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

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

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


( D ) Results of in vivo plasmid - to - plasmid transposition 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 .

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 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

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 ) .

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(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 .

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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 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 .

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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 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 ) .

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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 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 .

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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.


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 .

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

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 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 .

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

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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 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 .

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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 , and KLF4 in PC3 - LN4 EDC3 KO - C60 cells , 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 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 .

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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 ) 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 , and analysed by autoradiography . The lower panel shows a FLAG immunoblot of the 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 ) Sequence conservation scores of the phosphosites and quantitative agreement between mTOR consensus motif and the sequence around the phosphosites of TFEB .

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


( 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 .

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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 cell that co ‐ expresses TFEB - GFP and mRFP - Rab7 ( green and red in the 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 ‐ and Rab7 ‐ positive lysosomes from the 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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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

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


( 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 ) .

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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 ) 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 ) 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 .

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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). 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 ) 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 .

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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 ) 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 ) 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 .

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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.


( I - J ) A schematic representation of the experiment to separate Extracellular Vesicles ( EVs ) on OptiPrep @ density gradient ( left panel ) . Densities of fractions 1 - 10 are plotted and a best - fit curve is drawn ( right upper panel ) . CD63 levels in individual fractions of Fed and Starved cells were detected by western blots to confirm the presence of exosomes of Fed and Straved cells ( right lower panel ) . Mean Ct values of miR - 122 in RNAs isolated from Exosome enriched ( 8 - 9 ) vs Non - exosomal ( 1 - 3 ) fractions were analyzed and plotted ( J ) ( mean + / - s . e . m . , n = 3 ) .

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(I-J) A schematic representation of the experiment to separate Extracellular Vesicles (EVs) on OptiPrep@ density gradient (left panel). Densities of fractions 1-10 are plotted and a best-fit curve is drawn (right upper panel). CD63 levels in individual fractions of Fed and Starved cells were detected by western blots to confirm the presence of exosomes of Fed and Straved cells (right lower panel). Mean Ct values of miR-122 in RNAs isolated from Exosome enriched (8-9) vs Non-exosomal (1-3) fractions were analyzed and plotted (J) (mean +/- s.e.m., n=3).


( K ) Effect of Starvation on apoptic cell numbers in Huh7 cells . Western Blot detection of apoptosis marker Cytochrome C in cellular and in EV associated fractions of Fed and Starved Huh 7 cells . β - Actin and CD63 are used as loading control for cellular and EV associated fraction respectively ( left panel ) . TUNEL positive cells in Fed and Starved Huh7 cells . TUNEL assays of Fed and Starved Huh 7 cells were performed and TUNEL positive cells were quantified . Fed , but DNase treated , cells were used as positive control ( + ve control ) ( right panel ) .

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(K) Effect of Starvation on apoptic cell numbers in Huh7 cells. Western Blot detection of apoptosis marker Cytochrome C in cellular and in EV associated fractions of Fed and Starved Huh 7 cells. β-Actin and CD63 are used as loading control for cellular and EV associated fraction respectively (left panel). TUNEL positive cells in Fed and Starved Huh7 cells. TUNEL assays of Fed and Starved Huh 7 cells were performed and TUNEL positive cells were quantified. Fed, but DNase treated, cells were used as positive control (+ve control) (right panel).


( L ) Effect of thapsigargain ( TG ) treatment of Huh7 cells on cellular and EV associated miR - 122 level . A schematic representation of the experiment ( upper panel ) . miR - 122 levels were measured by qRT - PCR in total cellular RNA and in EVs released by the treated cells . Values were normalized either against U6 snRNA or protein content of EVs ( mean + / - s . e . m . , n = 3 ) ( lower panels ) for cellular and EV associated RNA respectively .

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(L) Effect of thapsigargain (TG) treatment of Huh7 cells on cellular and EV associated miR-122 level. A schematic representation of the experiment (upper panel). miR-122 levels were measured by qRT-PCR in total cellular RNA and in EVs released by the treated cells. Values were normalized either against U6 snRNA or protein content of EVs (mean+/- s.e.m., n=3) (lower panels) for cellular and EV associated RNA respectively.


( M ) Effect of TG on cellular level of eIF2 - a and its phosphorylated form , and Ago2 and CD63 in EVs . b - Actin was used as loading control for cellular samples .

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(M) Effect of TG on cellular level of eIF2-a and its phosphorylated form, and Ago2 and CD63 in EVs. b-Actin was used as loading control for cellular samples.


( A ) Relative amount of miRNAs bound to HuR that are immunoprecipitated either from Fed or Starved Huh7 cells . Immunoprecipitated materials were separated into two equal halves to do western blot analysis of HuR and relative quantification of associated miR - 122 and miR - 21 were done by quantitative RT - PCR ( mean + / - s . e . m . , n = 3 ) . GFP used as negative control to confer the specificity of the anti - HuR3A2 antibody in immunoprecipitating HuR . The miR - 122 content in the GFP - antibody immunoprecipitated materials was too low to get detected reliably using the qRT - PCR .

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(A) Relative amount of miRNAs bound to HuR that are immunoprecipitated either from Fed or Starved Huh7 cells. Immunoprecipitated materials were separated into two equal halves to do western blot analysis of HuR and relative quantification of associated miR-122 and miR-21 were done by quantitative RT-PCR (mean +/- s.e.m., n=3). GFP used as negative control to confer the specificity of the anti-HuR3A2 antibody in immunoprecipitating HuR. The miR-122 content in the GFP-antibody immunoprecipitated materials was too low to get detected reliably using the qRT-PCR.


( B - C ) Effect of HuR depletion on cellular and EV content of miRNAs in Huh7 cells . Schemes of HuR depletion experiments in Huh7 cells ( B ) . In siHuR treated cells , depletion of HuR was monitored by western blotting ( mean + / - s . e . m . , n = 4 )

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(B-C) Effect of HuR depletion on cellular and EV content of miRNAs in Huh7 cells. Schemes of HuR depletion experiments in Huh7 cells (B). In siHuR treated cells, depletion of HuR was monitored by western blotting (mean+/- s.e.m., n=4)


( B - C ) Effect of HuR depletion on cellular and EV content of miRNAs in Huh7 cells . Relative levels of miRNAs were measured in EVs released from siCon or siHuR treated cells by qRT - PCR . Cellular miRNA contents were normalized against U6 snRNA . Levels of each miRNA in control siRNA ( siCon ) treated set were taken as unit . ( C ) . CAT - 1 and Aldolase mRNA levels of siHuR and siCon treated Fed Huh7 cells were quantified by qRT - PCR and normalized against GAPDH mRNA .

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(B-C) Effect of HuR depletion on cellular and EV content of miRNAs in Huh7 cells. Relative levels of miRNAs were measured in EVs released from siCon or siHuR treated cells by qRT-PCR. Cellular miRNA contents were normalized against U6 snRNA. Levels of each miRNA in control siRNA (siCon) treated set were taken as unit. (C). CAT-1 and Aldolase mRNA levels of siHuR and siCon treated Fed Huh7 cells were quantified by qRT-PCR and normalized against GAPDH mRNA.