( A ) BN - PAGE and SDS - PAGE of HeLa + HA - Parkin cells treated with 1 µM of each of ABT - 737 and S63485 for 3 h ( BH3 ) , antimycin A and oligomycin ( AO ) for 2 h or a combination of the two . Experiment was performed in the presence of 10 µM QVD . oph and immunoblots are representative of three independent experiments .

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(A) BN-PAGE and SDS-PAGE of HeLa+HA-Parkin cells treated with 1 µM of each of ABT-737 and S63485 for 3 h (BH3), antimycin A and oligomycin (AO) for 2 h or a combination of the two. Experiment was performed in the presence of 10 µM QVD.oph and immunoblots are representative of three independent experiments.


( B ) UBA enrichment of ubiquitinated proteins from HeLa + HA - Parkin cells , BAX - / - BAK - / - ( DKO ) HeLa cells or wild - type ( WT ) HeLa cells expressing HA - Parkin in response to 1 µM of each of ABT - 737 and S63485 for 3 h ( BH3 ) in the presence of QVD . oph ( 10 μM ) . Representative of three independent experiments .

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(B) UBA enrichment of ubiquitinated proteins from HeLa+HA-Parkin cells, BAX-/- BAK-/- (DKO) HeLa cells or wild-type (WT) HeLa cells expressing HA-Parkin in response to 1 µM of each of ABT-737 and S63485 for 3 h (BH3) in the presence of QVD.oph (10 μM). Representative of three independent experiments.


( C ) HeLa cells expressing HA - Parkin and Cas9 were transduced with sgRNA targeting PINK1 or a non - targeting sgRNA control , treated with AO ( 2 h ) to stabilise PINK1 expression and immunoblotted for PINK1 .

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(C) HeLa cells expressing HA-Parkin and Cas9 were transduced with sgRNA targeting PINK1 or a non-targeting sgRNA control, treated with AO (2 h) to stabilise PINK1 expression and immunoblotted for PINK1.


( D ) Parkin activity induced by apoptotic mitochondrial damage is PINK1 - dependent . UBA enrichment of ubiquitinated proteins from HeLa cells generated in response to 1 µM of each of ABT - 737 and S63485 for 3 h ( BH3 ) in the presence of Q - VD . oph ( 10 μM ) . * , ubiquitinated protein . Representative of two independent experiments .

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(D) Parkin activity induced by apoptotic mitochondrial damage is PINK1-dependent. UBA enrichment of ubiquitinated proteins from HeLa cells generated in response to 1 µM of each of ABT-737 and S63485 for 3 h (BH3) in the presence of Q-VD.oph (10 μM). *, ubiquitinated protein. Representative of two independent experiments.


( B ) Non - reducing SDS - PAGE and immunoblot of recombinant BAK and BAK - Ub following treatment with 25 mM

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(B) Non-reducing SDS-PAGE and immunoblot of recombinant BAK and BAK-Ub following treatment with 25 mM TCEP


( C ) Timecourse monitoring fluorescence increase on liposomes following addition of 4 µM BID - BH3 peptide to 50 nM BAK or BAK - Ub in the presence or absence of 25 mM TCEP . Data is mean + / - SD of three independent experiments .

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(C) Timecourse monitoring fluorescence increase on liposomes following addition of 4 µM BID-BH3 peptide to 50 nM BAK or BAK-Ub in the presence or absence of 25 mM TCEP. Data is mean +/- SD of three independent experiments.


( D ) BN - PAGE of BAK and BAK - Ub on liposomes following incubation with 4 µM BID - BH3 peptide in the presence or absence of 25 mM TCEP , immunoblotted for BAK . Data representative of three independent experiments . Graphs shows densitometric analysis of the BAK tetramer ( 4x ) from three or two independent experiments . Error bars represent mean + / - SD for n = 3 or mean and range for n = 2 .

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(D) BN-PAGE of BAK and BAK-Ub on liposomes following incubation with 4 µM BID-BH3 peptide in the presence or absence of 25 mM TCEP, immunoblotted for BAK. Data representative of three independent experiments. Graphs shows densitometric analysis of the BAK tetramer (4x) from three or two independent experiments. Error bars represent mean +/- SD for n=3 or mean and range for n=2.


( A ) Immunoblot of whole cell lysates of MEFs stably expressing ΔCys variants of BAK .

(A) Immunoblot of whole cell lysates of MEFs stably expressing ΔCys variants of BAK.


( B ) Conjugation of ubiquitin to BAK K113C impairs BAK apoptotic function . Mitochondrially - enriched membranes from MEFs expressing BAK or BAK K113C were cross - linked ( CuPhe ) with UbG76C and treated with cBID for 30 min at 30 ° C . Supernatant ( S ) and pellet ( P ) fractions were then immunoblotted for BAK under non - reducing conditions , or cytochrome c or ubiquitin under reducing conditions . Representative of three independent experiments . Note that the incorporation of recombinant UbG76C on higher molecular weight proteins is likely due to incomplete reduction on reducing SDS - PAGE .

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(B) Conjugation of ubiquitin to BAK K113C impairs BAK apoptotic function. Mitochondrially-enriched membranes from MEFs expressing BAK or BAK K113C were cross-linked (CuPhe) with UbG76C and treated with cBID for 30 min at 30°C. Supernatant (S) and pellet (P) fractions were then immunoblotted for BAK under non-reducing conditions, or cytochrome c or ubiquitin under reducing conditions. Representative of three independent experiments. Note that the incorporation of recombinant UbG76C on higher molecular weight proteins is likely due to incomplete reduction on reducing SDS-PAGE.


( C ) Parkin - mediated ubiquitination limits BAK activation . HeLa + HA - Parkin cells were treated with antimycin A and oligomycin ( AO ) for 2 h prior to induction of apoptosis with 1 µM of each of the BH3 mimetics ABT - 737 and S63485 ( BH3 mim ) for 1 h . Intracellular flow cytometry was performed using the conformation - specific anti - BAK antibody G317 - 2 . Representative histograms are shown and the fold increase in mean fluorescence intensity ( MFI ) is plotted from three independent experiments with error bars showing SD . Data are normalised to untreated control .

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(C) Parkin-mediated ubiquitination limits BAK activation. HeLa+HA-Parkin cells were treated with antimycin A and oligomycin (AO) for 2 h prior to induction of apoptosis with 1 µM of each of the BH3 mimetics ABT-737 and S63485 (BH3 mim) for 1 h. Intracellular flow cytometry was performed using the conformation-specific anti-BAK antibody G317-2. Representative histograms are shown and the fold increase in mean fluorescence intensity (MFI) is plotted from three independent experiments with error bars showing SD. Data are normalised to untreated control.


( D ) Parkin - mediated ubiquitination limits BAK apoptotic activity . BAK - / - BAX - / - HeLa cells expressing Parkin and BAK were treated with AO ( 2 h ) prior to isolation of mitochondria and treatment with recombinant cBID and analysis of cytochrome c release . Immunoblots are representative of three independent experiments . Graph shows densitometric analysis of three independent experiments or cytochrome c in the supernatant fraction . Error bars represent mean + / - SD .

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(D) Parkin-mediated ubiquitination limits BAK apoptotic activity. BAK-/-BAX-/- HeLa cells expressing Parkin and BAK were treated with AO (2 h) prior to isolation of mitochondria and treatment with recombinant cBID and analysis of cytochrome c release. Immunoblots are representative of three independent experiments. Graph shows densitometric analysis of three independent experiments or cytochrome c in the supernatant fraction. Error bars represent mean +/- SD.


( E ) Parkin activity limits cytochrome c release . HeLa cells or HeLa cells expressing Parkin were treated and analysed Immunoblots are representative of three independent experiments . Ubiquitinated BAK indicated ( arrow ) . Graph shows densitometric analysis of three independent experiments showing cytochrome c release ( supernatant / supernatant + membrane ) relative to 100 nM cBID as 100 % release . Error bars represent mean + / - SD .

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(E) Parkin activity limits cytochrome c release. HeLa cells or HeLa cells expressing Parkin were treated and analysed Immunoblots are representative of three independent experiments. Ubiquitinated BAK indicated (arrow). Graph shows densitometric analysis of three independent experiments showing cytochrome c release (supernatant/supernatant+membrane) relative to 100 nM cBID as 100% release. Error bars represent mean +/- SD.


( D ) A fluorescence anisotropy experiment demonstrated direct binding of the Pxp3 motif with purified Pex5 protein ( Kd = 9 . 5 + / - 2 . 9 µM ) , in strength similar to the binding of the known PTS1 motif from Idp3 ( Kd = 5 . 5 + / - 0 . 5 µM ) . The plots represent the mean of three independent experiments , and the error bars represent the standard deviation .

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(D) A fluorescence anisotropy experiment demonstrated direct binding of the Pxp3 motif with purified Pex5 protein (Kd = 9.5 +/-2.9 µM), in strength similar to the binding of the known PTS1 motif from Idp3 (Kd = 5.5 +/- 0.5 µM). The plots represent the mean of three independent experiments, and the error bars represent the standard deviation.


( F ) Subcellular fractionations followed by enzymatic activity assays show that untagged , native , Glr1 is active in peroxisomes , in addition to its known activity in mitochondria . Fum1 and Pox2 were used as markers for mitochondrial , and peroxisomal fractions , respectively .

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(F) Subcellular fractionations followed by enzymatic activity assays show that untagged, native, Glr1 is active in peroxisomes, in addition to its known activity in mitochondria. Fum1 and Pox2 were used as markers for mitochondrial, and peroxisomal fractions, respectively.


( H ) Yeast - 2 - hybrid assay shows Glr1 interacts in vivo with both Pex5 full - length and Pex5 N ' domain .

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(H) Yeast-2-hybrid assay shows Glr1 interacts in vivo with both Pex5 full-length and Pex5 N' domain.


( B ) Metabolomic analysis focused on strains with over - expression or deletion of the AYT1 gene shows a significant reduction or accumulation , respectively , in acetyl - glutamate compared to the matched control strain of each mutant . Only metabolites changing significantly in at least one of the two conditions are presented . D - EIP is D - erythro - 1 - ( Imidazole - 4 - yl ) glycerol 3 - phosphate .

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(B) Metabolomic analysis focused on strains with over-expression or deletion of the AYT1 gene shows a significant reduction or accumulation, respectively, in acetyl-glutamate compared to the matched control strain of each mutant. Only metabolites changing significantly in at least one of the two conditions are presented. D-EIP is D-erythro-1-(Imidazole-4-yl) glycerol 3-phosphate.


( D ) A growth assay in a condition that does not induce high levels of acetyl - CoA in peroxisomes ( glucose ) shows that all strains grow similarly to the control until stationary phase when peroxisomes become vital . In stationary phase , the over - expression ( OE ) of AYT1 grows to a lower density than the control strain , plausibly due to burdening of peroxisomal functions . ( E ) A growth assay in a condition expected to elevate acetyl - glutamate levels in peroxisomes ( oleate as a sole carbon source and glutamate as a nitrogen source ) demonstrates that the AYT1 overexpressing strain grows faster and to a higher density than the control . Data information : The growth assays in D and E were done in three biologically independent replicates and error bars were plotted . Note that some error bars are shorter than the symbol , hence are not visible in the graph .

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(D) A growth assay in a condition that does not induce high levels of acetyl-CoA in peroxisomes (glucose) shows that all strains grow similarly to the control until stationary phase when peroxisomes become vital. In stationary phase, the over-expression (OE) of AYT1 grows to a lower density than the control strain, plausibly due to burdening of peroxisomal functions. (E) A growth assay in a condition expected to elevate acetyl-glutamate levels in peroxisomes (oleate as a sole carbon source and glutamate as a nitrogen source) demonstrates that the AYT1 overexpressing strain grows faster and to a higher density than the control. Data information: The growth assays in D and E were done in three biologically independent replicates and error bars were plotted. Note that some error bars are shorter than the symbol, hence are not visible in the graph.


( G ) A β - oxidation activity assay of Δlpx1 , Δfsh3 , and Δlpx1Δfsh3 strains supplemented with labeled 8 carbon - or 18 carbon - free fatty acids in media supplemented with 0 . 5 % glucose shows a significant reduction in β - oxidation activity compared to the wild type and the two single mutants , suggesting an overlapping role for Fsh3 with Lpx1 . This assay was done in three biologically independent replicates . Bars represent the mean and error bars represent the standard deviation .

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(G) A β-oxidation activity assay of Δlpx1, Δfsh3, and Δlpx1Δfsh3 strains supplemented with labeled 8 carbon- or 18 carbon-free fatty acids in media supplemented with 0.5% glucose shows a significant reduction in β-oxidation activity compared to the wild type and the two single mutants, suggesting an overlapping role for Fsh3 with Lpx1.This assay was done in three biologically independent replicates. Bars represent the mean and error bars represent the standard deviation.


( E ) Western blot analysis of Fbp1 - HA levels in mutants Δpex3 ( no peroxisomes ) , Δpex5 ( abolished targeting of GID subunits to peroxisomes ) , and Δpex7 ( no β - oxidation due to loss of Pot1 targeting ) demonstrates that two hours after the transition from glucose to ethanol ( gluconeogenic conditions ) , control and Δpex7 cells upregulated Fbp1 - HA levels , while for Δpex3 and Δpex5 cells Fbp1 - HA levels remain low . Antibodies were used against the HA tag ( for Fbp1 ) and Actin as a loading control .

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(E) Western blot analysis of Fbp1-HA levels in mutants Δpex3 (no peroxisomes), Δpex5 (abolished targeting of GID subunits to peroxisomes), and Δpex7 (no β-oxidation due to loss of Pot1 targeting) demonstrates that two hours after the transition from glucose to ethanol (gluconeogenic conditions), control and Δpex7 cells upregulated Fbp1-HA levels, while for Δpex3 and Δpex5 cells Fbp1-HA levels remain low. Antibodies were used against the HA tag (for Fbp1) and Actin as a loading control.


A . Volcano plots of genes that are differentially expressed in infected cells relative to mock - infected cells at 12 hpi and 24 hpi in ileum organoids . The statistical significance ( - log10 of the adjusted p - value ) is shown as a function of the log2 fold change . MAST tests were used to generate P ‐ values , bonferroni multiple hypotheses correction was used to compute FDR values . Labeled dots in all panels are gene names of selected differentially expressed genes between the compared two populations .

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A. Volcano plots of genes that are differentially expressed in infected cells relative to mock-infected cells at 12 hpi and 24 hpi in ileum organoids. The statistical significance (-log10 of the adjusted p-value) is shown as a function of the log2 fold change. MAST tests were used to generate P‐values, bonferroni multiple hypotheses correction was used to compute FDR values. Labeled dots in all panels are gene names of selected differentially expressed genes between the compared two populations.


F . Multiplex in situ RNA hybridization of ACE2 and SARS - CoV - 2 of mock - infected and infected 2D ileum organoids at 12 hpi and 24 hpi . White arrows point at SARS - CoV - 2 - infected cells . A representative image is shown .

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F. Multiplex in situ RNA hybridization of ACE2 and SARS-CoV-2 of mock-infected and infected 2D ileum organoids at 12 hpi and 24 hpi. White arrows point at SARS-CoV-2-infected cells. A representative image is shown.


Volcano plots displaying the genes that are differentially expressed in immature enterocytes 2 upon SARS - CoV - 2 infection of colon organoids . infected vs . mock infected cells The statistical significance ( - log10 adjusted p - value ) is shown as a function of the log2 fold change . MAST tests were used to generate P ‐ values , bonferroni multiple hypotheses correction was used to compute FDR values . Labeled dots in blue in all panels are gene names of selected differentially expressed genes between the compared two populations . Labeled dots in red in all panels are gene corresponding to interferon if detected .

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Volcano plots displaying the genes that are differentially expressed in immature enterocytes 2 upon SARS-CoV-2 infection of colon organoids. infected vs. mock infected cells The statistical significance (-log10 adjusted p-value) is shown as a function of the log2 fold change. MAST tests were used to generate P‐values, bonferroni multiple hypotheses correction was used to compute FDR values. Labeled dots in blue in all panels are gene names of selected differentially expressed genes between the compared two populations. Labeled dots in red in all panels are gene corresponding to interferon if detected.


A . Human ileum - derived organoids were seeded in 2D on iBIDi chamber slides . 12 and 24 hpi cells were fixed and the amount of SARS - CoV - 2 infected cells ( red ) and the induction of ISG15 ( white ) was analyzed by single molecule RNA FISH . Nuclei were visualized with DAPI ( blue ) . White arrows indicate SARS - CoV - 2 positive cells . N = 3 biological samples . Representative image is shown . Scale bar = 100 um .

A. Human ileum-derived organoids were seeded in 2D on iBIDi chamber slides. 12 and 24 hpi cells were fixed and the amount of SARS-CoV-2 infected cells (red) and the induction of ISG15 (white) was analyzed by single molecule RNA FISH. Nuclei were visualized with DAPI (blue). White arrows indicate SARS-CoV-2 positive cells. N=3 biological samples. Representative image is shown. Scale bar=100 um.


D . T84 IRF3 knock - out cells were infected with SARS - CoV - 2 or human astrovirus 1 at an MOI = 3 . 24 hpi , cells were incubated in the presence or absence of 2000 IU / mL of IFNβ1 . 12 h post - treatment RNA was harvested and the induction of ISG15 was analyzed by q - RT - PCR and normalized to the housekeeping gene TBP . N = 3 biological replicates . Error bar indicates standard deviation . Statistics were determined by unpaired t - test .

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D. T84 IRF3 knock-out cells were infected with SARS-CoV-2 or human astrovirus 1 at an MOI=3. 24 hpi, cells were incubated in the presence or absence of 2000 IU/mL of IFNβ1. 12 h post-treatment RNA was harvested and the induction of ISG15 was analyzed by q-RT-PCR and normalized to the housekeeping gene TBP. N=3 biological replicates. Error bar indicates standard deviation. Statistics were determined by unpaired t-test.


E . T84 IRF3 knock - out cells were infected with SARS - CoV - 2 or human astrovirus 1 at an MOI = 3 . 24 hpi , cells were fixed and stained for either the SARS - CoV - 2 N protein or for human astrovirus 1 ( HAstV1 ) capsid protein . N = 3 biological replicates . Representative image is shown . Scale bar = 100 um .

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E. T84 IRF3 knock-out cells were infected with SARS-CoV-2 or human astrovirus 1 at an MOI=3. 24 hpi, cells were fixed and stained for either the SARS-CoV-2 N protein or for human astrovirus 1 (HAstV1) capsid protein. N=3 biological replicates. Representative image is shown. Scale bar=100 um.


G . T84 MX - 1 mcherry were infected with SARS - CoV - 2 at an MOI = 3 or MOI = 1 . 24 hpi cells were incubated in the presence or absence of 2000 IU / mL of IFNβ1 . 12 h post - treatment cells were fixed and stained for SARS - CoV - 2 N protein . The number of MX - 1 positive , SARS - CoV - 2 positive and double positive cells was quantified . N = 3 biological replicates were performed . Error bar indicates standard deviation .

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G. T84 MX-1 mcherry were infected with SARS-CoV-2 at an MOI=3 or MOI=1. 24 hpi cells were incubated in the presence or absence of 2000 IU/mL of IFNβ1. 12 h post-treatment cells were fixed and stained for SARS-CoV-2 N protein. The number of MX-1 positive, SARS-CoV-2 positive and double positive cells was quantified. N=3 biological replicates were performed. Error bar indicates standard deviation.


( A ) Confocal microscopy of GFP - TRIM33 - overexpressing mESCs and differentiated cells . Scale bar = 5 µm . ( B ) Line scans of GFP - TRIM33 and DAPI fluorescence imaging in each cell context at the position depicted by the white line ( from left to right ) . These line scans were processed by plot profile using image J software . ( C ) FRAP assays showing recovery time of normalized fluorescence signals for GFP - TRIM33 puncta in mESCs . Photobleaching occurs at t = 5s . Data are the mean ± S . D . of three biological experiments .

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(A) Confocal microscopy of GFP-TRIM33-overexpressing mESCs and differentiated cells. Scale bar = 5 µm. (B) Line scans of GFP-TRIM33 and DAPI fluorescence imaging in each cell context at the position depicted by the white line (from left to right). These line scans were processed by plot profile using image J software. (C) FRAP assays showing recovery time of normalized fluorescence signals for GFP-TRIM33 puncta in mESCs. Photobleaching occurs at t = 5s. Data are the mean ± S.D. of three biological experiments.


( D ) Sphericity analysis and 3D imaging by NIS - Elements 3D tools of TRIM33 puncta in live mESCs . Panels show the xz , xy , and yz planes , from left to right . Scale bar = 1 µm . Data information : All data are from three biological replicates .

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(D) Sphericity analysis and 3D imaging by NIS-Elements 3D tools of TRIM33 puncta in live mESCs. Panels show the xz, xy, and yz planes, from left to right. Scale bar = 1 µm. Data information: All data are from three biological replicates.


( F ) Western blot analysis of TRIM33 interactions with PML in lysates of HEK293T cells co - transfected with plasmids encoding Flag - PML and GFP - TRIM33 . GFP - TRIM33 was immunoprecipitated with anti - GFP affinity beads and immune complexes were detected using antibodies targeting Flag and GFP . Protein inputs were detected using antibodies against Flag and GFP in the same amount of cell lysates . ( n = 3 ) . Data information : All data are from three biological replicates .

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(F) Western blot analysis of TRIM33 interactions with PML in lysates of HEK293T cells co-transfected with plasmids encoding Flag-PML and GFP-TRIM33. GFP-TRIM33 was immunoprecipitated with anti-GFP affinity beads and immune complexes were detected using antibodies targeting Flag and GFP. Protein inputs were detected using antibodies against Flag and GFP in the same amount of cell lysates. (n = 3). Data information: All data are from three biological replicates.


( G ) Lysates from mESCs and differentiated cells were immunoprecipitated with anti - PML antibody , and protein complexes and inputs were analyzed by immunoblotting with antibodies against TRIM33 and PML . ( n = 3 ) . Data information : All data are from three biological replicates .

(G) Lysates from mESCs and differentiated cells were immunoprecipitated with anti-PML antibody, and protein complexes and inputs were analyzed by immunoblotting with antibodies against TRIM33 and PML. (n = 3). Data information: All data are from three biological replicates.


( H ) Lysates from HEK293T cells co - transfected with plasmids encoding either full length Flag - TRIM33 or the Flag - TRIM33 - RBCC domain alone and full - length HA - PML or the HA - PML - RBCC domain alone immunoprecipitated with anti - Flag affinity beads . Protein complexes were detected using antibodies against Flag and HA . The protein inputs were detected using antibodies against Flag and HA in the same amount of cell lysates . ( n = 3 ) . Data information : All data are from three biological replicates .

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

(H) Lysates from HEK293T cells co-transfected with plasmids encoding either full length Flag-TRIM33 or the Flag-TRIM33-RBCC domain alone and full-length HA-PML or the HA-PML-RBCC domain alone immunoprecipitated with anti-Flag affinity beads. Protein complexes were detected using antibodies against Flag and HA. The protein inputs were detected using antibodies against Flag and HA in the same amount of cell lysates. (n = 3). Data information: All data are from three biological replicates.


( A ) Representative images of immunofluorescence staining of PML in WT and Pml null mESCs . Scale bar = 5 µm .

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 images of immunofluorescence staining of PML in WT and Pml null mESCs. Scale bar = 5 µm.


( B ) Representative confocal microscopy images of mESCs overexpressing mCherry - PML or GFP - PML ( top ) . Representative confocal microscopy images of mESCs overexpressing mCherry - EV or GFP - EV ( bottom ) . Scale bar = 5 µm . ( C ) FRAP assays quantifying normalized fluorescence recovery of GFP - PML signal in mESCs . Photobleaching occurs at t = 5s . Data are the mean ± S . D . of four biological experiments .

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

(B) Representative confocal microscopy images of mESCs overexpressing mCherry-PML or GFP-PML (top). Representative confocal microscopy images of mESCs overexpressing mCherry-EV or GFP-EV (bottom). Scale bar = 5 µm. (C) FRAP assays quantifying normalized fluorescence recovery of GFP-PML signal in mESCs. Photobleaching occurs at t = 5s. Data are the mean ± S.D. of four biological experiments.


( D , G , I , Representative images of co - localization assays with mCherry - PML and GFP - TRIM33 in mESCs ( D ) , differentiated cells ( G ) , NaAsO2 - treated mESCs ( 200 µM for 2 hr ) ( I ) Scale bar = 5 µm . ( E , H , J Line scans of GFP - TRIM33 and mCherry - PML fluorescence imaging in each cell context at the position depicted by the white line ( from left to right ) . These line scans were processed by plot profile using image J software . ( F ) Column graph of the numbers of GFP - TRIM33 puncta and mCherry - PML NBs in mESCs .

O O O O O O O O O O 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

(D, G, I, Representative images of co-localization assays with mCherry-PML and GFP-TRIM33 in mESCs (D), differentiated cells (G), NaAsO2-treated mESCs (200 µM for 2 hr) (I) Scale bar = 5 µm. (E, H, J Line scans of GFP-TRIM33 and mCherry-PML fluorescence imaging in each cell context at the position depicted by the white line (from left to right). These line scans were processed by plot profile using image J software. (F) Column graph of the numbers of GFP-TRIM33 puncta and mCherry-PML NBs in mESCs.


K ) Representative images of co - localization assays with GFP - TRIM33 in mESCs Pml null ( K ) . Scale bar = 5 µm . L ) Line scans of GFP - TRIM33 fluorescence imaging in each cell context at the position depicted by the white line ( from left to right ) . These line scans were processed by plot profile using image J software . ( M ) Column graph of the numbers of GFP - TRIM33 puncta and DAPI in mESCs .

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

K) Representative images of co-localization assays with GFP-TRIM33 in mESCs Pml null (K). Scale bar = 5 µm. L) Line scans of GFP-TRIM33 fluorescence imaging in each cell context at the position depicted by the white line (from left to right). These line scans were processed by plot profile using image J software. (M) Column graph of the numbers of GFP-TRIM33 puncta and DAPI in mESCs.


( N ) Scatter plot of the numbers of GFP - TRIM33 puncta ( per cell ) in WT and Pml KO mESCs . ( n = 20 ) . ( O ) Scatter plot of the volume of GFP - TRIM33 puncta ( per cell ) in WT and Pml KO mESCs . ( n = 20 ) . Data information : In panels ( N , O , 20 individual puncta with three biological experiments were analyzed . Significant differences were determined by t - tests . ( * p < 0 . 05 , * * * * p < 0 . 0001 ) .

(N) Scatter plot of the numbers of GFP-TRIM33 puncta (per cell) in WT and Pml KO mESCs. (n = 20). (O) Scatter plot of the volume of GFP-TRIM33 puncta (per cell) in WT and Pml KO mESCs. (n = 20). Data information: In panels (N, O, 20 individual puncta with three biological experiments were analyzed. Significant differences were determined by t-tests. (*p < 0.05, ****p < 0.0001).


( P ) Representative images of immunofluorescence staining of PML in WT and Trim33 null mESCs . Scale bar = 10 µm . ( Q ) Scatter plot of the numbers of PML NBs ( per cell ) in WT and Trim33 KO mESCs . ( n = 20 ) . ( R ) Scatter plot of the proportion of PML NBs ( per cell ) in WT and Trim33 KO mESCs . ( n = 20 ) . Data information : In panels Q and R ) , 20 individual puncta with three biological experiments were analyzed . Significant differences were determined by t - tests . ( * p < 0 . 05 , * * * * p < 0 . 0001 ) .

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

(P) Representative images of immunofluorescence staining of PML in WT and Trim33 null mESCs. Scale bar = 10 µm. (Q) Scatter plot of the numbers of PML NBs (per cell) in WT and Trim33 KO mESCs. (n = 20). (R) Scatter plot of the proportion of PML NBs (per cell) in WT and Trim33 KO mESCs. (n = 20). Data information: In panels Q and R), 20 individual puncta with three biological experiments were analyzed. Significant differences were determined by t-tests. (*p < 0.05, ****p < 0.0001).


( A ) Blood ammonia in 8 - 9 - week - old C57BL / 6J wild - type ( WT ) mice ( n = 8 ) and Becn1F121A mice with constitutive activation of autophagy ( n = 8 ) at baseline and 30 min after i . p . injection of NH4Cl ( 10 mmol / kg ) . * * p < 0 . 01 ( Unpaired t - test ) . ns : not statistically significant difference .

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 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

(A) Blood ammonia in 8-9-week-old C57BL/6J wild-type (WT) mice (n=8) and Becn1F121A mice with constitutive activation of autophagy (n=8) at baseline and 30 min after i.p. injection of NH4Cl (10 mmol/kg). **p<0.01 (Unpaired t-test). ns: not statistically significant difference.


( B ) Urinary orotic acid of 12 - week - old spf - ash mice treated with TB - 1 ( 15 mg / kg , i . p ) or vehicle at various times as indicated by the arrows ( n = 5 mice / group ) . * p < 0 . 05 ( Two - way ANOVA ) .

O O O O B-MEASURED_VAR I-MEASURED_VAR 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

(B) Urinary orotic acid of 12-week-old spf-ash mice treated with TB-1 (15 mg/kg, i.p) or vehicle at various times as indicated by the arrows (n=5 mice/group). *p<0.05 (Two-way ANOVA).


( C ; D ) Western blotting and densitometric quantifications of autophagy markers ( LC3II : autophagosomes ; p62 and NBR1 : cargo receptors ) in livers of spf - ash mice harvested after ten days of treatment with TB - 1 or vehicle . β - actin was used as loading control . n = 5 mice / group . * * p < 0 . 01 , * p < 0 . 05 ( Unpaired t - test ) .

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

(C; D) Western blotting and densitometric quantifications of autophagy markers (LC3II: autophagosomes; p62 and NBR1: cargo receptors) in livers of spf-ash mice harvested after ten days of treatment with TB-1 or vehicle. β-actin was used as loading control. n=5 mice/group. **p<0.01, *p<0.05 (Unpaired t-test).


( E ) Survival curves of spf - ash mice fed with a high protein diet ( HPD ) for ten days and treated with TB - 1 alone or combined with scavenger drug and L - Arginine , or treated with scavenger drug and L - Arginine , or left untreated . WT control were included ( n = 5 / group ) . * * * p < 0 . 001 ( Log - rank Mantel - Cox test ) . Data information : Treatments Scavenger ( Na - benzoate 250 mg / kg / day , i . p . ) and L - arginine ( L - Arg , 250 mg / kg / day , i . p . ) ; TB - 1 ( 15 mg / kg every 2 days , i . p ) . WT mice were age - , gender - and strain ( C3H ) - matched . All values are shown as averages ± S . E . M .

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

(E) Survival curves of spf-ash mice fed with a high protein diet (HPD) for ten days and treated with TB-1 alone or combined with scavenger drug and L-Arginine, or treated with scavenger drug and L-Arginine, or left untreated. WT control were included (n=5/group). ***p<0.001 (Log-rank Mantel-Cox test). Data information: Treatments Scavenger (Na-benzoate 250 mg/kg/day, i.p.) and L-arginine (L-Arg, 250 mg/kg/day, i.p.); TB-1 (15 mg/kg every 2 days, i.p). WT mice were age-, gender- and strain (C3H)-matched. All values are shown as averages ± S.E.M.


( F ) Blood ammonia levels determined after 4 days under HPD ( n = 5 mice / group ) ; * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( One - way ANOVA ) . Data information : Treatments Scavenger ( Na - benzoate 250 mg / kg / day , i . p . ) and L - arginine ( L - Arg , 250 mg / kg / day , i . p . ) ; TB - 1 ( 15 mg / kg every 2 days , i . p ) . WT mice were age - , gender - and strain ( C3H ) - matched . All values are shown as averages ± S . E . M .

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 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 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 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

(F) Blood ammonia levels determined after 4 days under HPD (n=5 mice/group); ***p<0.001, **p<0.01, *p<0.05 (One-way ANOVA). Data information: Treatments Scavenger (Na-benzoate 250 mg/kg/day, i.p.) and L-arginine (L-Arg, 250 mg/kg/day, i.p.); TB-1 (15 mg/kg every 2 days, i.p). WT mice were age-, gender- and strain (C3H)-matched. All values are shown as averages ± S.E.M.


( A ) Survival curves of AslNeo / Neo mice and age - matched wild - type ( WT ) controls treated with TB - 1 ( 15 mg / kg , i . p . , every 48 hours starting at day 10 of age ) or vehicle . WT + Vehicle n = 8 ; WT + TB - 1 n = 8 ; AslNeo / Neo + Vehicle n = 20 ; AslNeo / Neo + TB - 1 n = 16 . * p < 0 . 05 ( Log - rank Mantel - Cox test ) . Data information : All values are shown as averages ± S . E . M . ns : not statistically significant difference .

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 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 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

(A) Survival curves of AslNeo/Neo mice and age-matched wild-type (WT) controls treated with TB-1 (15 mg/kg, i.p., every 48 hours starting at day 10 of age) or vehicle. WT + Vehicle n=8; WT + TB-1 n=8; AslNeo/Neo + Vehicle n=20; AslNeo/Neo + TB-1 n=16. *p<0.05 (Log-rank Mantel-Cox test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


( B ) Isotopic enrichment of 15N - labeled urea in blood , 20 min after i . p . injection of 15NH4Cl tracer ( 4 mmol / kg ) in AslNeo / Neo mice treated with TB - 1 ( n = 15 ) or vehicle ( n = 9 ) . * p < 0 . 05 ( Unpaired t - test ) . Data information : All values are shown as averages ± S . E . M . ns : not statistically significant difference .

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 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

(B) Isotopic enrichment of 15N-labeled urea in blood, 20 min after i.p. injection of 15NH4Cl tracer (4 mmol/kg) in AslNeo/Neo mice treated with TB-1 (n=15) or vehicle (n=9). *p<0.05 (Unpaired t-test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


( C ) Representative Western blotting bands of LC3 , p62 and NBR1 in livers of AslNeo / Neo mice treated with TB - 1 or vehicle . GAPDH and β - actin were used as loading controls . ( D ) Densitometric quantifications . AslNeo / Neo + Vehicle n = 5 ; AslNeo / Neo + TB - 1 n = 9 . * * p < 0 . 01 , * p < 0 . 05 ( Unpaired t - test ) . Data information : All values are shown as averages ± S . E . M . ns : not statistically significant difference .

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 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

(C) Representative Western blotting bands of LC3, p62 and NBR1 in livers of AslNeo/Neo mice treated with TB-1 or vehicle. GAPDH and β-actin were used as loading controls. (D) Densitometric quantifications. AslNeo/Neo + Vehicle n=5; AslNeo/Neo + TB-1 n=9. **p<0.01, *p<0.05 (Unpaired t-test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


( E ) Argininosuccinate in dried blood spots ( DBS ) of WT and AslNeo / Neo mice injected with TB - 1 or vehicle ( n = 4 - 14 mice / group ) . * * * p < 0 . 001 , * p < 0 . 05 ( One - way ANOVA ) . Data information : All values are shown as averages ± S . E . M . ns : not statistically significant difference .

O O O B-MEASURED_VAR 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

(E) Argininosuccinate in dried blood spots (DBS) of WT and AslNeo/Neo mice injected with TB-1 or vehicle (n=4-14 mice/group). ***p<0.001, *p<0.05 (One-way ANOVA). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


( F ) Isotopic enrichment of 15N - labeled argininosuccinate in livers of AslNeo / Neo mice treated with TB - 1 ( n = 11 ) or vehicle ( n = 5 ) . * p < 0 . 05 ( Unpaired t - test ) . Data information : All values are shown as averages ± S . E . M . ns : not statistically significant difference .

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

(F) Isotopic enrichment of 15N-labeled argininosuccinate in livers of AslNeo/Neo mice treated with TB-1 (n=11) or vehicle (n=5). *p<0.05 (Unpaired t-test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


( A ) Hematoxylin and eosin ( H & E , upper panels ) and periodic acid Schiff ( PAS , lower panels ) staining of liver samples harvested from wild - type ( WT ) and AslNeo / Neo mice treated with TB - 1 or vehicle . Scale bars : 500 µm .

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

(A) Hematoxylin and eosin (H&E, upper panels) and periodic acid Schiff (PAS, lower panels) staining of liver samples harvested from wild-type (WT) and AslNeo/Neo mice treated with TB-1 or vehicle. Scale bars: 500 µm.


( B ) Computational analysis of PAS staining ( n ≥ 4 mice / group ) . * * p < 0 . 01 , * p < 0 . 05 ( One - way ANOVA ) . Data information : All values are shown as averages ± S . E . M .

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

(B) Computational analysis of PAS staining (n≥4 mice/group). **p<0.01, *p<0.05 (One-way ANOVA). Data information: All values are shown as averages ± S.E.M.


( C ) Quantification of hepatic glycogen in vehicle ( n = 6 ) - and TB - 1 - treated AslNeo / Neo mice ( n = 12 ) compared to WT ( n = 5 ) controls . * * p < 0 . 01 , * p < 0 . 05 ( One - way ANOVA ) . Data information : All values are shown as averages ± S . E . M .

O O O O O O B-MEASURED_VAR 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

(C) Quantification of hepatic glycogen in vehicle (n=6)- and TB-1-treated AslNeo/Neo mice (n=12) compared to WT (n=5) controls. **p<0.01, *p<0.05 (One-way ANOVA). Data information: All values are shown as averages ± S.E.M.


( D ) Representative Western blotting bands and densitometric quantification of PYGL in livers of WT and AslNeo / Neo mice treated with either with TB - 1 or vehicle ( n = 4 mice / group ) . * p < 0 . 05 ( One - way ANOVA ) . GAPDH was used as loading control . Data information : All values are shown as averages ± S . E . M .

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

(D) Representative Western blotting bands and densitometric quantification of PYGL in livers of WT and AslNeo/Neo mice treated with either with TB-1 or vehicle (n=4 mice/group). *p<0.05 (One-way ANOVA). GAPDH was used as loading control. Data information: All values are shown as averages ± S.E.M.


( E ) Representative electron microscopy images of liver samples harvested from WT and AslNeo / Neo mice treated with TB - 1 or vehicle . Scale bar : 900 nm .

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

(E) Representative electron microscopy images of liver samples harvested from WT and AslNeo/Neo mice treated with TB-1 or vehicle. Scale bar: 900 nm.


( A ) Representative electron microscopy images of liver samples harvested from wild - type ( WT ) and AslNeo / Neo mice treated with TB - 1 or vehicle . False color on the images indicates glycogen within the nuclei . Scale bar : 1 μm . ( B ) Quantification of nuclei containing glycogen ( approx . 180 nuclei were analyzed in total per condition , n = 3 mice / group ) .

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

(A) Representative electron microscopy images of liver samples harvested from wild-type (WT) and AslNeo/Neo mice treated with TB-1 or vehicle. False color on the images indicates glycogen within the nuclei. Scale bar: 1 μm. (B) Quantification of nuclei containing glycogen (approx. 180 nuclei were analyzed in total per condition, n=3 mice/group).


A . Tryptophan - fluorescence time course associated to Na + ( blue ) and K + ( black ) binding , respectively , to detergent - purified apo EAAT1CRYST at pH 7 . 4 . Horizontal and vertical scale bars represent time and ∆ F / F0 , respectively . B . Tryptophan - fluorescence time course associated to L - asp binding in presence of 1 mM ( blue ) and absence ( black ) of Na + , respectively , at pH 9 .

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

A. Tryptophan-fluorescence time course associated to Na+ (blue) and K+ (black) binding, respectively, to detergent-purified apo EAAT1CRYST at pH 7.4. Horizontal and vertical scale bars represent time and ∆F/F0, respectively. B. Tryptophan-fluorescence time course associated to L-asp binding in presence of 1 mM (blue) and absence (black) of Na+, respectively, at pH 9. C


C . Na + binding isotherms of EAAT1CRYST at pH 7 . 4 in the presence ( pink ) and absence ( blue ) of UPCH101 . Mutation D400380N at Na3 strongly impairs Na + binding ( green ) . Solid lines represent fits of Hill equation .

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 B-CONTROLLED_VAR 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

C. Na+ binding isotherms of EAAT1CRYST at pH 7.4 in the presence (pink) and absence (blue) of UPCH101. Mutation D400380N at Na3 strongly impairs Na+ binding (green). Solid lines represent fits of Hill equation.


A . Na + - induced L - glutamate uptake by purified EAAT1CRYST reconstituted in liposomes loaded with K + . Choline ( Ch + ) condition was subtracted to the Na + condition .

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

A. Na+-induced L-glutamate uptake by purified EAAT1CRYST reconstituted in liposomes loaded with K+. Choline (Ch+) condition was subtracted to the Na+ condition.


B . Log - log plots of Na + ( red , n = 19 ) and L - asp ( blue , n = 27 ) KD , as a function of pH at [ Na + ] = 0 . 5 mM . Na + ( n = 10 ) and L - asp ( n = 7 ) titration data at pH 7 . 4 and 9 . 0 , respectively , are also reported in Fig . 1 . Solid lines are fits of straight lines .

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B. Log-log plots of Na+ (red, n=19) and L-asp (blue, n=27) KD, as a function of pH at [Na+]=0.5 mM. Na+ (n=10) and L-asp (n=7) titration data at pH 7.4 and 9.0, respectively, are also reported in Fig. 1. Solid lines are fits of straight lines.


C . Log - log plots of L - asp KD for EAAT1CRYST - E406386Q ( light blue , n = 24 ) and EAAT1CRYST - Y405385F ( dark blue , n = 18 ) , as a function of pH at [ Na + ] = 0 . 5 mM . .

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C. Log-log plots of L-asp KD for EAAT1CRYST-E406386Q (light blue, n=24) and EAAT1CRYST-Y405385F (dark blue, n=18), as a function of pH at [Na+]=0.5 mM..


A . Purified EAAT1CRYST takes up L - glutamate in liposomes loaded with K + or Rb + , but not with choline ( Ch + ) , or with Ch + and Ca2 + . Plots depict data as in Fig . 3A .

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A. Purified EAAT1CRYST takes up L-glutamate in liposomes loaded with K+ or Rb+, but not with choline (Ch+), or with Ch+ and Ca2+. Plots depict data as in Fig. 3A.


B . Ca2 + binding isotherms of EAAT1CRYST at pH = 7 . 4 in the presence ( pink ) and absence ( blue ) of UPCH101 . Mutation D400380N at Na3 strongly impairs Ca2 + binding ( green ) . Symbols represent average and s . e . m . values of at least three independent experiments . Solid lines represent fits of Hill equation and dashed line is the fit in Fig . 1C ( blue line ) corresponding to Na + binding .

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B. Ca2+ binding isotherms of EAAT1CRYST at pH=7.4 in the presence (pink) and absence (blue) of UPCH101. Mutation D400380N at Na3 strongly impairs Ca2+ binding (green). Symbols represent average and s.e.m. values of at least three independent experiments. Solid lines represent fits of Hill equation and dashed line is the fit in Fig. 1C (blue line) corresponding to Na+ binding.


C . Log - log plot of L - asp KD as a function of Ca2 + ( n = 13 ) .

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C. Log-log plot of L-asp KD as a function of Ca2+ (n=13).


A Detection of the activation of IRE1α , PERK and ATF6 UPR branches by immunoblotting in control ( shCtrl ) and Fam20C stable knockdown ( shFAM20C ) HepG2 cells treated with or without 5 μM Tg for 30 min . ATF6 - FL , full - length ATF6 ; ATF6 - N , N - terminal cleavage product of ATF6 ; Asterisk , unspecific background bands . Fam20C knockdown was verified by protein immunoblotting of Concanavalin A - Sepharose ( Con A ) - enriched culture medium . Ponceau staining was shown as a loading control .

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A Detection of the activation of IRE1α, PERK and ATF6 UPR branches by immunoblotting in control (shCtrl) and Fam20C stable knockdown (shFAM20C) HepG2 cells treated with or without 5 μM Tg for 30 min. ATF6-FL, full-length ATF6; ATF6-N, N-terminal cleavage product of ATF6; Asterisk, unspecific background bands. Fam20C knockdown was verified by protein immunoblotting of Concanavalin A-Sepharose (Con A)-enriched culture medium. Ponceau staining was shown as a loading control.


B ( Left ) Detection of spliced XBP1 ( S ) and unspliced XBP1 ( U ) mRNA in shCtrl and shFAM20C HepG2 cells treated with or without 5 μM Tg for 1 h . ( Right ) Quantification of XBP1 mRNA splicing levels . Data information : data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( two - tailed Student ' s t - test ) .

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B (Left) Detection of spliced XBP1 (S) and unspliced XBP1 (U) mRNA in shCtrl and shFAM20C HepG2 cells treated with or without 5 μM Tg for 1 h. (Right) Quantification of XBP1 mRNA splicing levels. Data information: data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (two-tailed Student's t-test).


C ( Left ) Detection of XBP1 mRNA splicing in shFAM20C HepG2 cells expressing RNAi - resistant codon - altered Fam20C wild - type ( WT ) or its inactive D478A mutant ( DA ) treated with or without 5 μM Tg for 1 h . Fam20C expression levels were shown by protein immunoblotting . ( Right ) Quantification of XBP1 mRNA splicing levels . Data information : data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( two - tailed Student ' s t - test ) .

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C (Left) Detection of XBP1 mRNA splicing in shFAM20C HepG2 cells expressing RNAi-resistant codon-altered Fam20C wild-type (WT) or its inactive D478A mutant (DA) treated with or without 5 μM Tg for 1 h. Fam20C expression levels were shown by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data information: data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (two-tailed Student's t-test).


( Left ) Detection of XBP1 mRNA splicing in shCtrl and shFAM20C HepG2 cells ( D ) treated with or without 2 μg / ml Tm for 8 h . ( Right ) Quantification of XBP1 mRNA splicing levels . Data information : data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( two - tailed Student ' s t - test ) .

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(Left) Detection of XBP1 mRNA splicing in shCtrl and shFAM20C HepG2 cells (D) treated with or without 2 μg/ml Tm for 8 h. (Right) Quantification of XBP1 mRNA splicing levels. Data information: data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (two-tailed Student's t-test).


Detection of XBP1 mRNA splicing in shFAM20C HepG2 cells expressing Fam20C WT or DA ( E ) treated with or without 2 μg / ml Tm for 8 h . ( Right ) Quantification of XBP1 mRNA splicing levels . Data information : data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( two - tailed Student ' s t - test ) .

Detection of XBP1 mRNA splicing in shFAM20C HepG2 cells expressing Fam20C WT or DA (E) treated with or without 2 μg/ml Tm for 8 h. (Right) Quantification of XBP1 mRNA splicing levels. Data information: data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (two-tailed Student's t-test).


F Volcano plot depicting the log2 of fold change versus - log10 ( p - value ) for Fam20C - interacting proteins in Tg - treated HepG2 cells . The red dots represent proteins showing statistically significant increase of binding with Fam20C ( log2 fold change ≥ 1 ; - log10 ( p - value ) ≥ 1 . 3 ) ; the blue dots represent proteins showing statistically significant decrease of binding with Fam20C ( log2 fold change ≤ - 1 ; - log10 ( p - value ) ≥ 1 . 3 ) ; the green dot is Fam20C .

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F Volcano plot depicting the log2 of fold change versus - log10 (p-value) for Fam20C-interacting proteins in Tg-treated HepG2 cells. The red dots represent proteins showing statistically significant increase of binding with Fam20C (log2 fold change ≥ 1; - log10 (p-value) ≥ 1.3); the blue dots represent proteins showing statistically significant decrease of binding with Fam20C (log2 fold change ≤ - 1; - log10 (p-value) ≥ 1.3); the green dot is Fam20C.


G HepG2 cells expressing FLAG - tagged Fam20C were treated with or without 5 μM Tg for 30 min . FLAG - immunoprecipitates were analyzed by PDI immunoblotting .

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G HepG2 cells expressing FLAG-tagged Fam20C were treated with or without 5 μM Tg for 30 min. FLAG-immunoprecipitates were analyzed by PDI immunoblotting.


A Representative MS / MS fragmentation spectrum of tryptic phosphorylated PDI peptide ( Ile351 - Lys370 ) depicting PDI Ser357 phosphorylation enriched from HepG2 cells treated with 5 μM Tg for 10 min .

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A Representative MS/MS fragmentation spectrum of tryptic phosphorylated PDI peptide (Ile351-Lys370) depicting PDI Ser357 phosphorylation enriched from HepG2 cells treated with 5 μM Tg for 10 min.


C Time - dependent incorporation of phosphate group into PDI WT or S357A catalyzed by recombinant Fam20C . Phosphorylated PDI was determined by anti - pS357 - PDI immunoblotting on both regular and phostag gels .

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C Time-dependent incorporation of phosphate group into PDI WT or S357A catalyzed by recombinant Fam20C. Phosphorylated PDI was determined by anti-pS357-PDI immunoblotting on both regular and phostag gels.


D Phosphorylated PDI catalyzed by Fam20C were analyzed by dual - color immunoblotting with anti - PDI ( red ) and anti - pS357 - PDI ( green ) antibodies . The yellow signal depicts the phosphorylated PDI species .

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D Phosphorylated PDI catalyzed by Fam20C were analyzed by dual-color immunoblotting with anti-PDI (red) and anti-pS357-PDI (green) antibodies. The yellow signal depicts the phosphorylated PDI species.


Detection of pS357 - PDI in HepG2 cells treated with 5 μM Tg ( E ) for indicated times .

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Detection of pS357-PDI in HepG2 cells treated with 5 μM Tg (E) for indicated times.


Detection of pS357 - PDI in HepG2 cells treated with 2 . 5 μg / ml Tm ( F ) for indicated times .

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Detection of pS357-PDI in HepG2 cells treated with 2.5 μg/ml Tm (F) for indicated times.


Detection of pS357 - PDI in HepG2 cells treated with 1 mM DTT ( G ) for indicated times .

Detection of pS357-PDI in HepG2 cells treated with 1 mM DTT (G) for indicated times.


( Left ) Detection of p - PDI in WT and PDI KO HepG2 cells ( H ) treated with 5 μM Tg for indicated times by protein immunoblotting on both regular and phostag gels . ( Right ) Quantification of p - PDI percentage based on band intensities on phostag gels . Data information : Data were shown as mean ± SEM of three ( H ) independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - way ANOVA , the post hoc Tukey ' s HSD test ) .

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(Left) Detection of p-PDI in WT and PDI KO HepG2 cells (H) treated with 5 μM Tg for indicated times by protein immunoblotting on both regular and phostag gels. (Right) Quantification of p-PDI percentage based on band intensities on phostag gels. Data information: Data were shown as mean ± SEM of three (H) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA, the post hoc Tukey's HSD test).


( Left ) Detection of p - PDI in WT and Fam20C KO HeLa cells ( I ) treated with 5 μM Tg for indicated times by protein immunoblotting on both regular and phostag gels . ( Right ) Quantification of p - PDI percentage based on band intensities on phostag gels . Data information : Data were shown as mean ± SEM of four ( I ) independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - way ANOVA , the post hoc Tukey ' s HSD test ) .

(Left) Detection of p-PDI in WT and Fam20C KO HeLa cells (I) treated with 5 μM Tg for indicated times by protein immunoblotting on both regular and phostag gels. (Right) Quantification of p-PDI percentage based on band intensities on phostag gels. Data information: Data were shown as mean ± SEM of four (I) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA, the post hoc Tukey's HSD test).


A Fluorescent photomicrographs of HepG2 cells co - transfected with PDI - mEmerald - KDEL ( green ) and Fam20C - mApple ( red ) . The cells were treated with DMSO ( Upper ) or 5 μM Tg ( Middle ) for 30 min . After Tg treatment , the cells were washed with PBS and then returned to culture medium for 60 min ( Lower ) . GM130 was immunostained ( blue ) as a cis - Golgi marker . Scale bar = 10 μm .

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A Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple (red). The cells were treated with DMSO (Upper) or 5 μM Tg (Middle) for 30 min. After Tg treatment, the cells were washed with PBS and then returned to culture medium for 60 min (Lower). GM130 was immunostained (blue) as a cis-Golgi marker. Scale bar = 10 μm.


B Pearson correlation coefficient calculated Data were shown as mean ± SEM of N > 80 cells from four independent experiments . * * * p < 0 . 001 ( one - way ANOVA , the post hoc Tukey ' s HSD test ) .

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B Pearson correlation coefficient calculated Data were shown as mean ± SEM of N > 80 cells from four independent experiments. ***p < 0.001 (one-way ANOVA, the post hoc Tukey's HSD test).


C Detection of pS357 - PDI and UPR signaling in HepG2 cells after Tg treatment and washout for indicated times .

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C Detection of pS357-PDI and UPR signaling in HepG2 cells after Tg treatment and washout for indicated times.


D HepG2 cells were treated with 25 ng / μl CHX for indicated times and analyzed by immunoblotting for pS357 - PDI and UPR signaling . 5 μM Tg or DMSO was added 30 min before cell harvest .

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D HepG2 cells were treated with 25 ng/μl CHX for indicated times and analyzed by immunoblotting for pS357-PDI and UPR signaling. 5 μM Tg or DMSO was added 30 min before cell harvest.


E Fluorescent photomicrographs of HepG2 cells co - transfected with PDI - mEmerald - KDEL ( green ) and Fam20C - mApple ( red ) pretreated with 25 ng / μl CHX for 7 . 5 h , followed by introduction of DMSO ( Upper ) or 5 μM Tg ( Lower ) for 30 min , respectively . GM130 was immunostained ( blue ) as a cis - Golgi marker . Scale bar = 10 μm .

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E Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple (red) pretreated with 25 ng/μl CHX for 7.5 h, followed by introduction of DMSO (Upper) or 5 μM Tg (Lower) for 30 min, respectively. GM130 was immunostained (blue) as a cis-Golgi marker. Scale bar = 10 μm.


F Fluorescent photomicrographs of HepG2 cells co - transfected with PDI - mEmerald - KDEL ( green ) and Fam20C - mApple - KDEL ( red ) treated with DMSO ( Upper ) or 5 μM Tg ( Lower ) for 30 min , respectively . Scale bar = 10 μm .

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F Fluorescent photomicrographs of HepG2 cells co-transfected with PDI-mEmerald-KDEL (green) and Fam20C-mApple-KDEL (red) treated with DMSO (Upper) or 5 μM Tg (Lower) for 30 min, respectively. Scale bar = 10 μm.


G HepG2 cells were transfected with empty vector ( - ) , FLAG - tagged Fam20C or Fam20C - KDEL followed by introduction without or with 5 μM Tg for 30 min , respectively . PDI phosphorylation was detected by protein immunoblotting on both regular and phostag gels . p - PDI percentage was quantified based on band intensities on phostag gels .

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G HepG2 cells were transfected with empty vector (-), FLAG-tagged Fam20C or Fam20C-KDEL followed by introduction without or with 5 μM Tg for 30 min, respectively. PDI phosphorylation was detected by protein immunoblotting on both regular and phostag gels. p-PDI percentage was quantified based on band intensities on phostag gels.


B The time course of the distance between the geometrical centers of domains a and a ′ and the angle among domains b , b ′ and a ′ from the simulation of pS357 - PDI ( red ) and PDI ( black ) ( Yang et al . , 2014 ) .

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B The time course of the distance between the geometrical centers of domains a and a′ and the angle among domains b, b′ and a′ from the simulation of pS357-PDI (red) and PDI (black) (Yang et al., 2014).


C ( Left ) ANS fluorescence spectra of PDI WT and S357E . Controls with ANS alone was shown . A . U . , arbitrary units . ( Right ) Enhancement factor were quantified . Data information : All data were shown as mean ± SEM of three independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) .

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C (Left) ANS fluorescence spectra of PDI WT and S357E. Controls with ANS alone was shown. A.U., arbitrary units. (Right) Enhancement factor were quantified. Data information: All data were shown as mean ± SEM of three independent experiments. **p < 0.01, ***p < 0.001 (two-tailed Student's t-test).


D Limited proteolysis of PDI WT ( Left ) and S357E ( Right ) proteins by proteinase K ( Pro K ) for indicated times . Main digested fragments were indicated on the right margin . Band abb ' x * is a digested product with cleavage after Met356 in the x - linker region ( Wang et al . , 2010 ) .

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D Limited proteolysis of PDI WT (Left) and S357E (Right) proteins by proteinase K (Pro K) for indicated times. Main digested fragments were indicated on the right margin. Band abb'x* is a digested product with cleavage after Met356 in the x-linker region (Wang et al., 2010).


E shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 30 min , and in some aliquots of cells Tg was removed for 30 min . Cell lysates were subjected to proteinase K digestion for 3 min and immunoblotting analysis .

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E shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 30 min, and in some aliquots of cells Tg was removed for 30 min. Cell lysates were subjected to proteinase K digestion for 3 min and immunoblotting analysis.


F HepG2 cells expressing HA - tagged PDI and FLAG - tagged Fam20C with different ratios of plasmid concentrations were subjected to proteinase K digestion for 21 min and immunoblotting analysis .

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F HepG2 cells expressing HA-tagged PDI and FLAG-tagged Fam20C with different ratios of plasmid concentrations were subjected to proteinase K digestion for 21 min and immunoblotting analysis.


Chaperone activities of PDI WT and S357E ( G , H ) or phosphorylated PDI WT and S357A by Fam20C for indicated times ( I Denatured and reduced GAPDH ( G , I ) and rhodanese ( H , were used as substrates , respectively . The chaperone activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . , * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) . In ( I , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) .

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Chaperone activities of PDI WT and S357E (G, H) or phosphorylated PDI WT and S357A by Fam20C for indicated times (I Denatured and reduced GAPDH (G, I) and rhodanese (H, were used as substrates, respectively. The chaperone activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. , **p < 0.01, ***p < 0.001 (two-tailed Student's t-test). In (I , ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).


Chaperone activities of phosphorylated PDI WT and S357A by Fam20C for indicated times J ) . Denatured and reduced rhodanese J ) were used as substrates , respectively . The chaperone activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) .

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Chaperone activities of phosphorylated PDI WT and S357A by Fam20C for indicated times J). Denatured and reduced rhodanese J) were used as substrates, respectively. The chaperone activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).


Reductase activities of PDI WT and S357E ( K ) measured by insulin reduction assay . The activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) .

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Reductase activities of PDI WT and S357E (K) measured by insulin reduction assay. The activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. **p < 0.01, ***p < 0.001 (two-tailed Student's t-test).


Isomerase activities of PDI WT and S357E ( L ) measured by scrambled RNase A reactivation assay . The activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . , * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) .

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Isomerase activities of PDI WT and S357E (L) measured by scrambled RNase A reactivation assay. The activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. , **p < 0.01, ***p < 0.001 (two-tailed Student's t-test).


Reductase activities of phosphorylated PDI WT and S357A by Fam20C for indicated times ( M ) measured by insulin reduction assay . The activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) .

Reductase activities of phosphorylated PDI WT and S357A by Fam20C for indicated times (M) measured by insulin reduction assay. The activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. , ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).


Isomerase activities of phosphorylated PDI WT and S357A by Fam20C for indicated times ( N ) measured by scrambled RNase A reactivation assay . The activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) .

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Isomerase activities of phosphorylated PDI WT and S357A by Fam20C for indicated times (N) measured by scrambled RNase A reactivation assay. The activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. , ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).


B Fluorescent photomicrographs of HepG2 cells expressing AgHaloER ( TMR - labeled , red ) . Endogenous PDI was immunostained ( green ) as an ER marker . Scale bar = 10 μm .

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B Fluorescent photomicrographs of HepG2 cells expressing AgHaloER (TMR-labeled, red). Endogenous PDI was immunostained (green) as an ER marker. Scale bar = 10 μm.


C Confocal live cell imaging of HepG2 cells expressing AgHaloER labeled by P1 ( green ) and TMR ( red ) , followed by introduction of DMSO or 5 μM Tg for 30 min . Scale bar = 10 μm .

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C Confocal live cell imaging of HepG2 cells expressing AgHaloER labeled by P1 (green) and TMR (red), followed by introduction of DMSO or 5 μM Tg for 30 min. Scale bar = 10 μm.


D Confocal live cell imaging of WT and PDI KO HepG2 cells co - transfected with AgHaloER labeled by P1 ( green ) and TMR ( red ) and empty vector ( - ) , PDI WT , S357E or S357A . Scale bar = 10 μm .

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D Confocal live cell imaging of WT and PDI KO HepG2 cells co-transfected with AgHaloER labeled by P1 (green) and TMR (red) and empty vector (-), PDI WT, S357E or S357A. Scale bar = 10 μm.


E Confocal live cell imaging of shCtrl and shFAM20C HepG2 cells expressing AgHaloER labeled by P1 ( green ) and TMR ( red ) . Scale bar = 10 μm .

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E Confocal live cell imaging of shCtrl and shFAM20C HepG2 cells expressing AgHaloER labeled by P1 (green) and TMR (red). Scale bar = 10 μm.


F ( Upper ) Cell viabilities of WT and PDI KO HepG2 cells co - transfected with AgHaloER and empty vector ( - ) , PDI WT , S357E or S357A . Cells were treated by 5 μM Tg for indicated times and visualized by crystal violet staining . ( Lower ) Quantification of surviving fraction of cells . Data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus WT HepG2 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) .

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F (Upper) Cell viabilities of WT and PDI KO HepG2 cells co-transfected with AgHaloER and empty vector (-), PDI WT, S357E or S357A. Cells were treated by 5 μM Tg for indicated times and visualized by crystal violet staining. (Lower) Quantification of surviving fraction of cells. Data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus WT HepG2 (two-way ANOVA, the post hoc Tukey's HSD test).


A ( Left ) Detection of XBP1 mRNA splicing in WT and PDI KO HepG2 cells treated with or without 5 μM Tg for 1 h . PDI KO was verified by protein immunoblotting . ( Right ) Quantification of XBP1 mRNA splicing levels . Data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 ( two - tailed Student ' s t - test ) .

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A (Left) Detection of XBP1 mRNA splicing in WT and PDI KO HepG2 cells treated with or without 5 μM Tg for 1 h. PDI KO was verified by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data were shown as mean ± SEM of three independent experiments. *p < 0.05 (two-tailed Student's t-test).


B ( Left ) Detection of XBP1 mRNA splicing in PDI KO HepG2 cells expressing PDI WT or its mutants treated with or without 5 μM Tg for 1 h . PDI expression levels were shown by protein immunoblotting . ( Right ) Quantification of XBP1 mRNA splicing levels . Data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( one - way ANOVA , the post hoc Tukey ' s HSD test ) .

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B (Left) Detection of XBP1 mRNA splicing in PDI KO HepG2 cells expressing PDI WT or its mutants treated with or without 5 μM Tg for 1 h. PDI expression levels were shown by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (one-way ANOVA, the post hoc Tukey's HSD test).


C Detection of IRE1α , PERK and ATF6 activation by immunoblotting in PDI KO HepG2 cells expressing PDI S537A or S357E treated with 5 μM Tg for 30 min . Asterisk indicates unspecific background band .

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C Detection of IRE1α, PERK and ATF6 activation by immunoblotting in PDI KO HepG2 cells expressing PDI S537A or S357E treated with 5 μM Tg for 30 min. Asterisk indicates unspecific background band.


D HepG2 cells were treated without or with 5 μM Tg for 1 h . Endogenous IRE1α - immunoprecipitates were analyzed by PDI and BiP immunoblotting .

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D HepG2 cells were treated without or with 5 μM Tg for 1 h. Endogenous IRE1α-immunoprecipitates were analyzed by PDI and BiP immunoblotting.


E HepG2 cells expressing FLAG - tagged IRE1α were treated without or with 5 μM Tg for 1 h . An aliquot of Tg - treated cells was then washed with culture medium and chased for 4 h . FLAG - immunoprecipitates were analyzed by PDI and BiP immunoblotting .

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E HepG2 cells expressing FLAG-tagged IRE1α were treated without or with 5 μM Tg for 1 h. An aliquot of Tg-treated cells was then washed with culture medium and chased for 4 h. FLAG-immunoprecipitates were analyzed by PDI and BiP immunoblotting.


F shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 1 h . Endogenous IRE1α - immunoprecipitates were analyzed by PDI and BiP immunoblotting .

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F shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 1 h. Endogenous IRE1α-immunoprecipitates were analyzed by PDI and BiP immunoblotting.


G Co - immunoprecipitation of FLAG - tagged IRE1α and HA - tagged PDI in HepG2 cells expressing V5 - tagged Fam20C WT or DA .

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G Co-immunoprecipitation of FLAG-tagged IRE1α and HA-tagged PDI in HepG2 cells expressing V5-tagged Fam20C WT or DA.


H Co - immunoprecipitation of FLAG - tagged IRE1α , HA - tagged PDI WT or S357A in HepG2 cells expressing V5 - tagged Fam20C .

H Co-immunoprecipitation of FLAG-tagged IRE1α, HA-tagged PDI WT or S357A in HepG2 cells expressing V5-tagged Fam20C.


I Co - immunoprecipitation of HA - tagged PDI and FLAG - tagged IRE1αNLD in HepG2 cells .

I Co-immunoprecipitation of HA-tagged PDI and FLAG-tagged IRE1αNLD in HepG2 cells.


J Recombinant GST - PDI was phosphorylated by Fam20C protein for 2 h , and then used to pulldown His - IRE1αNLD protein , as visualized by Coomassie blue staining . Successful phosphorylation of GST - PDI was verified by pS357 - PDI immunoblotting .

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J Recombinant GST-PDI was phosphorylated by Fam20C protein for 2 h, and then used to pulldown His-IRE1αNLD protein, as visualized by Coomassie blue staining. Successful phosphorylation of GST-PDI was verified by pS357-PDI immunoblotting.


K Co - immunoprecipitation of FLAG - tagged IRE1αNLD WT or its mutant C109 / 148 / 332S with HA - tagged PDI ( Left ) or MYC - tagged P5 ( Right ) in HepG2 cells .

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K Co-immunoprecipitation of FLAG-tagged IRE1αNLD WT or its mutant C109/148/332S with HA-tagged PDI (Left) or MYC-tagged P5 (Right) in HepG2 cells.


L Co - immunoprecipitation of FLAG - tagged IRE1α and HA - tagged PDI WT or its binding mutant W128I / L403W in HepG2 cells expressing V5 - tagged Fam20C or not .

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L Co-immunoprecipitation of FLAG-tagged IRE1α and HA-tagged PDI WT or its binding mutant W128I/L403W in HepG2 cells expressing V5-tagged Fam20C or not.


B Sanger sequencing results of Pdi + / + ( WT ) and PdiS359A / S359A ( KI ) mice .

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B Sanger sequencing results of Pdi+/+ (WT) and PdiS359A/S359A (KI) mice.


C ( Left ) Detection of Xbp1 mRNA splicing in mice liver after injection of Tm ( 50 ng / g , i . p . ) for indicated times . Each lane represents independent animals . ( Right ) Quantification of Xbp1 mRNA splicing levels at indicated times . Data information : All data were shown as mean ± SEM from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) .

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C (Left) Detection of Xbp1 mRNA splicing in mice liver after injection of Tm (50 ng/g, i.p.) for indicated times. Each lane represents independent animals. (Right) Quantification of Xbp1 mRNA splicing levels at indicated times. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-tailed Student's t-test).


Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0 . 2 μM Tg for 2 h ( D )

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Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0.2 μM Tg for 2 h (D)


Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0 . 5 μg / ml Tm for 2 h ( E ) .

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Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0.5 μg/ml Tm for 2 h (E).


Detection of Bip ( F ) , Pdi ( G ) , Erdj4 ( H ) mRNA levels by real - time PCR in the liver of the same animals after 24 h of Tm injection . Data information : All data were shown as mean ± SEM from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) .

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Detection of Bip (F), Pdi (G), Erdj4 (H) mRNA levels by real-time PCR in the liver of the same animals after 24 h of Tm injection. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).


Detection of Edem1 ( I ) , Il6 ( J ) , Chop ( K ) mRNA levels by real - time PCR in the liver of the same animals after 24 h of Tm injection . Data information : All data were shown as mean ± SEM from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) .

Detection of Edem1 (I), Il6 (J), Chop (K) mRNA levels by real-time PCR in the liver of the same animals after 24 h of Tm injection. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).


L Detection of serum alanine transaminase ( ALT ) levels of the same animals as in ( C ) after 24 h of Tm injection . Data information : All data were shown as mean ± SEM from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) .

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L Detection of serum alanine transaminase (ALT) levels of the same animals as in (C) after 24 h of Tm injection. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).


M Hematoxylin and eosin staining of liver tissue the same animals as in ( C ) after 24 h of Tm injection ( magnification × 400 ) . Immune cell infiltration was indicated with a black arrow . Scale bar = 50 μm . Representative images were shown from five animals per group analyzed .

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M Hematoxylin and eosin staining of liver tissue the same animals as in (C) after 24 h of Tm injection (magnification × 400). Immune cell infiltration was indicated with a black arrow. Scale bar = 50 μm. Representative images were shown from five animals per group analyzed.


N ( Left ) TUNEL assay of the same liver as in ( M ) . Scale bar = 40 μm ; Zoom box , scale bar = 5 μm . ( Right ) Quantification of apoptotic cells . 10 random fields were counted for each TUNEL - stained tissue sample . Data information : All data were shown as mean ± SEM from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) .

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N (Left) TUNEL assay of the same liver as in (M). Scale bar = 40 μm; Zoom box, scale bar = 5 μm. (Right) Quantification of apoptotic cells. 10 random fields were counted for each TUNEL-stained tissue sample. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-tailed Student's t-test).


Naïve CD4 + T cells were isolated from the spleen and LNs of WT , Stim2fl / fl Cd4Cre ( S2 ) , Orai1fl / fl Cd4Cre ( O1 ) and Stim1fl / fl Cd4Cre ( S1 ) mice and differentiated into Th1 , Th17 and iTreg cells . ( A ) Fura - 2 loaded cells were stimulated with 0 . 3 μM ionomycin to induce SOCE . Representative Ca2 + traces ( left ) and peak Ca2 + levels ( right ) . Data are from the mean ± SEM of 4 mice per group .

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Naïve CD4+ T cells were isolated from the spleen and LNs of WT, Stim2fl/fl Cd4Cre (S2), Orai1fl/fl Cd4Cre (O1) and Stim1fl/fl Cd4Cre (S1) mice and differentiated into Th1, Th17 and iTreg cells. (A) Fura-2 loaded cells were stimulated with 0.3 μM ionomycin to induce SOCE. Representative Ca2+ traces (left) and peak Ca2+ levels (right). Data are from the mean ± SEM of 4 mice per group.


( B ) Induction of colitis in Rag1 - / - mice by adoptive transfer of naive CD4 + T cells from wildtype , Stim2fl / fl Cd4Cre , Orai1fl / fl Cd4Cre and Stim1fl / fl Cd4Cre mice . Weight curves of recipient Rag1 - / - mice following injection of naive CD4 + T cells . Data are from two independent experiments with 8 recipient mice per group .

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(B) Induction of colitis in Rag1-/- mice by adoptive transfer of naive CD4+ T cells from wildtype, Stim2fl/fl Cd4Cre, Orai1fl/fl Cd4Cre and Stim1fl/fl Cd4Cre mice. Weight curves of recipient Rag1-/- mice following injection of naive CD4+ T cells. Data are from two independent experiments with 8 recipient mice per group.


( C ) H & E stains of colon tissues and histological inflammation scores of Rag1 - / - mice 8 weeks after T cell transfer . Data are from one representative experiment with 5 mice per group .

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(C) H&E stains of colon tissues and histological inflammation scores of Rag1-/- mice 8 weeks after T cell transfer. Data are from one representative experiment with 5 mice per group.


( D - E ) Representative flow cytometry plots and frequencies of donor CD4 + T cells producing IFNγ , IL - 17A , TNF or expressing Foxp3 that were isolated from the mesenteric LNs of recipient Rag1 - / - mice . Data are from 2 independent experiments with 3 - 8 mice per group .

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(D-E) Representative flow cytometry plots and frequencies of donor CD4+ T cells producing IFNγ, IL-17A, TNF or expressing Foxp3 that were isolated from the mesenteric LNs of recipient Rag1-/- mice. Data are from 2 independent experiments with 3-8 mice per group.


( A , B ) Ca2 + influx in human lamina propria CD4 + and CD8 + T cells from 3 IBD patient samples analyzed by flow cytometry . ( A ) Experimental design . ( B ) Ca2 + influx rates in T cells pre - incubated for 4 h in the presence of 15 - 1000 nM BTP2 , which was present until data acquisition . SOCE was induced by stimulation with the sarco - endoplasmic reticulum Ca2 + ATPase ( SERCA ) inhibitor thapsigargin ( TG ) in Ca2 + free buffer followed by the addition of 2 mM Ca2 + . Bar graphs show the mean ± SEM of Ca2 + influx rates after Ca2 + readdition from 1 experiment ( n = 3 IBD patients ) .

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(A,B) Ca2+ influx in human lamina propria CD4+ and CD8+ T cells from 3 IBD patient samples analyzed by flow cytometry. (A) Experimental design. (B) Ca2+ influx rates in T cells pre-incubated for 4 h in the presence of 15-1000 nM BTP2, which was present until data acquisition. SOCE was induced by stimulation with the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor thapsigargin (TG) in Ca2+ free buffer followed by the addition of 2 mM Ca2+. Bar graphs show the mean ± SEM of Ca2+ influx rates after Ca2+ readdition from 1 experiment (n=3 IBD patients).


( D ) viSNE plots of CD45 + CD3 + LPMCs stimulated with 20 ng / ml PMA and 1 µg / ml ionomycin for 4 hours in the presence of 15 - 1000 nM BTP2 . viSNE plots are colored according to the expression level of IL - 2 ( blue : low , red : high ) and are representative of one CD patient .

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(D) viSNE plots of CD45+CD3+ LPMCs stimulated with 20 ng/ml PMA and 1 µg/ml ionomycin for 4 hours in the presence of 15-1000 nM BTP2. viSNE plots are colored according to the expression level of IL-2 (blue: low, red: high) and are representative of one CD patient.


( E ) Dose - response curves of the frequencies of cytokine producing CD4 + and CD8 + T cells after PMA / ionomycin stimulation for 4h in the presence of increasing doses of BTP2 . The frequencies of cytokine producing cells were normalized to control samples treated with PMA / ionomycin alone . Bar graphs show the mean ± SEM of 5 CD patient samples .

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(E) Dose-response curves of the frequencies of cytokine producing CD4+ and CD8+ T cells after PMA/ionomycin stimulation for 4h in the presence of increasing doses of BTP2. The frequencies of cytokine producing cells were normalized to control samples treated with PMA/ionomycin alone. Bar graphs show the mean ± SEM of 5 CD patient samples.


( F ) Heatmaps representing the median fold change of cytokine and cell surface marker expression in CD45 + CD3 + LPMCs stimulated for 4h with PMA / ionomycin in the presence of BTP2 ; data are normalized to PMA / ionomycin treatment alone .

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(F) Heatmaps representing the median fold change of cytokine and cell surface marker expression in CD45+CD3+ LPMCs stimulated for 4h with PMA/ionomycin in the presence of BTP2; data are normalized to PMA/ionomycin treatment alone.