow ( YFP ) cyan ( CFP ) ) overexpressed in COS - 7 . Co - expressed constructs are indicated . Graph represents energy transfer efficiencies from acceptor photobleaching , depicted as bleach corrected values ( subtraction of ClC - 4CFP alone ) . Mean values ± s . e . m . N = 20 ( ClC - 3YFP / ClC - 4CFP ) ; 33 ( ClC - 3YFP / ClC - 3CFP ) ; 26 ( ClC - 4YFP / ClC - 4CFP ) ; 42 ( ClC - 3YFP / Lamp - 1CFP ) . * * * p < 0 . 0005 ( two - unpaired t )

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

(B) FRET experiments show homo- and heteromerization of ClC-3 and ClC-4 constructs (fused to yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)) overexpressed in COS-7 cells. Co-expressed constructs are indicated. Graph represents energy transfer efficiencies from acceptor photobleaching, depicted as bleach corrected values (subtraction of ClC-4CFP alone). Mean values ± s.e.m. N = 20 (ClC-3YFP / ClC-4CFP); 33 (ClC-3YFP / ClC-3CFP); 26 (ClC-4YFP / ClC-4CFP); 42 (ClC-3YFP / Lamp-1CFP) cells. *** p < 0.0005 (two-tailed unpaired t test)