( 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

(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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 the protein level in tissues of mouse liver tumor 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 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 .

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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 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 ) .

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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 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 .

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

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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 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 ) .

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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 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 .

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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 ) 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 .

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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 patients ' paired tissues from TCGA database . The relative expression ratio of tumor to non - tumor 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 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 .

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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 patients stratified by TFAM expression . ( F Kaplan - Meier curves for overall survival of HCC patients stratified by nuclear 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 ) the recurrence of HCC patients stratified by TFAM expression . G ) the recurrence of HCC patients stratified by nuclear 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 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 .

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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 images of NeuN ( green ) and Npas4 ( red ) immunostaining performed 1h after the 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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

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 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 .

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

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 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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 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 .

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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 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 .

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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 and 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 percentage of GFP + cells obtained at day 15 , from CT and 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 .

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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 and ( F ) downregulated in RA + compared to CT + at day 31 . Fold change > 2 .

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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 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 .

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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 ) and RA - treated ( RA ) groups at 1 Hz .

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


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 .

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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 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 .

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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 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 ) .

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 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 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 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 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 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

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 ) and COUP - TFII ( D ) immunofluorescence at day 31 in AM ( top ) and 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 and 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 O B-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 and 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 O B-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 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 .

O O O O O O O O O O O O B-MEASURED_VAR O O O O B-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 O O O O B-MEASURED_VAR O 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 ) and KCNJ3 ( right ) in GFP + pools of VM and AM CMs at day 31 , as well as in ventricles and 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and AM CMs in the absence , presence and 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 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 and AM in the CCh , to activate IK , ACh and 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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(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 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 .

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

(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 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 .

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

O 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 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 ) .

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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 , 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 .

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

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 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 .

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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 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 ) .

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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 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 .

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

(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 and - 5 of membrane fractions of WT ( + / + ) , Clcn3unc / unc ( unc / unc ) , Clcn3unc / + ( unc / + ) and 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 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 ) .

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(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 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 .

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(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 - 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 )

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(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)


( C ) Immunolabelling shows subcellular localization of hClC - 4 ( top , green in merge ) , hClC - 3 ( middle , green in merge ) and hClC - 3unc ( bottom , green in merge ) of transiently transfected COS - 7 cells , in comparison to either PDI or Lamp - 1 ( both red ) as marker for the ER and late endosomes / lysosomes , respectively .

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(C) Immunolabelling shows subcellular localization of hClC-4 (top, green in merge), hClC-3 (middle, green in merge) and hClC-3unc (bottom, green in merge) of transiently transfected COS-7 cells, in comparison to either PDI or Lamp-1 (both red) as marker for the ER and late endosomes/lysosomes, respectively.


( D ) Co - localization of hClC - 3 and hClC - 4 in cytoplasmic vesicles of COS - 7 cells transfected with hClC - 4 cDNA ( hemagglutinin ( HA ) tagged ) together with either hClC - 3 or hClC - 3unc . Immunostaining used antibodies against ClC - 3 and HA tag ( green and red in merge , respectively ) .

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(D) Co-localization of hClC-3 and hClC-4 in cytoplasmic vesicles of COS-7 cells transfected with hClC-4 cDNA (hemagglutinin (HA) tagged) together with either hClC-3 or hClC-3unc. Immunostaining used antibodies against ClC-3 and HA tag (green and red in merge, respectively).


( A ) Clcn3unc / unc / Clcn4 - / - and Clcn3 - / - mice died within 3 - 4 weeks after birth . Approximately 20 % of the animals survived in either line ( Clcn3unc / unc / Clcn4 - / - , n = 136 and Clcn3 - / - , n = 187 ) . All Clcn3 - / - / Clcn4 - / - mice ( n = 4 ) died within 1 - 2 weeks after birth .

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(A) Clcn3unc/unc/Clcn4-/- and Clcn3-/- mice died within 3-4 weeks after birth. Approximately 20% of the animals survived in either line (Clcn3unc/unc/Clcn4-/-, n = 136 and Clcn3-/-, n = 187). All Clcn3-/-/Clcn4-/- mice (n= 4) died within 1-2 weeks after birth.


( B ) Nissl - stained paraffin sections show progressive neuronal cell loss ( arrows ) that begins in hippocampal CA1 region of P14 Clcn3unc / unc / Clcn4 - / - mice and results in a complete loss of the hippocampus at P28 . Neurodegeneration progresses slower in Clcn3 - / - mice Stobrawa et al , 2001 ( ) ( scale bar : 200 µm ) .

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(B) Nissl-stained paraffin sections show progressive neuronal cell loss (arrows) that begins in hippocampal CA1 region of P14 Clcn3unc/unc/Clcn4-/- mice and results in a complete loss of the hippocampus at P28. Neurodegeneration progresses slower in Clcn3-/- mice Stobrawa et al, 2001() (scale bar: 200 µm).


( C ) Semi - thin sections of P28 retinae revealed degeneration of photoreceptor cells in the outer nuclear layer and outer and inner segment of Clcn3unc / unc / Clcn4 - / - , but not of Clcn3unc / unc or Clcn4 - / - mice ( scale bar : 50 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 .

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(C) Semi-thin sections of P28 retinae revealed degeneration of photoreceptor cells in the outer nuclear layer and outer and inner segment of Clcn3unc/unc/Clcn4-/-, but not of Clcn3unc/unc or Clcn4-/- mice (scale bar: 50 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.


( A ) GFP antibodies immunolabel VenusClC - 3 ( green ) in the somata of CA1 pyramidal neurons of Clcn3ven / ven , but not Clcn3 + / + brain sections . sp , stratum pyramidale ( scale bar : 20 µm ) .

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(A) GFP antibodies immunolabel VenusClC-3 (green) in the somata of CA1 pyramidal neurons of Clcn3ven/ven, but not Clcn3+/+ brain sections. sp, stratum pyramidale (scale bar: 20 µm).


( B ) Co - immunolabelling of VenusClC - 3 ( green ) with ClC - 4 ( red ) in the CA3 region of Clcn3ven / ven mice ( scale bar : 20 µm ) . sl , stratum lucidum ; sp , stratum pyramidale .

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(B) Co-immunolabelling of VenusClC-3 (green) with ClC-4 (red) in the CA3 region of Clcn3ven/ven mice (scale bar: 20 µm). sl, stratum lucidum; sp, stratum pyramidale.


( C ) Co - immunostaining of VenusClC - 3 ( green ) with EEA1 ( red , left panel ) , Lamp - 1 ( red , middle panel ) or synaptophysin ( red , right panel ) in either CA1 or CA3 region of Clcn3ven / ven mice . Arrows indicate structures of overlap ( scale bar : 5 µm ) .

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(C) Co-immunostaining of VenusClC-3 (green) with EEA1 (red, left panel), Lamp-1 (red, middle panel) or synaptophysin (red, right panel) in either CA1 or CA3 region of Clcn3ven/ven mice. Arrows indicate structures of overlap (scale bar: 5 µm).


( D ) GFP antibody immunolabels VenusClC - 3 ( green ) in neurites of cultured neurons . EEA1 and VGLUT1 are co - stained ( both in red ; left and right panel , respectively ) . The dendritic marker MAP2 is stained in blue ( right panel ) ( scale bars : 10 µm ) . DNA stained with DAPI .

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(D) GFP antibody immunolabels VenusClC-3 (green) in neurites of cultured neurons. EEA1 and VGLUT1 are co-stained (both in red; left and right panel, respectively). The dendritic marker MAP2 is stained in blue (right panel) (scale bars: 10 µm). DNA stained with DAPI.


( E ) Immuno - blot analysis of fractions collected during preparation of SVs from brain of Clcn3ven / ven mice . VenusClC - 3 ( detected with GFP antibody ) co - purified with SV marker protein synaptophysin .

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(E) Immuno-blot analysis of fractions collected during preparation of SVs from brain of Clcn3ven/ven mice. VenusClC-3 (detected with GFP antibody) co-purified with SV marker protein synaptophysin.


( F ) Fluorescence of immobilized Venus - tagged SVs imaged with TIRF microscopy . Scale bar , 2 µm .

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(F) Fluorescence of immobilized Venus-tagged SVs imaged with TIRF microscopy. Scale bar, 2 µm.


( G , H ) Co - localization between the Venus - tagged vesicles with antibodies against VGLUT1 and synaptophysin was analyzed . ( G ) 11 . 4 ± 0 . 6 ( SD ) of VGLUT1 - labeled SVs and 8 . 2 ± 2 ( SD ) of synaptophysin - labeled SVs showed Venus fluorescence ( n = 6 ) . ( H ) 65 ± 5 . 4 ( SD ) and 79 . 4 ± 3 . 8 ( SD ) of Venus - tagged SVs showed co - localization with VGLUT1 and synaptophysin , respectively ( n = 6 ) .

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(G, H) Co-localization between the Venus-tagged vesicles with antibodies against VGLUT1 and synaptophysin was analyzed. (G) 11.4 ± 0.6 (SD) of VGLUT1-labeled SVs and 8.2 ± 2 (SD) of synaptophysin-labeled SVs showed Venus fluorescence (n=6). (H) 65 ± 5.4 (SD) and 79.4 ± 3.8 (SD) of Venus-tagged SVs showed co-localization with VGLUT1 and synaptophysin, respectively (n=6).


( I ) Co - immunolabelling of VenusClC - 3 ( green ) with synaptophysin ( red ) of immobilized SVs imaged with TIRF microscopy . Vesicle in quadrant delimited by dashed line is shown in higher magnification in merge . Scale bar , 2 µm .

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(I) Co-immunolabelling of VenusClC-3 (green) with synaptophysin (red) of immobilized SVs imaged with TIRF microscopy. Vesicle in quadrant delimited by dashed line is shown in higher magnification in merge. Scale bar, 2 µm.


( A ) Immunoblots for ClC - 3 , ClC - 4 , VGLUT1 , VGLUT2 , VGAT , GluR4 and GABAA - R - α1 of whole lysates from brain of Clcn3 + / + and Clcn3 - / - mice at 2 , 6 and 12 weeks of age .

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(A) Immunoblots for ClC-3, ClC-4, VGLUT1, VGLUT2, VGAT, GluR4 and GABAA-R-α1 of whole lysates from brain of Clcn3+/+ and Clcn3-/- mice at 2, 6 and 12 weeks of age.


( B ) Acidification of synaptic vesicle LP2 fractions from Clcn3unc / unc and Clcn3 + / + mice at one year of age ( upper panel , n = 2 animals each and ≥ 3 measurements per animal ) , 10 - weeks - old Clcn4 - / - and WT mice ( middle panel , n = 3 animals each and ≥ 3 measurements per animal ) , and 4 - 6 - weeks - old Clcn3unc / unc / Clcn4 - / - and control mice ( lower panel , 6 animals each with ≥ 2 measurements per animal ) . A decrease in fluorescence reflects acidification . The protonophore FCCP dissipated the pH gradient . Mean values ± s . e . m .

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(B) Acidification of synaptic vesicle LP2 fractions from Clcn3unc/unc and Clcn3+/+ mice at one year of age (upper panel, n= 2 animals each and ≥3 measurements per animal), 10-weeks-old Clcn4-/- and WT mice (middle panel, n= 3 animals each and ≥3 measurements per animal), and 4-6-weeks-old Clcn3unc/unc/Clcn4-/- and control mice (lower panel, 6 animals each with ≥2 measurements per animal). A decrease in fluorescence reflects acidification. The protonophore FCCP dissipated the pH gradient. Mean values ± s.e.m.


( C ) Quantification of ATP - induced acidification of LP2 fractions derived from 2 , 6 , and 10 - 12 weeks - old Clcn3 - / - compared to wild - type mice . Acidification measured by acridine orange fluorescence in the presence of 60 mM KCl . At least 2 animals in at least 2 independent experiments were pooled . Each measurement was performed at least 3 times . Mean ± s . e . m . is shown . * p < 0 . 05 , * * * p < 0 . 0005 ( two - tailed unpaired t test ) .

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(C) Quantification of ATP-induced acidification of LP2 fractions derived from 2, 6, and 10-12 weeks-old Clcn3-/- compared to wild-type mice. Acidification measured by acridine orange fluorescence in the presence of 60 mM KCl. At least 2 animals in at least 2 independent experiments were pooled. Each measurement was performed at least 3 times. Mean ± s.e.m. is shown.* p < 0.05, *** p < 0.0005 (two-tailed unpaired t test).


( D ) Immunoblot revealed strong reduction of VGLUT1 levels in the homogenate ( H ) and LP2 fraction of Clcn3unc / unc / Clcn4 - / - mice .

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(D) Immunoblot revealed strong reduction of VGLUT1 levels in the homogenate (H) and LP2 fraction of Clcn3unc/unc/Clcn4-/- mice.


( E ) Endosomal pH of Clcn3 + / + and Clcn3 - / - primary hippocampal neurons determined with a pH - sensitive transferrin conjugate . N = 5 independent cell lines with at least 2 live cell dishes per cell line with about 10 images each and ≥ 10 Tfn - positive compartments per image and genotype were analysed . Mean ± s . e . m . is shown .

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(E) Endosomal pH of Clcn3+/+ and Clcn3-/- primary hippocampal neurons determined with a pH-sensitive transferrin conjugate. N= 5 independent cell lines with at least 2 live cell dishes per cell line with about 10 images each and ≥10 Tfn-positive compartments per image and genotype were analysed. Mean ± s.e.m. is shown.


Miniature inhibitory postsynaptic currents ( mIPSCs ) measured in CA1 pyramidal neurons in different mouse models at P14 - P16 . ( A ) example mIPSCs of WT ( left ) and Clcn3 - / - neurons .

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Miniature inhibitory postsynaptic currents (mIPSCs) measured in CA1 pyramidal neurons in different mouse models at P14-P16. (A) example mIPSCs of WT (left) and Clcn3-/- neurons.


Mean values ± s . e . m . and cumulative histogram of amplitude ( top ) and frequency / interevent - interval ( bottom ) of mIPSCs in different mouse - models as indicated . Several cells of 3 mice of each genotype were analyzed . Compared genotypes were siblings . No difference in amplitude or frequency / interevent - interval distribution of mIPSCs was found in ( B ) Clcn3 - / - and Clcn3 + / + ( Clcn3 + / + : 16 cells , Clcn3 - / - : 15 cells ; mean amplitude : p > 0 . 8 , t test ; mean frequency : p > 0 . 4 , MW - test ) , in ( C ) Clcn3unc / unc and Clcn3 + / + ( Clcn3 + / + : 21 cells , Clcn3unc / unc : 13 cells ; mean amplitude : p > 0 . 7 ; mean frequency : p = 0 . 3 , t test ) , and in ( D ) Clcn4 - / - and Clcn3 + / + ( Clcn3 + / + : 18 cells , Clcn4 - / - : 18 cells ; mean amplitude : p > 0 . 3 , t - test ; mean frequency : p = 0 . 6 , MW - test ) .

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Mean values ± s.e.m. and cumulative histogram of amplitude (top) and frequency/interevent-interval (bottom) of mIPSCs in different mouse-models as indicated . Several cells of 3 mice of each genotype were analyzed. Compared genotypes were siblings. No difference in amplitude or frequency/ interevent-interval distribution of mIPSCs was found in (B) Clcn3-/- and Clcn3+/+ (Clcn3+/+: 16 cells, Clcn3-/-: 15 cells; mean amplitude: p > 0.8, t test; mean frequency: p > 0.4, MW-test), in (C) Clcn3unc/unc and Clcn3+/+ (Clcn3+/+: 21 cells, Clcn3unc/unc: 13 cells; mean amplitude: p > 0.7; mean frequency: p = 0.3, t test), and in (D) Clcn4-/- and Clcn3+/+ (Clcn3+/+: 18 cells, Clcn4-/-: 18 cells; mean amplitude: p > 0.3, t-test; mean frequency: p = 0.6, MW-test).


( E ) No difference in mean amplitude ( p > 0 . 5 , t test ) , in cumulative amplitude and mean frequency of mIPSCs ( p > 0 . 1 , t test ) was found in Clcn3unc / unc / Clcn4 - / - and Clcn3 + / + / Clcn4 - / - . The interevent - interval distribution was shifted to the right , indicating a reduced frequency of mIPSCs in Clcn3unc / unc / Clcn4 - / - ( KS - test , p < 0 , 05 ) ( Clcn3 + / + / Clcn4 - / - : 16 cells , Clcn3unc / unc / Clcn4 - / - : 12 cells ) .

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(E) No difference in mean amplitude (p > 0.5, t test), in cumulative amplitude and mean frequency of mIPSCs (p > 0.1, t test) was found in Clcn3unc/unc/Clcn4-/- and Clcn3+/+/Clcn4-/-. The interevent-interval distribution was shifted to the right, indicating a reduced frequency of mIPSCs in Clcn3unc/unc/Clcn4-/- (KS-test, p < 0,05) (Clcn3+/+/Clcn4-/-: 16 cells, Clcn3unc/unc/Clcn4-/-: 12 cells).


( F , G ) miniature excitatory postsynaptic currents ( mEPSCs ) . ( F ) example traces of mEPSCs in WT and Clcn3 - / - neurons ( G ) No significant difference between Clcn3unc / unc / Clcn4 - / - and Clcn3 + / + / Clcn4 - / - mEPSCs in mean amplitudes ( p > 0 . 9 , t test ) or in the distribution of amplitudes ( Clcn4 - / - : 12 cells , 2 mice ; Clcn3unc / unc / Clcn4 - / - : 8 cells , 2 mice ) .

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

(F,G) miniature excitatory postsynaptic currents (mEPSCs). (F) example traces of mEPSCs in WT and Clcn3-/- neurons (G) No significant difference between Clcn3unc/unc/Clcn4-/- and Clcn3+/+/Clcn4-/- mEPSCs in mean amplitudes (p > 0.9, t test) or in the distribution of amplitudes (Clcn4-/-: 12 cells, 2 mice; Clcn3unc/unc/Clcn4-/-: 8 cells, 2 mice).


( H ) Primary hippocampal neurons co - transfected with ClC - 3 and synaptobrevin ( syb2 ) , both luminally tagged with pHluorin or the pH - sensitive mOrange Ramirez et al , 2012 ( ) , respectively , were stimulated by 200 action potentials ( APs ) at 20 Hz ( indicated by bar ) . Vesicle exocytosis was monitored by fluorescence increase upon exposure to extracellular neutral pH . Note that only Syb2 and not ClC - 3 shows signs of exocytosis . Traces were corrected for photobleaching . The initial decay of the pHluorin signal is probably due to the high light exposure , which forces the fluorophores into a transient dark - state Dean et al , 2011 ( ) . Averaged fluorescence traces ( n = 10 ) , error bars , s . e . m .

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

(H) Primary hippocampal neurons co-transfected with ClC-3 and synaptobrevin (syb2), both luminally tagged with pHluorin or the pH-sensitive mOrange Ramirez et al, 2012(), respectively, were stimulated by 200 action potentials (APs) at 20 Hz (indicated by bar). Vesicle exocytosis was monitored by fluorescence increase upon exposure to extracellular neutral pH. Note that only Syb2 and not ClC-3 shows signs of exocytosis. Traces were corrected for photobleaching. The initial decay of the pHluorin signal is probably due to the high light exposure, which forces the fluorophores into a transient dark-state Dean et al, 2011(). Averaged fluorescence traces (n=10), error bars, s.e.m.


B Pim1 , 2 , or 3 and GFP - EDC3 expression vectors were transfected into HEK - 293T . GFP - Trap Nano - body beads ( GFP - Trap ) were used to immunoprecipitate ( IP ) GFP - EDC3 , and the associated proteins were analyzed by Western blotting with the indicated Abs .

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

B Pim1, 2, or 3 and GFP-EDC3 expression vectors were transfected into HEK-293T. GFP-Trap Nano-body beads (GFP-Trap) were used to immunoprecipitate (IP) GFP-EDC3, and the associated proteins were analyzed by Western blotting with the indicated Abs.


B HEK - 293T cells were transfected with Pim1 and each EDC3 domain ( Fig . 2A ) fused to the C - terminus of the GFP - tagged vector . The individual fragments were immunoprecipitated with GFP - Trap beads and analyzed by Western blotting with the indicated Abs . EDC3 wild type ( W ) , F1 , F2 , F3 , F4 were EDC3 fragments vector fused to GFP . The C1 lane was only transfected with the GFP Flag vector while C2 contained EDC3 wild type fused to GFP and no Pim1 but empty vector control ( pcDNA3 ) .

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

B HEK-293T cells were transfected with Pim1 and each EDC3 domain (Fig. 2A) fused to the C-terminus of the GFP-tagged vector. The individual fragments were immunoprecipitated with GFP-Trap beads and analyzed by Western blotting with the indicated Abs. EDC3 wild type (W), F1, F2, F3, F4 were EDC3 fragments vector fused to GFP. The C1 lane was only transfected with the GFP Flag vector while C2 contained EDC3 wild type fused to GFP and no Pim1 but empty vector control (pcDNA3).