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 .

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

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 .

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

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 .

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 B-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 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 B-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-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

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 .

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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 .

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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 ( 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 ( 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 .

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

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 .

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

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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 .

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 .

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

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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 .

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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 .

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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 .

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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 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 .

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(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 .

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(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 .

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(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 .

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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 .

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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 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 ) .

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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 . 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 .

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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 - 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 )

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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 ) hClC - 3unc ( bottom , green in merge ) of transiently transfected COS - 7 , in comparison to either PDI or Lamp - 1 ( both red ) as marker for ER 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 hClC - 4 in vesicles of COS - 7 transfected with hClC - 4 cDNA ( hemagglutinin ( HA ) tagged ) together with either hClC - 3 or hClC - 3unc . Immunostaining used antibodies against ClC - 3 HA tag ( green red in merge , 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).


( A ) Clcn3unc / unc / Clcn4 - / - Clcn3 - / - mice died within 3 - 4 after birth . Approximately 20 % of survived in either ( Clcn3unc / unc / Clcn4 - / - , n = 136 Clcn3 - / - , n = 187 ) . Clcn3 - / - / Clcn4 - / - mice ( n = 4 ) died within 1 - 2 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 loss ( arrows ) that begins in hippocampal CA1 region of P14 Clcn3unc / unc / Clcn4 - / - mice results in a complete loss of 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 in outer layer outer 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 layer ; OPL , outer plexiform layer ; INL , inner layer ; IPL , inner plexiform layer ; GCL , ganglion 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 somata of CA1 pyramidal neurons of Clcn3ven / ven , but not Clcn3 + / + brain sections . sp , stratum pyramidale ( scale bar : 20 µm ) .

(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 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 VGLUT1 are co - stained ( both in red ; left right panel , respectively ) . dendritic marker MAP2 is stained in blue ( right panel ) ( scale bars : 10 µm ) . stained with DAPI .

(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 of fractions collected during preparation of SVs from brain of Clcn3ven / ven mice . VenusClC - 3 ( detected with GFP antibody ) co - purified with SV marker 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 ) of immobilized Venus - tagged SVs imaged with TIRF microscopy . Scale bar , 2 µm .

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


( G , H ) Co - localization between Venus - tagged vesicles with antibodies against VGLUT1 synaptophysin was analyzed . ( G ) 11 . 4 ± 0 . 6 ( SD ) of VGLUT1 - labeled SVs 8 . 2 ± 2 ( SD ) of synaptophysin - labeled SVs showed Venus ( n = 6 ) . ( H ) 65 ± 5 . 4 ( SD ) 79 . 4 ± 3 . 8 ( SD ) of Venus - tagged SVs showed co - localization with VGLUT1 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 is shown in higher magnification in merge . Scale bar , 2 µm .

(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 GABAA - R - α1 of whole lysates from brain of Clcn3 + / + Clcn3 - / - mice at 2 , 6 12 of .

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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 Clcn3 + / + mice at one year of ( upper panel , n = 2 each ≥ 3 measurements per ) , 10 - - old Clcn4 - / - WT mice ( middle panel , n = 3 each ≥ 3 measurements per ) , 4 - 6 - - old Clcn3unc / unc / Clcn4 - / - control mice ( lower panel , 6 each with ≥ 2 measurements per ) . A decrease in reflects acidification . protonophore FCCP dissipated 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 ) of ATP - induced acidification of LP2 fractions derived from 2 , 6 , 10 - 12 - old Clcn3 - / - compared to wild - type mice . Acidification measured by acridine orange in presence of 60 mM KCl . At least 2 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 - unpaired t ) .

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

(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 ) 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 . cumulative histogram of amplitude ( top ) frequency / interevent - interval ( bottom ) of mIPSCs in different mouse - models as indicated . Several 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 - / - Clcn3 + / + ( Clcn3 + / + : 16 , Clcn3 - / - : 15 ; mean amplitude : p > 0 . 8 , t ; mean frequency : p > 0 . 4 , MW - ) , in ( C ) Clcn3unc / unc Clcn3 + / + ( Clcn3 + / + : 21 , Clcn3unc / unc : 13 ; mean amplitude : p > 0 . 7 ; mean frequency : p = 0 . 3 , t ) , in ( D ) Clcn4 - / - Clcn3 + / + ( Clcn3 + / + : 18 , Clcn4 - / - : 18 ; mean amplitude : p > 0 . 3 , t - ; mean frequency : p = 0 . 6 , MW - ) .

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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 ) , in cumulative amplitude mean frequency of mIPSCs ( p > 0 . 1 , t ) was found in Clcn3unc / unc / Clcn4 - / - Clcn3 + / + / Clcn4 - / - . interevent - interval distribution was shifted to right , indicating a reduced frequency of mIPSCs in Clcn3unc / unc / Clcn4 - / - ( KS - , p < 0 , 05 ) ( Clcn3 + / + / Clcn4 - / - : 16 , Clcn3unc / unc / Clcn4 - / - : 12 ) .

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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 Clcn3 - / - neurons ( G ) No significant difference between Clcn3unc / unc / Clcn4 - / - Clcn3 + / + / Clcn4 - / - mEPSCs in mean amplitudes ( p > 0 . 9 , t ) or in distribution of amplitudes ( Clcn4 - / - : 12 , 2 mice ; Clcn3unc / unc / Clcn4 - / - : 8 , 2 mice ) .

(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 ) hippocampal neurons co - transfected with ClC - 3 synaptobrevin ( syb2 ) , both luminally tagged with pHluorin or 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 increase upon exposure to extracellular neutral pH . Note that only Syb2 not ClC - 3 shows signs of exocytosis . Traces were corrected for photobleaching . initial decay of pHluorin signal is probably due to high exposure , which forces fluorophores into a transient - state Dean et al , 2011 ( ) . Averaged traces ( n = 10 ) , error bars , s . e . m .

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(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 GFP - EDC3 expression vectors were transfected into HEK - 293T . GFP - Trap Nano - beads ( GFP - Trap ) were used to immunoprecipitate ( IP ) GFP - EDC3 , associated were analyzed by Western blotting with indicated Abs .

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

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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).


F PC3 - LN4 CRISPR - Ctr Pim1 KO were treated with PIMi ( PIM447 , 3μM ) , AKTi ( GSK690693 , 5μM ) , or combination for 24 h . Extracts were subjected to Western blotting with indicated Abs .

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F PC3-LN4 CRISPR-Ctr and Pim1 KO cells were treated with PIMi (PIM447, 3μM), AKTi (GSK690693, 5μM), or the combination for 24 h. Extracts were subjected to Western blotting with the indicated Abs.


of EDC3 / DCP1a foci number from Control ( Ctrl ) , PIMi , AKTi AKTi + PIMi combination in ( A ) . Data information : data of ( B are presented as mean + / - ( n = 30 / treatment group ) . p values < 0 . 05 , calculated using unpaired student t - , are considered significant . p values > 0 . 05 not significant ( n . s . ) . analyses done included multiple independent fields .

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Quantification of EDC3/DCP1a foci number from Control (Ctrl), PIMi, AKTi and the AKTi + PIMi combination in (A). Data information: The quantification data of (B are presented as mean +/- SEM (n = 30 cells/treatment group). p values<0.05, calculated using unpaired student t-test, are considered significant. p values>0.05 not significant (n.s.). The analyses done included multiple independent fields.


D of DCP1a foci number for Control ( Ctrl ) , PIMi , AKTi , AKTi + PIMi combination in ( C ) . Data information : data of D ) are presented as mean + / - ( n = 30 / treatment group ) . p values < 0 . 05 , calculated using unpaired student t - , are considered significant . p values > 0 . 05 not significant ( n . s . ) . analyses done included multiple independent fields .

D Quantification of DCP1a foci number for Control (Ctrl), PIMi, AKTi, and AKTi + PIMi combination in (C). Data information: The quantification data of D) are presented as mean +/- SEM (n = 30 cells/treatment group). p values<0.05, calculated using unpaired student t-test, are considered significant. p values>0.05 not significant (n.s.). The analyses done included multiple independent fields.


A , B Western blot of whole - ( WCL ) P - ( PB ) purified from PC3 - LN4 stably expressing GFP - DCP1a used as a P - tag . P - bodies ( PB ) were isolated with GFP - Trap beads analyzed by Western blotting with indicated Abs .

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A, B Western blot of whole-cell (WCL) and P-body (PB) purified from PC3-LN4 cells stably expressing GFP-DCP1a used as a P-body tag. The P-bodies (PB) were isolated with GFP-Trap beads and analyzed by Western blotting with the indicated Abs.


A PC3 - LN4 Naïve CRISPR / Cas9 genome - edited EDC3 S161A ( SA ) , S161D ( Fredriksson et al . ) , EDC3 knock - out ( KO ) lines were plated ( 4000 / well in 24 well plates ) imaged every 12 h using phase contrast in IncuCyte ZOOM platform . graph shown was generated with IncuCyte Basic Software following 5 days of growth . experiments were done in triplicates . Values are mean + / - S . E . M . p values < 0 . 05 , calculated using unpaired student t - . Data information : invasion migration assays were performed in triplicates 6 different fields at 10x magnification were counted in 3 separate inserts . data are presented as mean + / - S . D . p values < 0 . 05 , calculated using unpaired student ' s t - .

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A PC3-LN4 Naïve and CRISPR/Cas9 genome-edited EDC3 S161A (SA), S161D (Fredriksson et al.), and EDC3 knock-out (KO) cell lines were plated (4000 cells/well in 24 well plates) and imaged every 12 h using phase contrast in the IncuCyte ZOOM platform. The graph shown was generated with IncuCyte Basic Software following 5 days of growth. The experiments were done in triplicates. Values are mean +/- S.E.M. p values<0.05, calculated using unpaired student t-test. Data information: All invasion and migration assays were performed in triplicates and 6 different fields at 10x magnification were counted in 3 separate inserts. The data are presented as the mean +/- S.D. p values<0.05, calculated using unpaired student's t-test.


H Relative mRNA expression of ITGA6 , ITGB1 , KLF4 in PC3 - LN4 EDC3 KO - C60 , containing reexpressed EDC3 WT or EDC3 S161A mutant cDNAs Data information : Real - time PCR

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H Relative mRNA expression of ITGA6, ITGB1, and KLF4 in PC3-LN4 EDC3 KO-C60 cells, containing reexpressed EDC3 WT or EDC3 S161A mutant cDNAs Data information: Real-time PCR data


J Western blot of extracted from PC3 - LN4 treated with DMSO or combination of Pim447 AZD5363 for 96 h using specified antibodies .

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J Western blot analysis of proteins extracted from PC3-LN4 cells treated with DMSO or the combination of Pim447 and AZD5363 for 96 h using the specified antibodies.


A Two independently derived CRISPR / Cas9 PC3 - LN4 EDC3 S161A mutants ( EDC3 SA # 1 , # 2 ) wild type were injected subcutaneously in flank ( 5X106 ) of SCID mice ( n = 8 / group ) , growth was monitored until control group reached 1000 mm3 . subcutaneous volume measurement ( mm3 ) was done as outlined in Materials Methods . Data are presented as mean ± , ( n = 8 / group ) .

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A Two independently derived CRISPR/Cas9 PC3-LN4 EDC3 S161A mutants (EDC3 SA#1, #2) and wild type cells were injected subcutaneously in the flank (5X106) of SCID mice (n=8/group), and tumor growth was monitored until the control group reached 1000 mm3. The subcutaneous tumor volume measurement (mm3) was done as outlined in Materials and Methods. Data are presented as mean ± SEM, (n = 8/group).


B Phosphorylated EDC3 were examined in normal immortalized breast lines , MCF10A MCF12A , human breast cancer lines HCC38 , HCC1954 , MDA - MB - 231 , MDA - MD - 468 , BT - 549 , MRKNU1 , mouse 4T1 by Western blotting with indicated Abs .

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B Phosphorylated EDC3 levels were examined in normal immortalized breast cell lines, MCF10A and MCF12A, human breast cancer cell lines HCC38, HCC1954, MDA-MB-231, MDA-MD-468, BT-549, MRKNU1, and mouse 4T1 by Western blotting with the indicated Abs.


C Dual immunohistochemistry of human breast with p - EDC3 Ab ( Fredriksson et al . ) total EDC3 ( brown ) . Scale bar : 50µm . D Dual immunohistochemistry of human normal resected breast tissue sections with p - EDC3 Ab ( Fredriksson et al . ) total EDC3 ( brown ) . Scale bar : 50µm .

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C Dual immunohistochemistry staining of human breast tumor with p-EDC3 Ab (Fredriksson et al.) and total EDC3 (brown). Scale bar: 50µm. D Dual immunohistochemistry staining of human normal resected breast tissue sections with p-EDC3 Ab (Fredriksson et al.) and total EDC3 (brown). Scale bar: 50µm.


( A ) Immunofluorescence of HEK ‐ 293T that express TFEB - 3 × FLAG , subjected to indicated treatments stained with antibodies against FLAG lysosomal marker LAMP2 . FLAG LAMP2 channels are in green red , respectively , in merge . DAPI ( blue ) is included in merge . Scale bars represent 10 μm .

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(A) Immunofluorescence of HEK‐293T cells that express TFEB-3 × FLAG, subjected to the indicated treatments and stained with antibodies against FLAG and the lysosomal marker LAMP2. The FLAG and LAMP2 channels are in green and red, respectively, in the merge. DAPI (blue) is included in the merge. Scale bars represent 10 μm.


( B ) of number of with TFEB - 3 × FLAG in four conditions in ( A ) . Each value represents mean ± s . d . from three independent fields with N = 300 .

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(B) Quantification of the number of cells with nuclear TFEB-3 × FLAG in the four conditions in (A). Each value represents mean±s.d. from three independent fields with N=300.


( C ) Immunofluorescence of HEK ‐ 293T treated as indicated stained with antibodies against endogenous TFEB lysosomal RagC ( green red , respectively , in merge ) . DAPI is included in merge . Scale bars represent 10 μm .

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(C) Immunofluorescence of HEK‐293T cells treated as indicated and stained with antibodies against endogenous TFEB and the lysosomal protein RagC (green and red, respectively, in the merge). DAPI is included in the merge. Scale bars represent 10 μm.


( D ) Immunoblotting of extracted from HeLa that express TFEB - 3 × FLAG treated with DMSO , chloroquine ( CQ ) or SalA , subjected to / fractionation blotted with antibody against FLAG to detect TFEB . H3 tubulin were used as markers , respectively . Blots are representative of triplicate experiments .

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(D) Immunoblotting of proteins extracted from HeLa cells that express TFEB-3 × FLAG treated with DMSO, chloroquine (CQ) or SalA, subjected to nuclear/cytosolic fractionation and blotted with antibody against FLAG to detect TFEB. H3 and tubulin were used as nuclear and cytosolic markers, respectively. Blots are representative of triplicate experiments.


( A ) Lysosomal stress inhibits mTOR signalling . Immunoblotting of extracts isolated from HeLa treated overnight as indicated . Membranes were probed with antibodies against p ‐ T202 / Y204 ‐ ERK1 / 2 , ERK1 / 2 , p ‐ T389 ‐ S6K , S6K to measure ERK mTORC1 activities .

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(A) Lysosomal stress inhibits mTOR signalling. Immunoblotting of protein extracts isolated from HeLa cells treated overnight as indicated. Membranes were probed with antibodies against p‐T202/Y204‐ERK1/2, ERK1/2, p‐T389‐S6K, and S6K to measure ERK and mTORC1 activities.


( B ) Torin 1 induces TFEB dephosphorylation translocation . FLAG immunoblotting of fractions isolated from TFEB - 3 × FLAG HeLa starved in acid ‐ free media subsequently stimulated as indicated for at least 3 h . Correct subcellular fractionation was verified with H3 tubulin antibodies .

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(B) Torin 1 induces TFEB dephosphorylation and nuclear translocation. FLAG immunoblotting of cytosolic and nuclear fractions isolated from TFEB-3 × FLAG HeLa cells starved in amino acid‐free media and subsequently stimulated as indicated for at least 3 h. Correct subcellular fractionation was verified with H3 and tubulin antibodies.


( C ) Effects of ERK mTOR inhibitors on TFEB translocation . TFEB - GFP HeLa were seeded in 96 ‐ well plates , cultured for 12 h , then treated with indicated concentrations of ERK inhibitor U0126 , or mTOR inhibitors Rapamycin , Torin 1 , Torin 2 . After 3 h at 37 ° C , were processed were acquired using OPERA automated confocal microscope ( Perkin ‐ Elmer ) . Scale bars represent 30 μm .

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(C) Effects of ERK and mTOR inhibitors on TFEB nuclear translocation. TFEB-GFP HeLa cells were seeded in 96‐well plates, cultured for 12 h, and then treated with the indicated concentrations of the ERK inhibitor U0126, or the mTOR inhibitors Rapamycin, Torin 1, and Torin 2. After 3 h at 37°C, cells were processed and images were acquired using the OPERA automated confocal microscope (Perkin‐Elmer). Scale bars represent 30 μm.


( D ) Dose - response curves of effects of ERK mTOR inhibitors on TFEBnuclear translocation . TFEB - GFPHeLa were seeded in 384 ‐ well plates , cultured for 12 h , treated with 10 different concentrations of ERK inhibitor U0126 , or mTOR inhibitors Rapamycin , Torin 1 , Torin 2 ranging from 2 . 54 nM to 50 μM . graph shows of translocation at different concentrations of each compound ( in log of ) . EC50 for each compound was calculated using Prism software ( see Materials methods for details ) .

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(D) Dose-response curves of the effects of ERK and mTOR inhibitors on TFEBnuclear translocation. TFEB-GFPHeLa cells were seeded in 384‐well plates, cultured for 12 h, and treated with 10 different concentrations of the ERK inhibitor U0126, or the mTOR inhibitors Rapamycin, Torin 1, and Torin 2 ranging from 2.54 nM to 50 μM. The graph shows the percentage of nuclear translocation at the different concentrations of each compound (in log of the concentration). The EC50 for each compound was calculated using Prism software (see Materials and methods for details).


( E ) Immunofluorescence of HEK ‐ 293T treated with DMSO or Torin 1 stained with antibodies against endogenous TFEB lysosomal RagC ( green red , respectively , in merge ) . DAPI is included in merge . Scale bars represent 10 μM .

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(E) Immunofluorescence of HEK‐293T cells treated with DMSO or Torin 1 and stained with antibodies against endogenous TFEB and the lysosomal protein RagC (green and red, respectively, in the merge). DAPI is included in the merge. Scale bars represent 10 μM.


( F ) Rag GTPase knockdown induces TFEB translocation . HeLa stably expressing TFEB - 3 × FLAG were infected with lentiviruses encoding Short hairpin ( Sh ‐ ) RNAs targeting luciferase ( control ) or RagC RagD mRNAs . In samples , 96 h post infection , were left untreated ( N = normal media ) , starved ( S = starved media ) or treated with Torin 1 ( T = Torin 1 ) for 4 h then subjected to / fractionation . TFEB localization was detected with a FLAG antibody , whereas tubulin H3 were used as controls for fraction , respectively ; of S6K phosphorylation were used to RagC RagD knockdown efficiency .

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(F) Rag GTPase knockdown induces TFEB nuclear translocation. HeLa cells stably expressing TFEB-3 × FLAG were infected with lentiviruses encoding Short hairpin (Sh‐) RNAs targeting luciferase (control) or RagC and RagD mRNAs. In all samples, 96 h post infection, cells were left untreated (N=normal media), starved (S=starved media) or treated with Torin 1 (T=Torin 1) for 4 h and then subjected to nuclear/cytosolic fractionation. TFEB localization was detected with a FLAG antibody, whereas tubulin and H3 were used as controls for the cytosolic and nuclear fraction, respectively; levels of S6K phosphorylation were used to test RagC and RagD knockdown efficiency.


( G ) Loss of mTORC2 does not affect TFEB phosphorylation . Mouse embryonic fibroblasts ( MEFs ) isolated from Sin1 − / − or control ( E14 . 5 ) were infected with a retrovirus encoding TFEB - 3 × FLAG ; 48 h post infection , were treated with Torin 1 ( T ) for 4 h where indicated , subjected to / fractionation immunoblotted for FLAG , tubulin , H3 .

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(G) Loss of mTORC2 does not affect TFEB phosphorylation. Mouse embryonic fibroblasts (MEFs) isolated from Sin1−/− or control embryos (E14.5) were infected with a retrovirus encoding TFEB-3 × FLAG; 48 h post infection, cells were treated with Torin 1 (T) for 4 h where indicated, subjected to nuclear/cytosolic fractionation and immunoblotted for FLAG, tubulin, and H3.


( H ) Binding of TFEB to mTORC1 . HEK ‐ 293T that express TFEB - 3 × FLAG were lysed subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR , mTORC1 subunit raptor mTORC2 components rictor Sin1 . FLAG - Rap2A served as negative control .

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(H) Binding of TFEB to mTORC1. HEK‐293T cells that express TFEB-3 × FLAG were lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR, the mTORC1 subunit raptor and the mTORC2 components rictor and Sin1. FLAG-Rap2A served as negative control.


( A ) Torin 1 induces S142 dephosphorylation . HeLa were treated as indicated total extracts were probed with a TFEB p ‐ S142 phosphoantibody with anti ‐ FLAG antibody . Disappearance of TFEB S142 phosphorylation upon starvation or Torin 1 treatment correlates with accumulation of TFEB in fraction .

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(A) Torin 1 induces S142 dephosphorylation. HeLa cells were treated as indicated and total and nuclear extracts were probed with a TFEB p‐S142 phosphoantibody and with anti‐FLAG antibody. Disappearance of TFEB S142 phosphorylation upon starvation or Torin 1 treatment correlates with accumulation of TFEB in the nuclear fraction.


( B ) mTORC1 in ‐ vitro kinase assays . Highly purified FLAG - S6K1 , TFEB - 3 × FLAG , or TFEBS142A - 3 × FLAG were incubated with radiolabelled ATP without kinase , with purified mTORC1 or with mTORC1 + Torin 1 , analysed by autoradiography . lower panel shows a FLAG immunoblot of substrates .

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(B) mTORC1 in‐vitro kinase assays. Highly purified FLAG-S6K1, TFEB-3 × FLAG, or TFEBS142A-3 × FLAG were incubated with radiolabelled ATP without kinase, with purified mTORC1 or with mTORC1+Torin 1, and analysed by autoradiography. The lower panel shows a FLAG immunoblot of the substrates.


( D ) conservation scores of phosphosites quantitative agreement between mTOR consensus motif around phosphosites of TFEB .

(D) Sequence conservation scores of the phosphosites and quantitative agreement between mTOR consensus motif and the sequence around the phosphosites of TFEB.


( E ) S142 S211 regulate TFEB localization . FLAGimmunostaining ( red ) of HeLa expressing serine ‐ to ‐ alanine mutated versions of TFEB - 3 × FLAG . Nuclei were stained with DAPI ( blue ) . Values are means of five fields containing at least 50 transfected . Student ' s t ‐ ( unpaired ) * * * P0 . 001 . Scale bars represent 30 μm .

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(E) S142 and S211 regulate TFEB localization. FLAGimmunostaining (red) of HeLa cells expressing serine‐to‐alanine mutated versions of TFEB-3 × FLAG. Nuclei were stained with DAPI (blue). Values are means of five fields containing at least 50 transfected cells. Student's t‐test (unpaired) ***P0.001. Scale bars represent 30 μm.


( A ) Spinning disk confocal image of a MEF that co ‐ expresses TFEB - GFP mRFP - Rab7 ( green red in merge , respectively ) .

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(A) Spinning disk confocal image of a MEF cell that co‐expresses TFEB-GFP and mRFP-Rab7 (green and red in the merge, respectively).


( B ) Time ‐ lapse of TFEB ‐ Rab7 ‐ positive lysosomes from boxed region in ( A ) . Time intervals are in seconds .

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(B) Time‐lapse of TFEB‐ and Rab7‐positive lysosomes from the boxed region in (A). Time intervals are in seconds.


( C ) Time ‐ lapse of Torin 1 treatment in a MEF expressing TFEB - GFP . Arrow indicates time of Torin 1 addition . Yellow arrowheads indicate Torin 1 ‐ induced lysosomal accumulation of TFEB - GFP . Time intervals are in minutes .

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(C) Time‐lapse analysis of Torin 1 treatment in a MEF cell expressing TFEB-GFP. Arrow indicates the time of Torin 1 addition. Yellow arrowheads indicate Torin 1‐induced lysosomal accumulation of TFEB-GFP. Time intervals are in minutes.


( D ) Immunofluorescence of HEK ‐ 293T expressing TFEB - 3 × FLAG , treated with DMSO ( top ) or Torin1 ( bottom ) stained with antibodies against FLAG mTOR ( green red in merge , respectively ; DAPI is in blue ) .

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(D) Immunofluorescence of HEK‐293T cells expressing TFEB-3 × FLAG, treated with DMSO (top) or Torin1 (bottom) and stained with antibodies against FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue).


( E ) FRAP of TFEB - GFP ‐ positive lysosomes from control MEFs ( blue ) or MEFs treated with Torin 1 ( red ) . Each data point represents mean ± s . d . from five independent spots .

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(E) FRAP analysis of TFEB-GFP‐positive lysosomes from control MEFs (blue) or MEFs treated with Torin 1 (red). Each data point represents mean±s.d. from five independent spots.


( F ) Time ‐ lapse of photobleaching recovery of TFEB - GFP ‐ positive lysosomes from control ‐ treated MEFs ( top ) or MEFs treated with Torin 1 ( bottom ) . Red arrowheads indicate time of photobleaching . Time intervals are in seconds .

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(F) Time‐lapse of photobleaching and fluorescence recovery of TFEB-GFP‐positive lysosomes from control‐treated MEFs (top) or MEFs treated with Torin 1 (bottom). Red arrowheads indicate time of photobleaching. Time intervals are in seconds.


( G ) Torin 1 increases binding of TFEB to mTORC1 . HEK ‐ 293T that express TFEB - 3 × FLAG along with HAGST ‐ Rap2A or HAGST ‐ RagsDN were treated with vehicle or with Torin1 , lysed subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR raptor . FLAG - Metap2 served as negative control .

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(G) Torin 1 increases binding of TFEB to mTORC1. HEK‐293T cells that express TFEB-3 × FLAG along with HAGST‐Rap2A or HAGST‐RagsDN were treated with vehicle or with Torin1, lysed and subjected to FLAG immunoprecipitation followed by immunoblotting for mTOR and raptor. FLAG-Metap2 served as negative control.


( H ) Immunofluorescence of HEK ‐ 293T that express TFEB - 3 × FLAG along with Rap2A ( top ) or RagsDN mutants ( bottom ) , treated with Torin 1 stained with antibodies against FLAG LAMP2 ( green red in merge , respectively ; DAPI is in blue ) . In , scale bars represent 10 μm .

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(H) Immunofluorescence of HEK‐293T cells that express TFEB-3 × FLAG along with Rap2A (top) or the RagsDN mutants (bottom), treated with Torin 1 and stained with antibodies against FLAG and LAMP2 (green and red in the merge, respectively; DAPI is in blue). In all images, scale bars represent 10 μm.


( A - C ) Immunofluorescence of HEK ‐ 293T that express TFEB - 3 × FLAG along with a control GTPase or indicated Rag mutants . were deprived of ( top ) or deprived then stimulated ( bottom ) for indicated times stained for FLAG mTOR ( green red in merge , respectively ; DAPI is in blue ) .

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(A-C) Immunofluorescence of HEK‐293T cells that express TFEB-3 × FLAG along with a control GTPase or the indicated Rag mutants. Cells were deprived of amino acids (top) or deprived and then stimulated (bottom) for the indicated times and stained for FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue).


( D ) of number of with TFEB from each condition in ( A - C ) .

(D) Quantification of the number of cells with nuclear TFEB from each condition in (A-C).


( E , F ) Immunofluorescence of HEK ‐ 293T that express TFEB - 3 × FLAG along with Rap2A ( E ) or RagsCA mutants ( F ) , subjected to indicated treatments stained with antibodies against FLAG mTOR ( green red in merge , respectively ; DAPI is in blue ) .

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(E, F) Immunofluorescence of HEK‐293T cells that express TFEB-3 × FLAG along with Rap2A (E) or the RagsCA mutants (F), subjected to the indicated treatments and stained with antibodies against FLAG and mTOR (green and red in the merge, respectively; DAPI is in blue).


( G ) of number of with TFEB from DMSO ‐ CQ ‐ treated fields in ( E ) ( F ) . In fields , scale bars represent 10 μm . In histograms , each value represents mean ± s . d . from three independent fields with N = 300 .

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(G) Quantification of the number of cells with nuclear TFEB from DMSO‐ and CQ‐treated fields in (E) and (F). In all fields, scale bars represent 10 μm. In all histograms, each value represents mean±s.d. from three independent fields with N=300.


( A ) Chloroquine treatment inhibits mTORC1 activity in hepatocytes . hepatocytes isolated from 2 ‐ month ‐ old Tcfebflox / flox ( control ) Tcfebflox / flox ; Alb ‐ Cre ( Tcfeb − / − ) mice were left untreated , or treated overnight with Torin 1 , U0126 , or Chloroquine . Subsequently , were lysed extracts were immunoblotted with indicated antibodies .

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(A) Chloroquine treatment inhibits mTORC1 activity in primary hepatocytes. Primary hepatocytes isolated from 2‐month‐old Tcfebflox/flox (control) and Tcfebflox/flox;Alb‐Cre(Tcfeb−/−) mice were left untreated, or treated overnight with Torin 1, U0126, or Chloroquine. Subsequently, cells were lysed and protein extracts were immunoblotted with the indicated antibodies.


( B , C ) TFEB mediates transcriptional response to chloroquine Torin 1 . Quantitative PCR ( qPCR ) of TFEB target genes in hepatocytes from control ( flox / flox ) Tcfeb − / − mice . were treated with Chloroquine ( left ) or Torin 1 ( right ) . graphs show relative increased expression in treated versus corresponding untreated samples . Values represent means ± s . d . of three independent hepatocyte preparations ( three mice / genotype ) . Student ' s t ‐ ( two ) * P ‐ value ⩽ 0 . 05 .

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(B, C) TFEB mediates the transcriptional response to chloroquine and Torin 1. Quantitative PCR (qPCR) of TFEB target genes in primary hepatocytes from control (flox/flox) and Tcfeb−/− mice. Cells were treated with Chloroquine (left) or Torin 1 (right). The graphs show the relative increased expression in the treated versus the corresponding untreated samples. Values represent means±s.d. of three independent hepatocyte preparations (three mice/genotype). Student's t‐test (two tailed) *P‐value ⩽0.05.


( A - B ) of miR - 122 in Huh7 released EVs either untreated ( Fed ) or subjected to starvation for metabolites including for 16h ( Starved ) . miR - 122 signals were detected by Northern blotting position of 32P - labelled Oligos that served as markers are shown in lane ( A ) . U6 snRNA was used as loading control .

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(A-B) Levels of miR-122 in Huh7 cells and released EVs either untreated (Fed) or subjected to starvation for metabolites including amino acids for 16h (Starved). miR-122 signals were detected by Northern blotting and position of the 32P-labelled Oligos that served as size markers are shown in the lane (A). U6 snRNA was used as loading control.


( A - B ) of miR - 122 in Huh7 released EVs either untreated ( Fed ) or subjected to starvation for metabolites including for 16h ( Starved ) . CAT - 1 were measured by qRT - PCR using GAPDH mRNA as control ( B ) . A scheme of experiment is shown in top panel in B .

(A-B) Levels of miR-122 in Huh7 cells and released EVs either untreated (Fed) or subjected to starvation for metabolites including amino acids for 16h (Starved). Cellular CAT-1 levels were measured by qRT-PCR using GAPDH mRNA as control (B). A scheme of experiment is shown in the top panel in B.


( C - D ) extracellular of different miRNAs EV associated in Fed Starved Huh7 . Relative changes in extracellular ( EV associated ) of miR - 122 miR - 24 in Fed or 8h Starved Huh7 were quantified by qRT - PCR plotted ( mean + / - s . e . m . , n = 3 ) . of miRNAs in Fed condition were taken as unit . Fold change ( with SD ) in level of miRNAs pre - miR - 122 measured were shown in bottom panel ( C ) . miRNA were normalized against U6 snRNA .

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(C-D) Cellular and extracellular levels of different miRNAs and EV associated proteins in Fed and Starved Huh7 cells. Relative changes in cellular and extracellular levels (EV associated) of miR-122 and miR-24 in Fed or 8h Starved Huh7 cells were quantified by qRT-PCR and plotted (mean+/- s.e.m., n=3). Levels of individual miRNAs in Fed condition were taken as unit. Fold change (with SD) in the cellular level of all miRNAs and pre-miR-122 measured were shown in the bottom panel (C). Cellular miRNA levels were normalized against U6 snRNA.


( C - D ) extracellular of different miRNAs EV associated in Fed Starved Huh7 . Western blot of different exosomal Ago2 in EVs isolated under Fed or Starved conditions . CD63 is an exosomal marker of HuR in EVs were quantified against respective CD63 . Mean of three independent measurements are plotted ( middle panel in D ) . Calnexin GAPDH are not detected in EVs but visible in extract . EV associated HA - HuR for Fed Starved Huh7 expressing HA - HuR were also detected in bottom panel in D .

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(C-D) Cellular and extracellular levels of different miRNAs and EV associated proteins in Fed and Starved Huh7 cells. Western blot analysis of different exosomal proteins and Ago2 in EVs isolated under Fed or Starved conditions. CD63 is an exosomal marker protein and levels of HuR in EVs were quantified against respective CD63 levels. Mean of three independent measurements are plotted (middle panel in D). Calnexin and GAPDH are not detected in EVs but visible in the cellular extract. EV associated HA-HuR levels for Fed and Starved Huh7 cells expressing HA-HuR were also detected in the bottom panel in D.


( E ) Effect of GW4869 treatment on miRNA content in Fed Starved . of miRNAs were measured by real - time normalized against U6 snRNA . Mean data are from three independent experiments . DMSO treatment was used as control for GW4869 treated .

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(E) Effect of GW4869 treatment on cellular miRNA content in Fed and Starved cells. Levels of miRNAs were measured by real-time quantification and normalized against U6 snRNA. Mean data are from three independent experiments. DMSO treatment was used as control for GW4869 treated cells.


( F ) CAT - 1 Aldolase mRNA expression in DMSO or GW4869 treated starved Huh7 . qRT - PCR techniques was adopted for using 18S rRNA values for normalization ( mean + / - s . e . m . , n = 3 ) .

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(F) CAT-1 and Aldolase mRNA expression in DMSO or GW4869 treated starved Huh7 cells. qRT-PCR techniques was adopted for quantification using 18S rRNA values for normalization (mean +/- s.e.m., n=3).


( G ) Effect of siSMPD2 treatment on miR - 122 content of EVs from Fed Starved Huh7 . EVs from Fed Starved Huh7 , depleted for SMPD2 , were isolated miR - 122 content were measured by qRT - PCR normalized against content of EVs . Fold change in EV associated miR - 122 upon starvation both for control siSMPD2 treated were shown above respective bars .

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(G) Effect of siSMPD2 treatment on miR-122 content of EVs from Fed and Starved Huh7 cells. EVs from Fed and Starved Huh7 cells, depleted for SMPD2, were isolated and miR-122 content were measured by qRT-PCR and normalized against protein content of EVs. Fold change in EV associated miR-122 levels upon starvation both for control and siSMPD2 treated cells were shown above the respective bars.


( H ) Effect of siSMPD2 treatment on CAT - 1 mRNA content ( left panel ) miR - 122 ( right panel ) in Huh7 . RNAs , from Fed Starved Huh7 , depleted for SMPD2 , were isolated miR - 122 CAT - 1 mRNA contents were measured by qRT - PCR . miR - 122 contents were normalized against U6 snRNA . GAPDH mRNA was used for normalization of CAT - 1 mRNA ( mean + / - s . e . m . , n = 3 ) . miR - 122 for siCon treated ( Fed Starved ) were considered as units .

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(H) Effect of siSMPD2 treatment on CAT-1 mRNA content (left panel) and miR-122 levels (right panel) in Huh7 cells. RNAs, from Fed and Starved Huh7 cells, depleted for SMPD2, were isolated and miR-122 and CAT-1 mRNA contents were measured by qRT-PCR. miR-122 contents were normalized against U6 snRNA. GAPDH mRNA was used for normalization of CAT-1 mRNA levels (mean +/- s.e.m., n=3). miR-122 levels for siCon treated cells (Fed and Starved) were considered as units.