( g ) HeLa cells stably expressing FLAG - Parkin were transiently transfected with vectors for KikGR - FKBP38ΔN47 or KikGR - FKBP38ΔN47 ( N402K ) , exposed to UV , incubated in the absence or presence of CCCP ( 10 μM ) for 4 h , and then monitored for red and green KikGR fluorescence . Scale bar ( a , b , e , g ) , 10 μm .

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(g) HeLa cells stably expressing FLAG-Parkin were transiently transfected with vectors for KikGR-FKBP38ΔN47 or KikGR-FKBP38ΔN47(N402K), exposed to UV, incubated in the absence or presence of CCCP (10 μM) for 4 h, and then monitored for red and green KikGR fluorescence. Scale bar (a,b,e,g), 10 μm.


( a ) Wild - type ( Mfn2 + / + ) and Mfn2 knockout ( Mfn2 - / - ) MEFs stably expressing mCherry - Parkin were incubated in the absence or presence of CCCP ( 30 μM ) for 4 h , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c . The fluorescence of mCherry - Parkin was also monitored directly .

O O O O O O O O O O O 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

(a) Wild-type (Mfn2+/+) and Mfn2 knockout (Mfn2-/-) MEFs stably expressing mCherry-Parkin were incubated in the absence or presence of CCCP (30 μM) for 4 h, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to FKBP38 and to cytochrome c. The fluorescence of mCherry-Parkin was also monitored directly.


( b ) MEFs as in a but expressing EGFP - Parkin were treated with CCCP ( 30 μM ) for the indicated times and then subjected to immunoblot analysis . The asterisk indicates a nonspecific band .

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

(b) MEFs as in a but expressing EGFP-Parkin were treated with CCCP (30 μM) for the indicated times and then subjected to immunoblot analysis. The asterisk indicates a nonspecific band.


( c ) Wild - type MEFs stably expressing EGFP - Parkin and FLAG - VAP - A were incubated in the absence or presence of CCCP ( 30 μM ) or nocodazole ( 10 μM ) for 6 h as indicated , fixed , permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c , to FLAG and to FKBP38 . The fluorescence of EGFP - Parkin was also monitored directly . ( d ) Higher - magnification views of the boxed areas in ( c , e ) . Wild - type MEFs stably expressing FLAG - Parkin and EGFP - FKBP38 were incubated in the absence or presence of CCCP ( 30 μM ) for 10 h and then subjected to immuno - electron microscopic analysis with antibodies to GFP . Black arrowheads indicate the mitochondria . White arrowheads indicate small vesicle .

O O 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 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 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

(c) Wild-type MEFs stably expressing EGFP-Parkin and FLAG-VAP-A were incubated in the absence or presence of CCCP (30 μM) or nocodazole (10 μM) for 6 h as indicated, fixed, permeabilized and subjected to immunofluorescence analysis with antibodies to cytochrome c, to FLAG and to FKBP38. The fluorescence of EGFP-Parkin was also monitored directly. (d) Higher-magnification views of the boxed areas in (c,e). Wild-type MEFs stably expressing FLAG-Parkin and EGFP-FKBP38 were incubated in the absence or presence of CCCP (30 μM) for 10 h and then subjected to immuno-electron microscopic analysis with antibodies to GFP. Black arrowheads indicate the mitochondria. White arrowheads indicate small vesicle.


( f ) Wild - type ( Fkbp38 + / + ) and Fkbp38 knockout ( Fkbp38 − / − ) MEFs stably expressing FLAG - Parkin and either EGFP or EGFP - FKBP38 were incubated in the absence or presence of CCCP ( 30 μM ) for 24 h and then stained with allophycocyanin ( APC ) - labelled annexin V and propidium iodide ( PI ) for flow cytometric analysis of the percentage of apoptotic cells ( annexin V + and PI + cells ) . Data are means ± s . d . , from three independent experiments .

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(f) Wild-type (Fkbp38+/+) and Fkbp38 knockout (Fkbp38−/−) MEFs stably expressing FLAG-Parkin and either EGFP or EGFP-FKBP38 were incubated in the absence or presence of CCCP (30 μM) for 24 h and then stained with allophycocyanin (APC)-labelled annexin V and propidium iodide (PI) for flow cytometric analysis of the percentage of apoptotic cells (annexin V+ and PI+ cells). Data are means±s.d., from three independent experiments.


A Location of missense variants of PDIA1 and ERp57 identified in ALS cases . PDI primary structure : catalytic a and a ' domains containing the active site motif CXXC sequence ( dark purple and dark green ) , non - catalytic domains b and b ' containing ligand binding sites ( light purple and light green ) , and x - linker region ( light gray ) . The constructs used in this study contained a V5 - tag ( orange ) at the C - terminus that was inserted previous to the ER - retention signal ( dark gray ) .

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A Location of missense variants of PDIA1 and ERp57 identified in ALS cases. PDI primary structure: catalytic a and a' domains containing the active site motif CXXC sequence (dark purple and dark green), non-catalytic domains b and b' containing ligand binding sites (light purple and light green), and x-linker region (light gray). The constructs used in this study contained a V5-tag (orange) at the C-terminus that was inserted previous to the ER-retention signal (dark gray).


B Expression of PDI variants in zebrafish . Zebrafish embryos at 1 - 2 cell stage were injected with sense mRNA coding for the indicated PDIs ( PDIA1WT , PDIA1D292N and PDIA1R300H : 200 pg / embryo ; ERp57WT , ERp57D217N and ERp57Q481K : 80 pg / embryo ) . Protein expression was confirmed by Western blot analysis using anti - V5 antibody in total embryo extracts at 24 h post - fertilization ( hpf ) . Actin was used as a loading control . In the left panel 80 µg protein and in the right panel 120 µg protein extract were used .

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B Expression of PDI variants in zebrafish. Zebrafish embryos at 1-2 cell stage were injected with sense mRNA coding for the indicated PDIs (PDIA1WT, PDIA1D292N and PDIA1R300H: 200 pg/embryo; ERp57WT, ERp57D217N and ERp57Q481K: 80 pg/embryo). Protein expression was confirmed by Western blot analysis using anti-V5 antibody in total embryo extracts at 24 h post-fertilization (hpf). Actin was used as a loading control. In the left panel 80 µg protein and in the right panel 120 µg protein extract were used.


C and D , Motoneuron defects induced in zebrafish embryos after expression of the indicated ALS - linked PDI mutants and wild - type controls ( PDIA1WT and PDIA1R300H : 80 pg mRNA / embryo ; ERp57WT , ERp57D217N , ERp57D217N and ERp57Q481K : 30 pgtba mRNA / embryo ) . The most frequent global phenotypes induced by PDI injection are shown in lateral views of embryos at 24 hpf ( left column ) . Black arrows indicate the presence of curly tail and / or shorter axis phenotypes ( see details in Appendix Table S1 ) . Axon motoneuron morphology was visualized using confocal microscopy in lateral views of the trunk region in transgenic Tg ( Huc : Kaede ) zebrafish at 36 hpf ( middle and right columns ) . Images in the right column correspond to magnification views of the rectangular regions depicted in left and middle columns . Asterisks and arrows point to examples of increased axonal branching and reduced axonal length , respectively .

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C and D, Motoneuron defects induced in zebrafish embryos after expression of the indicated ALS-linked PDI mutants and wild-type controls (PDIA1WT and PDIA1R300H: 80 pg mRNA/embryo; ERp57WT, ERp57D217N, ERp57D217N and ERp57Q481K: 30 pgtba mRNA/embryo). The most frequent global phenotypes induced by PDI injection are shown in lateral views of embryos at 24 hpf (left column). Black arrows indicate the presence of curly tail and/or shorter axis phenotypes (see details in Appendix Table S1). Axon motoneuron morphology was visualized using confocal microscopy in lateral views of the trunk region in transgenic Tg(Huc:Kaede) zebrafish at 36 hpf (middle and right columns). Images in the right column correspond to magnification views of the rectangular regions depicted in left and middle columns. Asterisks and arrows point to examples of increased axonal branching and reduced axonal length, respectively.


C and D , Motoneuron defects induced in zebrafishembryos after expression of the indicated ALS - linked PDI mutants and wild - type controls ( PDIA1WT and PDIA1R300H : 80 pg mRNA / embryo ; ERp57WT , ERp57D217N , ERp57D217N and ERp57Q481K : 30 pgtba mRNA / embryo ) . The most frequent global phenotypes induced by PDI injection are shown in lateral views of embryos at 24 hpf ( left column ) . Black arrows indicate the presence of curly tail and / or shorter axis phenotypes ( see details in Appendix Table S1 ) . Axonmotoneuronmorphology was visualized using confocalmicroscopy in lateral views of the trunk region in transgenic Tg ( Huc : Kaede ) zebrafish at 36 hpf ( middle and right columns ) . Images in the right column correspond to magnification views of the rectangular regions depicted in left and middle columns . Asterisks and arrows point to examples of increased axonal branching and reduced axonal length , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and D, Motoneuron defects induced in zebrafishembryos after expression of the indicated ALS-linked PDI mutants and wild-type controls (PDIA1WT and PDIA1R300H: 80 pg mRNA/embryo; ERp57WT, ERp57D217N, ERp57D217N and ERp57Q481K: 30 pgtba mRNA/embryo). The most frequent global phenotypes induced by PDI injection are shown in lateral views of embryos at 24 hpf (left column). Black arrows indicate the presence of curly tail and/or shorter axis phenotypes (see details in Appendix Table S1). Axonmotoneuronmorphology was visualized using confocalmicroscopy in lateral views of the trunk region in transgenic Tg(Huc:Kaede) zebrafish at 36 hpf (middle and right columns). Images in the right column correspond to magnification views of the rectangular regions depicted in left and middle columns. Asterisks and arrows point to examples of increased axonal branching and reduced axonal length, respectively.


E and F Quantification of motoneuron axon length and axon branching in 36hpf Tg ( Huc : Kaede ) embryos injected with the indicated PDIA1 ( D ) and ERp57 ( E ) mutants .

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E and F Quantification of motoneuron axon length and axon branching in 36hpf Tg(Huc:Kaede) embryos injected with the indicated PDIA1 (D) and ERp57 (E) mutants.


G Touch - evoked escape responses of 48 hpf zebrafish embryos injected with ALS - linked PDI mutants ( Kabashi et al , 2011 ; Kabashi et al , 2009 ) . The number of touches necessary to evoke an escape response ( top ) and speed ( bottom , in mm / s ) of the escape response was determined for each condition . The total number of analyzed embryos is indicated in parenthesis . Experiments of D - F were performed in selected animals sharing normal overall embryo morphology and viability , to avoid unspecific effects of axial shortening or curvature in motoneuron morphology and / or animal behavior . Statistical analyses were performed using one - way ANOVA and Bonferroni ´ s post hoc tests . Mean ± SEM with only statistically significant p values are shown : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . Abbreviations : A ( anterior ) , D ( dorsal ) , P ( posterior ) , V ( ventral ) . Scale bars represent 500 m ( C and D : a , d . g , j ) , 200 m ( C and D : b , e , h , k ) , 100 m ( C and D : c , f , i , l ) , and 25 µm ( G - H ) .

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

G Touch-evoked escape responses of 48 hpf zebrafish embryos injected with ALS-linked PDI mutants (Kabashi et al, 2011; Kabashi et al, 2009). The number of touches necessary to evoke an escape response (top) and speed (bottom, in mm/s) of the escape response was determined for each condition. The total number of analyzed embryos is indicated in parenthesis. Experiments of D-F were performed in selected animals sharing normal overall embryo morphology and viability, to avoid unspecific effects of axial shortening or curvature in motoneuron morphology and/or animal behavior. Statistical analyses were performed using one-way ANOVA and Bonferroni´s post hoc tests. Mean ± SEM with only statistically significant p values are shown: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. Abbreviations: A (anterior), D (dorsal), P (posterior), V (ventral). Scale bars represent 500 m (C and D: a,d.g,j), 200 m (C and D: b,e,h,k), 100 m (C and D: c,f,i,l), and 25 µm (G-H).


A NSC34cells were transiently transfected with expression vectors for V5 - tagged wild - type and mutant PDIs . After 48 h , overexpressed PDI variants was assessed under reducing conditions in an 8 % SDS - PAGE . An antibody detecting total PDIA1 ( endogenous mousePDIA1 and exogenous humanV5 - tagged PDIA1 ) ( first panel ) . A second antibody detects only humanPDIA1 , therefore only V5 - tagged PDIA1 appears ( second panel ) . A mouse and human specific antibody was used to detect total ERp57 ( third panel ) . Anti - V5 was used to detect the exogenous PDI variants . Anti - β - actin was used as a loading control .

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A NSC34cells were transiently transfected with expression vectors for V5-tagged wild-type and mutant PDIs. After 48 h, overexpressed PDI variants was assessed under reducing conditions in an 8% SDS-PAGE. An antibody detecting total PDIA1 (endogenous mousePDIA1 and exogenous humanV5-tagged PDIA1) (first panel). A second antibody detects only humanPDIA1, therefore only V5-tagged PDIA1 appears (second panel). A mouse and human specific antibody was used to detect total ERp57 (third panel). Anti-V5 was used to detect the exogenous PDI variants. Anti-β-actin was used as a loading control.


B and C NSC34 cells were transiently transfected with the indicated PDIs constructs together with a GFP expression plasmid . Cell were then differentiated for 24 h in Neurobasal medium containing B27 supplement to induce cell differentiation . Increased neurite outgrowth is indicated with white arrow heads . B Quantification of the average primary neurite lengths was performed , all cells from three independent experiments were compiled . A minimum of 100 cells per experiment was analyzed . In addition , C the percentage of cells with neurites was quantified in the three independent experiments ( right panel ) .

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B and C NSC34 cells were transiently transfected with the indicated PDIs constructs together with a GFP expression plasmid. Cell were then differentiated for 24 h in Neurobasal medium containing B27 supplement to induce cell differentiation. Increased neurite outgrowth is indicated with white arrow heads. B Quantification of the average primary neurite lengths was performed, all cells from three independent experiments were compiled. A minimum of 100 cells per experiment was analyzed. In addition, C the percentage of cells with neurites was quantified in the three independent experiments (right panel).


D Primary rat ventral spinal cordneurons were prepared and after 4 daysin vitro ( DIV ) transfected with GFP alone or together with the indicated PDIs constructs . Cells were fixed at 10 DIV and SMI32 staining was performed to identify motoneurons . Images were taken and the total outgrowth of GFP and SMI32 positive cells was quantified . Results are compiled from 3 independent experiments .

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D Primary rat ventral spinal cordneurons were prepared and after 4 daysin vitro (DIV) transfected with GFP alone or together with the indicated PDIs constructs. Cells were fixed at 10 DIV and SMI32 staining was performed to identify motoneurons. Images were taken and the total outgrowth of GFP and SMI32 positive cells was quantified. Results are compiled from 3 independent experiments.


E Human motoneurons were differentiated from the human embryonic stem cell ( ESC ) HuESC 3 Hb9 : : GFP line . Differentiated neurons were transduced with lentivirus expressing GFP alone or together with PDIs - expressing plasmids . Transduced cells were cultured for another 10 days . To identify motoneurons after lentiviral transduction , immunocytochemistry analyses were performed . Quantification of neurite outgrowth in the different experimental conditions of GFP and HB9 positive neurons were obtained . Neurite number and assessment of the length of each neurite was performed in a similar manner as for primary rat motoneurons ( see methods ) . Four independent experiments were performed .

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E Human motoneurons were differentiated from the human embryonic stem cell (ESC) HuESC 3 Hb9::GFP line. Differentiated neurons were transduced with lentivirus expressing GFP alone or together with PDIs-expressing plasmids. Transduced cells were cultured for another 10 days. To identify motoneurons after lentiviral transduction, immunocytochemistry analyses were performed. Quantification of neurite outgrowth in the different experimental conditions of GFP and HB9 positive neurons were obtained. Neurite number and assessment of the length of each neurite was performed in a similar manner as for primary rat motoneurons (see methods). Four independent experiments were performed.


F NSC34 cell lines stably knocked down for PDIA1 or ERp57 were differentiated for 24 h in Neurobasal medium containing B27 supplement to induce cell differentiation . The percentage of cells with neurites was quantified in the three independent experiments . A minimum of 100 cells per experiment was analyzed . Statistical analyses were performed using one - way ANOVA and Bonferroni ´ s post hoc tests . Mean ± SEM with only statistically significant p values are shown : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 .

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F NSC34 cell lines stably knocked down for PDIA1 or ERp57 were differentiated for 24 h in Neurobasal medium containing B27 supplement to induce cell differentiation. The percentage of cells with neurites was quantified in the three independent experiments. A minimum of 100 cells per experiment was analyzed.Statistical analyses were performed using one-way ANOVA and Bonferroni´s post hoc tests. Mean ± SEM with only statistically significant p values are shown: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.


A PDI mutants form abnormal disulfide - dependent protein complexes . NSC34 cells were transiently transfected with expression vectors for V5 - tagged wild - type and mutant PDIA1 . After 48 h , differential disulfide - dependent interactions / aggregations of overexpressed PDI variants was assessed under reducing ( + DTT ) and non - reducing ( - DTT ) conditions in an 8 % SDS - PAGE . Anti - V5 was used for detection in Western blot .

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A PDI mutants form abnormal disulfide-dependent protein complexes. NSC34 cells were transiently transfected with expression vectors for V5-tagged wild-type and mutant PDIA1. After 48 h, differential disulfide-dependent interactions/aggregations of overexpressed PDI variants was assessed under reducing (+DTT) and non-reducing (-DTT) conditions in an 8% SDS-PAGE. Anti-V5 was used for detection in Western blot.


B Analysis of the PDIA1 structure to model the effects of the R300H mutation . The close association between Arg300 located to the b ' domain of PDIA1 with Trp396 located to the a ' domain adjacent to the active site motif CGHC ( designated as AS in yellow ) is shown in comparison to the mutated version of PDIA1R300H highlighting the same residues . A potential stabilization of the interaction between the b ' and a ' domain is shown that may be caused by the interaction of the imidazole rings of mutated His300 with Trp396 .

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B Analysis of the PDIA1 structure to model the effects of the R300H mutation. The close association between Arg300 located to the b' domain of PDIA1 with Trp396 located to the a' domain adjacent to the active site motif CGHC (designated as AS in yellow) is shown in comparison to the mutated version of PDIA1R300H highlighting the same residues. A potential stabilization of the interaction between the b' and a' domain is shown that may be caused by the interaction of the imidazole rings of mutated His300 with Trp396.


C ALS - linked PDIA1 variants were generated as recombinant proteins and then analyzed by circular dichroism ( CD ) . Averages for CD spectroscopic scans of PDIA1WT and mutants are shown .

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C ALS-linked PDIA1 variants were generated as recombinant proteins and then analyzed by circular dichroism (CD). Averages for CD spectroscopic scans of PDIA1WT and mutants are shown.


D Averages for CD spectroscopic thermal denaturation of recombinant PDIA1WT and mutants .

D Averages for CD spectroscopic thermal denaturation of recombinant PDIA1WT and mutants.


E Representative electrophoresis analysis of Proteinase K - treated recombinant PDIA1 variants . Mass spectrometric analysis of Proteinase K digested samples from total sample and from in gel trypsin digested protein samples ( arrows indicate band 1 and band 2 ) indicated differences in the removal of the x - linker region . Bottom panel : Ratios of band 1 to band 2 were quantified in 4 independent experiments .

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E Representative electrophoresis analysis of Proteinase K-treated recombinant PDIA1 variants. Mass spectrometric analysis of Proteinase K digested samples from total sample and from in gel trypsin digested protein samples (arrows indicate band 1 and band 2) indicated differences in the removal of the x-linker region. Bottom panel: Ratios of band 1 to band 2 were quantified in 4 independent experiments.


F Representative fluorescence spectra of recombinant PDIA1b ' xWT fragments and the equivalent ALS - linked mutants ( n = 6 ) . Ratios from the two peak areas and change compared to PDIA1 b ' xWT . Both mutants analyzed show a significant shift in peak position compared to wild - type , but in opposite directions , suggesting that PDIA1D292N shifts equilibrium towards the capped version ( x region over the binding pocket ) and the PDIA1R300H mutant towards the uncapped version ( Nguyen et al , 2008 ) .

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F Representative fluorescence spectra of recombinant PDIA1b'xWT fragments and the equivalent ALS-linked mutants (n=6). Ratios from the two peak areas and change compared to PDIA1 b'xWT. Both mutants analyzed show a significant shift in peak position compared to wild-type, but in opposite directions, suggesting that PDIA1D292N shifts equilibrium towards the capped version (x region over the binding pocket) and the PDIA1R300H mutant towards the uncapped version (Nguyen et al, 2008).


G The activity of recombinant PDIA1 was measured in vitro using a BPTI refolding assay following by mass spectrometry analysis . The percentages of different BPTI species was calculated ( 6H , fully reduced ; 1S , one disulfide bond ; 2S , two disulfide bonds ; 3S , three disulfide bonds ) at time point 2 . 5 min in four independent experiments .

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G The activity of recombinant PDIA1 was measured in vitro using a BPTI refolding assay following by mass spectrometry analysis. The percentages of different BPTI species was calculated (6H, fully reduced; 1S, one disulfide bond; 2S, two disulfide bonds; 3S, three disulfide bonds) at time point 2.5 min in four independent experiments.


H Measurement of H2O2 levels at the ER lumen of living NSC34 . Left panel : Reduced PDIs can be oxidized by oxidized ERO1Lα that then transfers electrons to molecular oxygen ( O2 ) , generating hydrogen peroxide ( H2O2 ) as a product of PDIs activity . Right panel : NSC34 cells were transiently co - transfected with ER luminal HyPer sensor and indicated PDIs . After 48 h the 490 / 420 nm fluorescence ratio was recorded for 2 min under basal conditions . Means and SEM derived from all cells per condition ( n = 55 - 74 ) monitored in four independent experiments are shown .

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

H Measurement of H2O2 levels at the ER lumen of living NSC34. Left panel: Reduced PDIs can be oxidized by oxidized ERO1Lα that then transfers electrons to molecular oxygen (O2), generating hydrogen peroxide (H2O2) as a product of PDIs activity. Right panel: NSC34 cells were transiently co-transfected with ER luminal HyPer sensor and indicated PDIs. After 48 h the 490/420 nm fluorescence ratio was recorded for 2 min under basal conditions. Means and SEM derived from all cells per condition (n=55-74) monitored in four independent experiments are shown.


I HEK293T cells were transfected with expression vectors for V5 - tagged wild - type and mutant PDIA1 , as well as empty vector . After 48 h , V5 - tagged proteins were immunoprecipitated and eluted with V5 peptide . The interaction with endogenous ERO1Lα was analyzed by western blot . The inputs and elutions are shown as control . Right panel : Quantification of the degree of interaction is presented .

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I HEK293T cells were transfected with expression vectors for V5-tagged wild-type and mutant PDIA1, as well as empty vector. After 48 h, V5-tagged proteins were immunoprecipitated and eluted with V5 peptide. The interaction with endogenous ERO1Lα was analyzed by western blot. The inputs and elutions are shown as control. Right panel: Quantification of the degree of interaction is presented.


A PDI mutants form abnormal disulfide - dependent protein complexes . NSC34 cells were transiently transfected with expression vectors for V5 - tagged wild - type and mutant ERp57 . After 48 h , differential disulfide - dependent interactions / aggregations of overexpressed PDI variants was assessed under reducing ( + DTT ) and non - reducing ( - DTT ) conditions in an 8 % SDS - PAGE . Anti - V5 was used for detection in Western blot .

A PDI mutants form abnormal disulfide-dependent protein complexes. NSC34 cells were transiently transfected with expression vectors for V5-tagged wild-type and mutant ERp57. After 48 h, differential disulfide-dependent interactions/aggregations of overexpressed PDI variants was assessed under reducing (+DTT) and non-reducing (-DTT) conditions in an 8% SDS-PAGE. Anti-V5 was used for detection in Western blot.


B ALS - linked ERp57 variants were generated as recombinant proteins and then analyzed by circular dichroism ( CD ) . Averages for CD spectroscopic scans of recombinant ERp57WT and mutants are shown .

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B ALS-linked ERp57 variants were generated as recombinant proteins and then analyzed by circular dichroism (CD). Averages for CD spectroscopic scans of recombinant ERp57WT and mutants are shown.


C A thermal denaturation curve of ERp57WT and mutants was performed .

C A thermal denaturation curve of ERp57WT and mutants was performed.


D Representative electrophoresis of Proteinase K treated ERp57 recombinant proteins .

D Representative electrophoresis of Proteinase K treated ERp57 recombinant proteins.


E Surface Plasmon Resonance was performed to monitor the affinity of recombinant ERp57 for recombinant CRT P - domain . KD values are expressed as percentage of ERp57WT . Values are derived from four independent experiments . For absolute KD values please see Appendix Table S2 .

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E Surface Plasmon Resonance was performed to monitor the affinity of recombinant ERp57 for recombinant CRT P-domain. KD values are expressed as percentage of ERp57WT. Values are derived from four independent experiments. For absolute KD values please see Appendix Table S2.


FHEK293T NSC34 were transfected with expression vectors for V5 - tagged wild - type and mutant ERp57 , as well as empty vector . After 48 h , V5 - tagged proteins were immunoprecipitated and eluted with V5 peptide . The interaction with endogenous calnexin ( CNX ) and calreticulin ( CRT ) was analyzed by Western blot . The inputs and elutions are shown . Right panel : Quantification of the degree of interaction is presented .

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FHEK293T NSC34 were transfected with expression vectors for V5-tagged wild-type and mutant ERp57, as well as empty vector. After 48 h, V5-tagged proteins were immunoprecipitated and eluted with V5 peptide. The interaction with endogenous calnexin (CNX) and calreticulin (CRT) was analyzed by Western blot. The inputs and elutions are shown. Right panel: Quantification of the degree of interaction is presented.


G The gain of an N - glycosylation site of ERp57D217N was predicted after the analysis of the protein sequence since the change of Asp217 to an Asn creates the NXT / S consensus sequence . Neuro2a cells were transfected with expression vectors for V5 - tagged wild - type and mutant ERp57 , as well as empty vector and treated with 1 µg / ml tunicamycin ( Tm ) for 20 h to inhibit N - glycosylation . Alternatively , protein extracts were digested with PNGase F and the possible removal of the N - glycosylation was analyzed by Western blot using anti - V5 antibody .

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G The gain of an N-glycosylation site of ERp57D217N was predicted after the analysis of the protein sequence since the change of Asp217 to an Asn creates the NXT/S consensus sequence. Neuro2a cells were transfected with expression vectors for V5-tagged wild-type and mutant ERp57, as well as empty vector and treated with 1 µg/ml tunicamycin (Tm) for 20 h to inhibit N-glycosylation. Alternatively, protein extracts were digested with PNGase F and the possible removal of the N-glycosylation was analyzed by Western blot using anti-V5 antibody.


A ERp57flox / flox mice were crossed with Nestin Cre transgenic mice to generate nervous system specific ERp57 deficient animals . The levels of ERp57 protein in the spinal cord were monitored by Western blot . ERp57WT ( n = 4 ) , ERp57Nes + / - ( n = 5 ) and ERp57Nes - / - ( n = 4 ) mice . HSP90 levels were determined as a loading control . Right panel : Quantification of ERp57 levels was performed relative to Hsp90 levels .

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A ERp57flox/flox mice were crossed with Nestin Cre transgenic mice to generate nervous system specific ERp57 deficient animals. The levels of ERp57 protein in the spinal cord were monitored by Western blot. ERp57WT (n=4), ERp57Nes+/- (n=5) and ERp57Nes-/- (n=4) mice. HSP90 levels were determined as a loading control. Right panel: Quantification of ERp57 levels was performed relative to Hsp90 levels.


B Body weight measurements were performed for indicated time points in ERp57WT ( n = 50 ) , ERp57Nes + / - ( n = 32 ) and ERp57Nes - / - ( n = 19 ) mice .

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B Body weight measurements were performed for indicated time points in ERp57WT (n=50), ERp57Nes+/- (n=32) and ERp57Nes-/- (n=19) mice.


C Rotarod performance was performed ERp57WT ( n = 20 ) , ERp57Nes + / - ( n = 15 ) and ERp57Nes - / - ( n = 8 ) mice .

C Rotarod performance was performed ERp57WT (n=20), ERp57Nes+/- (n=15) and ERp57Nes-/- (n=8) mice.


D Hanging test performance was assessed in ERp57WT ( n = 41 ) , ERp57Nes + / - ( n = 32 ) and ERp57Nes - / - ( n = 12 ) mice .

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D Hanging test performance was assessed in ERp57WT (n=41), ERp57Nes+/- (n=32) and ERp57Nes-/- (n=12) mice.


E Kaplan - Meier survival curve for ERp57Nes - / - mice ( N = 19 ) that prematurely died or had to be sacrificed because of health reasons between the ages 22 and 73 days . Mean survival of this subgroup of animals was 57 days . ERp57WT ( n = 50 ) and ERp57Nes + / - ( n = 32 ) mice are shown as a reference .

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E Kaplan-Meier survival curve for ERp57Nes-/- mice (N = 19) that prematurely died or had to be sacrificed because of health reasons between the ages 22 and 73 days. Mean survival of this subgroup of animals was 57 days. ERp57WT (n=50) and ERp57Nes+/- (n=32) mice are shown as a reference.


F Histological analysis of NeuN and GFAP staining was performed in spinal cord tissue from ERp57WT and ERp57Nes - / - mice in three animals per group using indirect immunofluorescence . The nucleus was stained with Hoechst . Representative images from one mouse per group are shown . Scale bar : 50 μm .

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F Histological analysis of NeuN and GFAP staining was performed in spinal cord tissue from ERp57WT and ERp57Nes-/- mice in three animals per group using indirect immunofluorescence. The nucleus was stained with Hoechst. Representative images from one mouse per group are shown. Scale bar: 50 μm.


G Stereological analysis of the spinal cord from ERp57WT ( n = 4 ) , ERp57Nes + / - ( n = 4 ) and ERp57Nes - / - ( n = 4 ) mice . Alternate series of sections from the spinal cord of the mice were either stained for Nissl ( top row images ) or processed for immunohistochemistry for the cholinergic cell marker Choline Acetyl Transferase ( ChAT , bottom row images ) . The nucleoli of the motoneurons , as stained in the Nissl series , were counted inside the motoneurons pools previously defined using the adjacent ChAT series ( contours not shown ) . Cell densities of the three genotypes are shown on the right plots . No significant differences were found between the genotypes . Scale bar represents 200 μm and 50 μm on large and inset images , respectively .

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G Stereological analysis of the spinal cord from ERp57WT (n = 4), ERp57Nes+/- (n = 4) and ERp57Nes-/- (n = 4) mice. Alternate series of sections from the spinal cord of the mice were either stained for Nissl (top row images) or processed for immunohistochemistry for the cholinergic cell marker Choline Acetyl Transferase (ChAT, bottom row images). The nucleoli of the motoneurons, as stained in the Nissl series, were counted inside the motoneurons pools previously defined using the adjacent ChAT series (contours not shown). Cell densities of the three genotypes are shown on the right plots. No significant differences were found between the genotypes. Scale bar represents 200 μm and 50 μm on large and inset images, respectively.


H Representative images of the spinal cord myelination of ERp57WT and ERp57Nes - / - mice .

H Representative images of the spinal cord myelination of ERp57WT and ERp57Nes-/- mice.


A Representative EMG recordings of ERp57WT ( n = 16 ) , ERp57Nes - / + ( n = 12 ) and ERp57Nes - / - ( n = 5 ) mice is presented . The presence of positive sharp waves ( PSW ) in ERp57Nes + / - and ERp57Nes - / - mice indicated muscle denervation .

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A Representative EMG recordings of ERp57WT (n=16), ERp57Nes-/+ (n=12) and ERp57Nes-/- (n=5) mice is presented. The presence of positive sharp waves (PSW) in ERp57Nes+/- and ERp57Nes-/- mice indicated muscle denervation.


B Analysis of NMJ and muscle morphologies were performed from ERp57WT ( n = 5 ) , ERp57Nes + / - ( n = 5 ) and ERp57Nes - / - ( n = 4 ) . Quantifications of endplate width , measuring the most ventral region of the diaphragms every 134 µm considering both sides of the innervation profile . Twenty to forty measurements per diaphragm were obtained per animal .

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B Analysis of NMJ and muscle morphologies were performed from ERp57WT (n=5), ERp57Nes+/- (n=5) and ERp57Nes-/- (n=4). Quantifications of endplate width, measuring the most ventral region of the diaphragms every 134 µm considering both sides of the innervation profile. Twenty to forty measurements per diaphragm were obtained per animal.


C Whole - mounted diaphragms from ERp57WT and ERp57Nes - / - mice were co - stained with anti - neurofilament ( red ) and α - BTX to reveal the postsynaptic densities ( green ) . 3D reconstructions ( lower panel ) of higher magnification . ERp57WT NMJs are fully innervated pretzel - like , whereas ERp57Nes - / - NMJs display less complex postsynaptic densities and an incomplete or aberrantly distributed innervation pattern ( Movies EV4 - 7 ) .

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C Whole-mounted diaphragms from ERp57WT and ERp57Nes-/- mice were co-stained with anti-neurofilament (red) and α-BTX to reveal the postsynaptic densities (green). 3D reconstructions (lower panel) of higher magnification. ERp57WT NMJs are fully innervated pretzel-like, whereas ERp57Nes-/- NMJs display less complex postsynaptic densities and an incomplete or aberrantly distributed innervation pattern (Movies EV4-7).


D Representative 3D reconstructions of z - stacks showing the five categories in which NMJs morphologies were grouped : Pretzel , fragmented , O shaped , C shaped , and dismantled NMJs . Histogram of the distribution frequency of > 200 different NMJ per animal is shown .

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D Representative 3D reconstructions of z-stacks showing the five categories in which NMJs morphologies were grouped: Pretzel, fragmented, O shaped, C shaped, and dismantled NMJs. Histogram of the distribution frequency of >200 different NMJ per animal is shown.


E Quantitative morphometry of > 60 NMJs in the different genotypes analyzed .

E Quantitative morphometry of >60 NMJs in the different genotypes analyzed.


A The levels of ERp57 protein in the muscle were monitored by Western blot . ERp57WT ( n = 4 ) , ERp57Nes + / - ( n = 5 ) and ERp57Nes - / - ( n = 4 ) mice . HSP90 was determined as loading control .

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A The levels of ERp57 protein in the muscle were monitored by Western blot. ERp57WT (n = 4), ERp57Nes+/- (n = 5) and ERp57Nes-/- (n = 4) mice. HSP90 was determined as loading control.


B Tibialis anterior muscles from ERp57WT ( n = 5 ) , ERp57Nes + / - ( n = 5 ) , and ERp57Nes - / - ( n = 4 ) mice were dissected and cryosectioned ( 20 µm ) for the analysis of several parameters of muscle physiology . Upper panel : hyaluronic acid ( HA ) staining was performed to assess the gross morphology of the muscle . Middle panel : Bismarck brown staining to monitor skeletal muscles fibrosis . Lower panel : Cryosections were then double stained with wheat germ agglutinin ( WGA ) to visualize plasmamembrane and nuclei . The absence of central nuclei in all genotypes suggests that ERp57 deficiency does not affect degeneration / regeneration cycles . Images are representative of each genotype and where acquired in the same anatomical region of the TA muscles .

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B Tibialis anterior muscles from ERp57WT (n = 5), ERp57Nes+/- (n = 5), and ERp57Nes-/- (n = 4) mice were dissected and cryosectioned (20 µm) for the analysis of several parameters of muscle physiology. Upper panel: hyaluronic acid (HA) staining was performed to assess the gross morphology of the muscle. Middle panel: Bismarck brown staining to monitor skeletal muscles fibrosis. Lower panel: Cryosections were then double stained with wheat germ agglutinin (WGA) to visualize plasmamembrane and nuclei. The absence of central nuclei in all genotypes suggests that ERp57 deficiency does not affect degeneration/regeneration cycles. Images are representative of each genotype and where acquired in the same anatomical region of the TA muscles.


C Tibialis anterior cryosections ( 20 µm ) from ERp57WT , ERp57Nes + / - , and ERp57Nes - / - mice were stained for NADH - TR activity to identify oxidative myofibers ( upper panel ) . Representative micrographs of transversal cryosections show variable proportions of light , intermediate and dark positive staining in the different genotypes : ( middle panel ) NADH - TR positive ( slow - twitch ) and negative ( fast - twitch ) myofibers were quantified as percentage of total for every genotype ( lower panel ) The mean diameter of slow - and fast - twitch muscle fibers was evaluated at the light microscope using the ImageJ software . Data represent the average ± SEM of > 300 fibers per animal . Scale bars represent 50 m in B and C .

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C Tibialis anterior cryosections (20 µm) from ERp57WT, ERp57Nes+/-, and ERp57Nes-/- mice were stained for NADH-TR activity to identify oxidative myofibers (upper panel). Representative micrographs of transversal cryosections show variable proportions of light, intermediate and dark positive staining in the different genotypes: (middle panel) NADH-TR positive (slow-twitch) and negative (fast-twitch) myofibers were quantified as percentage of total for every genotype (lower panel) The mean diameter of slow- and fast-twitch muscle fibers was evaluated at the light microscope using the ImageJ software. Data represent the average ± SEM of >300 fibers per animal.Scale bars represent 50 m in B and C.


A The levels of synaptic proteins SV2 , NMDAR2A , PSD95 and Synaptophysin in the cortex were monitored by Western blot . ERp57WT ( n = 4 ) , ERp57Nes + / - ( n = 5 ) and ERp57Nes - / - ( n = 4 ) mice . HSP90 and β - Actin were determined as loading controls .

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A The levels of synaptic proteins SV2, NMDAR2A, PSD95 and Synaptophysin in the cortex were monitored by Western blot. ERp57WT (n = 4), ERp57Nes+/- (n = 5) and ERp57Nes-/- (n = 4) mice. HSP90 and β-Actin were determined as loading controls.


B Quantification of SV2 monomer ( left panel ) and SV2 high molecular weight species ( right panel ) . β - Actin was used as the reference .

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B Quantification of SV2 monomer (left panel) and SV2 high molecular weight species (right panel). β-Actin was used as the reference.


Western blot detection of PAX6 isoforms in human fetal forebrain and retina . The antibody used targets the C terminus of PAX6 and can detect all the 3 isoforms at different molecular weight . The green arrow indicates the PAX6A & B isoforms . The red arrow indicates the PAX6D isoform . # 1 , # 2 are # 3 are from ~ 8 - week - old human embryos . # 4 is from ~ 12 - week - old human fetus .

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Western blot detection of PAX6 isoforms in human fetal forebrain and retina. The antibody used targets the C terminus of PAX6 and can detect all the 3 isoforms at different molecular weight. The green arrow indicates the PAX6A&B isoforms. The red arrow indicates the PAX6D isoform. #1, #2 are #3 are from ~8-week-old human embryos. #4 is from ~12-week-old human fetus.


qPCR quantification of overall PAX6 gene expression during human forebrain and retinal differentiation ( n = 3 biological replicates for each group ) . qPCR quantification of PAX6A & B isoform expression during human forebrain and retinal differentiation ( n = 3 biological replicates for each group ) . qPCR of PAX6D isoform expression during human forebrain and retinal differentiation ( n = 3 biological replicates for each group ) .

qPCR quantification of overall PAX6 gene expression during human forebrain and retinal differentiation (n=3 biological replicates for each group). qPCR quantification of PAX6A&B isoform expression during human forebrain and retinal differentiation (n=3 biological replicates for each group). qPCR of PAX6D isoform expression during human forebrain and retinal differentiation (n=3 biological replicates for each group).


Western blot detection of PAX6 isoforms in cells during forebrain and retina differentiation from hESCs . D10 indicates day 10 of differentiation . The green arrow indicates the PAX6A & B isoforms . The red arrow indicates the PAX6D isoform .

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Western blot detection of PAX6 isoforms in cells during forebrain and retina differentiation from hESCs. D10 indicates day 10 of differentiation. The green arrow indicates the PAX6A&B isoforms. The red arrow indicates the PAX6D isoform.


qPCR quantification of PAX6 expression in Wildtype and PAX6 KO during retinal differentiation ( n = 3 biological replicates for each group ) .

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qPCR quantification of PAX6 expression in Wildtype and PAX6 KO during retinal differentiation (n=3 biological replicates for each group).


Western blot detection of PAX6 in Wildtype and PAX6 KO cells at day 20 of retinal differentiation .

Western blot detection of PAX6 in Wildtype and PAX6 KO cells at day 20 of retinal differentiation.


Immunostaining of PAX6 in Wildtype and PAX6 KO cells . C ' - PAX6 antibody detects PAX6A , B and D . N ' - PAX6 antibody detects PAX6 A and B but not D . Scale bars : 50 µm .

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Immunostaining of PAX6 in Wildtype and PAX6 KO cells. C'-PAX6 antibody detects PAX6A, B and D. N'-PAX6 antibody detects PAX6 A and B but not D. Scale bars: 50 µm.


qPCR quantification of retinal related genes in Wildtype and PAX6 KO cells . Samples are collected at day 6 , 10 , 14 and 19 after the start of differentiation .

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qPCR quantification of retinal related genes in Wildtype and PAX6 KO cells. Samples are collected at day 6, 10, 14 and 19 after the start of differentiation.


qPCR verification of PAX6D KO efficiency . PAX6A & B and PAX6D isoforms were also evaluated by qPCR with isoform specific primers ( n = 3 biological replicates for each group ) .

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qPCR verification of PAX6D KO efficiency. PAX6A&B and PAX6D isoforms were also evaluated by qPCR with isoform specific primers (n=3 biological replicates for each group).


Western blot detection of PAX6 isoforms in wildtype and PAX6D KO cells .

Western blot detection of PAX6 isoforms in wildtype and PAX6D KO cells.


qPCR quantification of retinal gene VSX2 in PAX6D KO cells ( n = 3 biological replicates for each group ) . qPCR quantification of retinal gene SIX6 in PAX6D KO cells ( n = 3 biological replicates for each group ) .

qPCR quantification of retinal gene VSX2 in PAX6D KO cells (n=3 biological replicates for each group).   qPCR quantification of retinal gene SIX6 in PAX6D KO cells (n=3 biological replicates for each group).


Immunostaining of VSX2 in wildtype and PAX6D KO cells . Scale bars : 100 µm . Samples were from day 30 differentiating retinal cells .

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Immunostaining of VSX2 in wildtype and PAX6D KO cells. Scale bars:100 µm. Samples were from day 30 differentiating retinal cells.


Immunostaining of SOX1 and SOX2 in human fetal forebrain and retinal tissues . Scale bars : 50 µm .

Immunostaining of SOX1 and SOX2 in human fetal forebrain and retinal tissues. Scale bars:50 µm.


Flow cytometry analysis of day 7 cell cultures under retinal differentiation from wildtype , PAX6 KO and PAX6D KO cell lines . Flow cytometry analysis of day 20 cell cultures under retinal differentiation from wildtype , PAX6 KO and PAX6D KO cell lines .

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Flow cytometry analysis of day 7 cell cultures under retinal differentiation from wildtype, PAX6 KO and PAX6D KO cell lines. Flow cytometry analysis of day 20 cell cultures under retinal differentiation from wildtype, PAX6 KO and PAX6D KO cell lines.


Quantification of SOX1 - / SOX2 + ratio in panel C ( n = 3 biological replicates for each sample ) .

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Quantification of SOX1-/SOX2+ ratio in panel C (n=3 biological replicates for each sample).


qPCR quantification of SOX1 in wildtype , PAX6 KO and PAX6D KO cell at day 20 of retinal differentiation ( n = 3 biological replicates for each sample ) .

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qPCR quantification of SOX1 in wildtype, PAX6 KO and PAX6D KO cell at day 20 of retinal differentiation (n=3 biological replicates for each sample).


qPCR quantification of PAX6A and PAX6D gene expression under the treatment of DOX ( n = 3 biological replicates for each sample ) .

qPCR quantification of PAX6A and PAX6D gene expression under the treatment of DOX (n=3 biological replicates for each sample).


Western blot analysis of PAX6A and PAX6D protein expression under the treatment of DOX .

Western blot analysis of PAX6A and PAX6D protein expression under the treatment of DOX.


qPCR quantification of SOX1 gene expression in the PAX6A TetOn and PAX6D TetOn cells ( n = 3 biological replicates for each group ) .

qPCR quantification of SOX1 gene expression in the PAX6A TetOn and PAX6D TetOn cells (n=3 biological replicates for each group).


qPCR quantification of retinal marker expression in PAX6D TetOn cells w / o DOX treatment ( n = 3 biological replicates for each group ) .

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qPCR quantification of retinal marker expression in PAX6D TetOn cells w/o DOX treatment (n=3 biological replicates for each group).


Cell population composition analysis with SOX1 and SOX2 staining from flow cytometry . SOX1 / SOX2 double positive cells represent the neuroepithelia fate while the SOX1 negative and SOX2 positive cells represent the neuroretina fate .

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Cell population composition analysis with SOX1 and SOX2 staining from flow cytometry. SOX1/SOX2 double positive cells represent the neuroepithelia fate while the SOX1 negative and SOX2 positive cells represent the neuroretina fate.


Verification of PAX6D target genes using PAX6D TetOn cells . Two sets of genes ( retinal and non - retinal neural genes ) are directly regulated by PAX6D in opposite directions ( n = 3 biological replicates for each group ) .

Verification of PAX6D target genes using PAX6D TetOn cells. Two sets of genes (retinal and non-retinal neural genes) are directly regulated by PAX6D in opposite directions (n=3 biological replicates for each group).


qPCR quantification of WNT8B in PAX6D KO cells at multiple time points during retinal differentiation ( n = 3 biological replicates for each group ) .

qPCR quantification of WNT8B in PAX6D KO cells at multiple time points during retinal differentiation (n=3 biological replicates for each group).


qPCR quantification of retinal gene expression under the effect of WNT agonist CHIR99021 and WNT antagonist IWR1 on retinal differentiation ( n = 3 biological replicates for each group ) .

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qPCR quantification of retinal gene expression under the effect of WNT agonist CHIR99021 and WNT antagonist IWR1 on retinal differentiation (n=3 biological replicates for each group).


qPCR quantification of retinal gene expression in PAX6D KO cells treated with WNT antagonist IWR1 to rescue the retinal differentiation ( n = 3 biological replicates for each group ) .

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qPCR quantification of retinal gene expression in PAX6D KO cells treated with WNT antagonist IWR1 to rescue the retinal differentiation (n=3 biological replicates for each group).


Western blot of WNT8B and ACTIN in PAX6D KO cells with treatment of untreat , control shRNA lentivirus , WNT8B shRNA lentivirus .

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Western blot of WNT8B and ACTIN in PAX6D KO cells with treatment of untreat, control shRNA lentivirus, WNT8B shRNA lentivirus.


qPCR quantification of retinal marker expression in PAX6D KO cells treated with control shRNA lentivirus and WNT8B shRNA lentivirus ( n = 3 biological replicates for each group ) .

qPCR quantification of retinal marker expression in PAX6D KO cells treated with control shRNA lentivirus and WNT8B shRNA lentivirus (n=3 biological replicates for each group).


qPCR quantification of retinal gene expression in PAX6D TetOn cells when PAX6D was induced by DOX in the presence of WNT agonist CHIR99021 ( n = 3 biological replicates for each group ) .

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qPCR quantification of retinal gene expression in PAX6D TetOn cells when PAX6D was induced by DOX in the presence of WNT agonist CHIR99021 (n=3 biological replicates for each group).


B , C . Distribution of the m6A peaks across host mRNAs ( B ) and EBV mRNAs ( C ) was analyzed based on the MeRIP - seq data .

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B, C. Distribution of the m6A peaks across host mRNAs (B) and EBV mRNAs (C) was analyzed based on the MeRIP-seq data.


B . The BZLF1 m6A levels were measured using MeRIP - qPCR in CNE2EBV , Raji and B95 - 8 cells with latent EBV infection or lytic reactivation . TPA ( 30 ng / ml ) and NaB ( 2 mM ) were used to treat the cells for 24 h to reactivate EBV . The cellular RNA was harvested for MeRIP - qPCR assays . The fold enrichment was determined by calculating the 2 - ΔCt of the MeRIP sample relative to the input sample . The mean value of the results in mock - treated cells was normalized to 1 .

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B. The BZLF1 m6A levels were measured using MeRIP-qPCR in CNE2EBV, Raji and B95-8 cells with latent EBV infection or lytic reactivation. TPA (30 ng/ml) and NaB (2 mM) were used to treat the cells for 24 h to reactivate EBV. The cellular RNA was harvested for MeRIP-qPCR assays. The fold enrichment was determined by calculating the 2-ΔCt of the MeRIP sample relative to the input sample. The mean value of the results in mock-treated cells was normalized to 1.


B . The EBV infection efficiency in HK1 cells transfected with the indicated siRNAs was analyzed by FACS . The cells were infected with EBV - GFP virus at 24 h after siRNA transfection . The ratio of GFP - positive cells was quantified by FACS at 24 hpi . The mean value of the GFP - positive rates in the siNC transfected HK1 cells was normalized to 1 .

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B. The EBV infection efficiency in HK1 cells transfected with the indicated siRNAs was analyzed by FACS. The cells were infected with EBV-GFP virus at 24 h after siRNA transfection. The ratio of GFP-positive cells was quantified by FACS at 24 hpi. The mean value of the GFP-positive rates in the siNC transfected HK1 cells was normalized to 1.


M . The relative EBV production in C666 cells transfected with YTHDF1 - specific siRNAs ( siY1 - 1 and siY1 - 2 ) or the siNC control was measured by qPCR . The DNA from the cell - free culture supernatant was harvested at 24 h after siRNA transfection . The number of EBV copies in the supernatant of the cells was quantified by qPCR using specific primers targeting BamHI - W fragment region of EBV . The mean value of EBV copies in the siNC cells was normalized to 1 .

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M. The relative EBV production in C666 cells transfected with YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control was measured by qPCR. The DNA from the cell-free culture supernatant was harvested at 24 h after siRNA transfection. The number of EBV copies in the supernatant of the cells was quantified by qPCR using specific primers targeting BamHI-W fragment region of EBV. The mean value of EBV copies in the siNC cells was normalized to 1.


B . The relative mRNA levels of BZLF1 and BRLF1 in the latently infected or reactivated ( 24 h post NaB treatment ) CNE2EBV cells transfected with the YTHDF1 - specific siRNAs ( siY1 - 1 and siY1 - 2 ) or the siNC control . After 24 h of siRNA transfection , CNE2EBV cells were treated with NaB ( 2 mM ) or mock for 24 h , followed by qRT - PCR analysis . All the gene expression levels were normalized to the housekeeping gene ACTB .

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B. The relative mRNA levels of BZLF1 and BRLF1 in the latently infected or reactivated (24 h post NaB treatment) CNE2EBV cells transfected with the YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control. After 24 h of siRNA transfection, CNE2EBV cells were treated with NaB (2 mM) or mock for 24 h, followed by qRT-PCR analysis. All the gene expression levels were normalized to the housekeeping gene ACTB.


F . The protein level of YTHDF1 , BZLF1 and ACTB in CNE2EBV cells following EBV reactivation ( 24 h post - NaB treatment ) , EBV infected HK1 or NPEC2 - Bmi1 cells at 24 hpi . The whole - cell protein was extracted at same time as the RNA .

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F. The protein level of YTHDF1, BZLF1 and ACTB in CNE2EBV cells following EBV reactivation (24 h post-NaB treatment), EBV infected HK1 or NPEC2-Bmi1 cells at 24 hpi. The whole-cell protein was extracted at same time as the RNA.


D . The relative enrichment of the BZLF1 and BRLF1 mRNA in CNE2EBV cells with transfected with siYTHDF2 , siYTHDF3 or siNC siRNA following EBV reactivation . After 24 h of siRNA transfection , CNE2EBV cells were treated with TPA ( 30 ng / ml ) and NaB ( 2 mM ) for 24 h . The RIP assays were performed using YTHDF1 - specific antibody or IgG control in these cells . Fold enrichment was determined by calculating the 2 - ΔCt of the RIP sample relative to the input sample .

D. The relative enrichment of the BZLF1 and BRLF1 mRNA in CNE2EBV cells with transfected with siYTHDF2, siYTHDF3 or siNC siRNA following EBV reactivation. After 24 h of siRNA transfection, CNE2EBV cells were treated with TPA (30 ng/ml) and NaB (2 mM) for 24 h. The RIP assays were performed using YTHDF1-specific antibody or IgG control in these cells. Fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample.


E , F . The relative m6A and YTHDF1 enrichment of the BZLF1 ( E ) or BRLF1 ( F ) mRNAs measured in CNE2EBV cells transfected with the METTL14 - specific siRNAs ( siM14 ) or siNC control following EBV reactivation . After 24 h of siRNA transfection , CNE2EBV cells were treated with TPA ( 30 ng / ml ) and NaB ( 2 mM ) for 24 h , followed by MeRIP and RIP assays . The fold enrichment was determined by calculating the 2 - ΔCt of the RIP sample relative to the input sample .

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E, F. The relative m6A and YTHDF1 enrichment of the BZLF1 (E) or BRLF1 (F) mRNAs measured in CNE2EBV cells transfected with the METTL14-specific siRNAs (siM14) or siNC control following EBV reactivation. After 24 h of siRNA transfection, CNE2EBV cells were treated with TPA (30 ng/ml) and NaB (2 mM) for 24 h, followed by MeRIP and RIP assays. The fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample.


D . The interaction between YTHDF1 and DCP2 was detected by co - IP in 293T cells .

D. The interaction between YTHDF1 and DCP2 was detected by co-IP in 293T cells.


F . The interactions between endogenous YTHDF1 and ZAP , DDX17 or DCP2 were detected by endogenous IP assays in CNE2EBV cells transfected with YTHDF2 - , YTHDF3 - specific siRNAs or the siNC control . YTHDF1 , YTHDF2 , YTHDF3 , ZAP , DDX17 , and DCP2 were detected in the immunoprecipitated complexes using specific antibodies , respectively .

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F. The interactions between endogenous YTHDF1 and ZAP, DDX17 or DCP2 were detected by endogenous IP assays in CNE2EBV cells transfected with YTHDF2-, YTHDF3-specific siRNAs or the siNC control. YTHDF1, YTHDF2, YTHDF3, ZAP, DDX17, and DCP2 were detected in the immunoprecipitated complexes using specific antibodies, respectively.


F . The relative mRNA levels of BZLF1 and BRLF1 in C666 cells transfected with YTHDF1 - specific siRNAs and XRN1 - specific siRNAs . The C666 cells were co - transfected with YTHDF1 - specific siRNAs and XRN1 - specific siRNAs for 24 h . The mRNA levels were determined using qPCR .

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F. The relative mRNA levels of BZLF1 and BRLF1 in C666 cells transfected with YTHDF1-specific siRNAs and XRN1-specific siRNAs. The C666 cells were co-transfected with YTHDF1-specific siRNAs and XRN1-specific siRNAs for 24 h. The mRNA levels were determined using qPCR.


G . The relative enrichment of BZLF1 and BRLF1 mRNAs by ZAP , DDX17 , or DCP2 in CNE2EBV cells following EBV reactivation . CNE2EBV cells were transfected with plasmids encoding myc - ZAP , myc - DDX17 , myc - DCP2 , or vector control . At 24 h post transfection , the cells were treated with TPA ( 30 ng / ml ) and NaB ( 2 mM ) for 24 h . The RIP assays were performed using Myc - beads . The fold enrichment was determined by calculating the 2 - ΔCt of the RIP sample relative to the input sample .

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G. The relative enrichment of BZLF1 and BRLF1 mRNAs by ZAP, DDX17, or DCP2 in CNE2EBV cells following EBV reactivation. CNE2EBV cells were transfected with plasmids encoding myc-ZAP, myc-DDX17, myc-DCP2, or vector control. At 24 h post transfection, the cells were treated with TPA (30 ng/ml) and NaB (2 mM) for 24 h. The RIP assays were performed using Myc-beads. The fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample.


H . Determination of the EBV infection efficiency in HK1 cells transfected with the XRN1 - specific siRNA or the siNC control . After 24 h of siRNA transfection , HK1 cells were infected with EBV and analyzed by FACS at 24 hpi .

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H. Determination of the EBV infection efficiency in HK1 cells transfected with the XRN1-specific siRNA or the siNC control. After 24 h of siRNA transfection, HK1 cells were infected with EBV and analyzed by FACS at 24 hpi.


( a ) Subcellular compartments from U937 cell extracts were separated by sedimentation velocity on discontinuous sucrose gradient and probed for the indicated proteins by immunoblotting . Numbers represent fractions from the gradient ( 1 , top ; 5 , bottom ) .

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(a) Subcellular compartments from U937 cell extracts were separated by sedimentation velocity on discontinuous sucrose gradient and probed for the indicated proteins by immunoblotting. Numbers represent fractions from the gradient (1, top; 5, bottom).


( b ) Immunoblot analysis of cellular compartments after purification of mitochondria by Qproteome Mitochondria Isolation Kit .

(b) Immunoblot analysis of cellular compartments after purification of mitochondria by Qproteome Mitochondria Isolation Kit.


( c ) Immunoblot analysis of KDEL and IRGM distribution in different cellular fractions after purification of mitochondria as in b .

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(c) Immunoblot analysis of KDEL and IRGM distribution in different cellular fractions after purification of mitochondria as in b.


d ) Intracellular localization of IRGM analysed by confocal microscopy . Cells were stained with antibodies against the indicated proteins or vitally with MitoTracker Red ( MTR ) where indicated . Images on the right are merged from images on the left . A - L ; endogenous IRGM localization in HeLa cells relative to calnexin , MTR , cytochrome c and COX IV . M - O ; IRGM localization in primary human macrophages relative to mitochondria visualized with cytochrome c antibody . Scale bars , 5 μm .

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d) Intracellular localization of IRGM analysed by confocal microscopy. Cells were stained with antibodies against the indicated proteins or vitally with MitoTracker Red (MTR) where indicated. Images on the right are merged from images on the left. A-L; endogenous IRGM localization in HeLa cells relative to calnexin, MTR, cytochrome c and COX IV. M-O; IRGM localization in primary human macrophages relative to mitochondria visualized with cytochrome c antibody. Scale bars, 5 μm.


( e ) Analysis of IRGM ( green ) and COX IV ( red ) intensity as a function of distance along the line shown in the inset . Image in inset corresponds to panels J - L in d . Uncropped images of blots are shown in Supplementary Information , Fig . S8 .

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(e) Analysis of IRGM (green) and COX IV (red) intensity as a function of distance along the line shown in the inset. Image in inset corresponds to panels J-L in d. Uncropped images of blots are shown in Supplementary Information, Fig. S8.


( a ) Purified mitochondria were left untreated , or were treated with Proteinase K ( Pr . K ) following osmotic shock ( OS ) , or solubilization of the total protein using Triton X - 100 ( TX - 100 ) , as indicated . Samples were analysed by immunoblotting with antibodies against the indicated proteins .

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(a) Purified mitochondria were left untreated, or were treated with Proteinase K (Pr.K) following osmotic shock (OS), or solubilization of the total protein using Triton X-100 (TX-100), as indicated. Samples were analysed by immunoblotting with antibodies against the indicated proteins.


( b ) Mitochondrial preparations were subjected to membrane disruption by freeze - thaw cycles as indicated and accessibility of the indicated proteins to Proteinase K was examined by immunoblotting .

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(b) Mitochondrial preparations were subjected to membrane disruption by freeze-thaw cycles as indicated and accessibility of the indicated proteins to Proteinase K was examined by immunoblotting.


( c ) Immunoblot analysis of membranous organelles from U937 cells separated by isopycnic sucrose density - gradient centrifugation . Numbers represent fractions from the gradient ( 1 , top ; 12 , bottom ) . Uncropped images of blots are shown in Supplementary Information , Fig . S8 .

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(c) Immunoblot analysis of membranous organelles from U937 cells separated by isopycnic sucrose density-gradient centrifugation. Numbers represent fractions from the gradient (1, top; 12, bottom). Uncropped images of blots are shown in Supplementary Information, Fig. S8.


( a ) Cells were transfected with control siRNA or with siRNA specific to IRGM ( top ) and DRP1 ( bottom ) for 48 h and protein samples from these cells were then analysed by immunoblotting with anti - IRGM and anti - DRP1 antibodies . Actin was used as a loading control .

(a) Cells were transfected with control siRNA or with siRNA specific to IRGM (top) and DRP1 (bottom) for 48 h and protein samples from these cells were then analysed by immunoblotting with anti-IRGM and anti-DRP1 antibodies. Actin was used as a loading control.


( b ) HeLa cells were treated with siRNA specific to either IRGM or DRP1 as indicated and were then labelled with MTR and analysed by live - cell confocal microscopy . Scale bars , 5 μm .

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(b) HeLa cells were treated with siRNA specific to either IRGM or DRP1 as indicated and were then labelled with MTR and analysed by live-cell confocal microscopy. Scale bars, 5 μm.


( c ) Cells were treated with either control siRNA or with DRP1 , IRGM , ATG7 or BECN1 ( Beclin 1 ) siRNAs , labelled with MTR and the percentage of cells with mitochondrial morphologies ranging from normal , punctiform ( dots ) and elongated were quantified . Definition of mitochondrial morphologies and quantification criteria are given in Supplementary Information , Figure S2 .

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(c) Cells were treated with either control siRNA or with DRP1, IRGM, ATG7 or BECN1 (Beclin 1) siRNAs, labelled with MTR and the percentage of cells with mitochondrial morphologies ranging from normal, punctiform (dots) and elongated were quantified. Definition of mitochondrial morphologies and quantification criteria are given in Supplementary Information, Figure S2.


( d ) Cells were transfected with either control siRNA or with siRNA specific to MFN1 / MFN2 , IRGM or IRGM and MFN1 / MFN2 , and then labelled with MTR and analysed for mitochondrial morphologies . Data are means ± s . e . m . ( c , n = 3 ; d , n = 4 ) . Asterisk indicates P 0 . 05 , double asterisks indicate P 0 . 01 and dagger indicates P > 0 . 05 ( t - test ) .

(d) Cells were transfected with either control siRNA or with siRNA specific to MFN1/MFN2, IRGM or IRGM and MFN1/MFN2, and then labelled with MTR and analysed for mitochondrial morphologies. Data are means ± s.e.m. (c, n = 3; d, n = 4). Asterisk indicates P 0.05, double asterisks indicate P 0.01 and dagger indicates P > 0.05 (t-test).


( a ) U937 cells were transfected with siRNA specific to FIS1 , DRP1 or IRGM ( or control oligonucleotides ) as indicated , followed by plasmid encoding GFP - LC3 . Autophagy was induced by treatment with hIFN - γ . GFP - LC3 puncta in cells were imaged by confocal microscopy . Scale bars , 5 μm . ( b ) LC3 puncta per cell were quantified from cells treated as in a .

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(a) U937 cells were transfected with siRNA specific to FIS1, DRP1 or IRGM (or control oligonucleotides) as indicated, followed by plasmid encoding GFP-LC3. Autophagy was induced by treatment with hIFN-γ. GFP-LC3 puncta in cells were imaged by confocal microscopy. Scale bars, 5 μm. (b) LC3 puncta per cell were quantified from cells treated as in a.


( c ) U937 cells were transfected with siRNA specific to FIS1 , DRP1 or IRGM ( or control oligonucleotides ) as indicated , followed by plasmid encoding GFP - LC3 . Autophagy was induced by starvation . GFP - LC3 puncta in cells were imaged by confocal microscopy . Scale bars , 5 μm .

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(c) U937 cells were transfected with siRNA specific to FIS1, DRP1 or IRGM (or control oligonucleotides) as indicated, followed by plasmid encoding GFP-LC3. Autophagy was induced by starvation. GFP-LC3 puncta in cells were imaged by confocal microscopy. Scale bars, 5 μm.


( d ) U937 cells were transfected with either control siRNA or siRNA specific to MFN1 / MFN2 , endogenous LC3 puncta per cell were quantified .

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(d) U937 cells were transfected with either control siRNA or siRNA specific to MFN1/MFN2, endogenous LC3 puncta per cell were quantified.


( e ) U937 cells were transfected with the indicated siRNA and infected with BCG . Autophagy was induced by starvation and phagosome maturation was analysed using LysoTracker probe ( LTR ) as a reporter of acidification .

(e) U937 cells were transfected with the indicated siRNA and infected with BCG. Autophagy was induced by starvation and phagosome maturation was analysed using LysoTracker probe (LTR) as a reporter of acidification.


( f ) U937 cells were treated with the indicated siRNA and infected with virulent M . tuberculosis H37Rv . Autophagy was induced by starvation , or cells were kept in full medium , as indicated . Colony forming units were counted , and expressed as a percentage of the number of plated bacterial colonies from untreated cells , to assess bacterial survival . Data are means ± s . e . m . ( n = 6 ; two independent transfections , 6 independent infections ) . Asterisk indicates P 0 . 05 , and dagger indicates P > 0 . 05 ( t - test ) for the indicated data .

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(f) U937 cells were treated with the indicated siRNA and infected with virulent M. tuberculosis H37Rv. Autophagy was induced by starvation, or cells were kept in full medium, as indicated. Colony forming units were counted, and expressed as a percentage of the number of plated bacterial colonies from untreated cells, to assess bacterial survival. Data are means ± s.e.m. (n = 6; two independent transfections, 6 independent infections). Asterisk indicates P 0.05, and dagger indicates P > 0.05 (t-test) for the indicated data.


( b ) HeLa cells were transfected with plasmids encoding fluorescent protein - tagged IRGMb , IRGMd WT ( wild - type IRGMd ) or IRGMS47N mutant ( control cells ; vector expressing GFP only ) , and after 48 h were labelled with MTR and imaged by live - cell confocal microscopy . Scale bars , 5 μm .

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(b) HeLa cells were transfected with plasmids encoding fluorescent protein-tagged IRGMb, IRGMd WT (wild-type IRGMd) or IRGMS47N mutant (control cells; vector expressing GFP only), and after 48 h were labelled with MTR and imaged by live-cell confocal microscopy. Scale bars, 5 μm.


( c ) Fluorescence intensity analysis of green and red channels along a line drawn through two adjacent cells , one GFP - IRGMd positive and one GFP - IRGMd negative , as indicated in the inset .

(c) Fluorescence intensity analysis of green and red channels along a line drawn through two adjacent cells, one GFP-IRGMd positive and one GFP-IRGMd negative, as indicated in the inset.


( d ) Quantification of GFP + MTR + cells from an experiment performed as in b .

(d) Quantification of GFP+ MTR+ cells from an experiment performed as in b.


( e ) Nitrocellulose filters spotted with the indicated lipids were incubated with GST or GST - IRGM . Binding was detected by immunoblotting .

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(e) Nitrocellulose filters spotted with the indicated lipids were incubated with GST or GST-IRGM. Binding was detected by immunoblotting.


( f ) Agarose beads ( control beads ) and cardiolipin - bound agarose beads ( CL beads ) were incubated with GST or GST - IRGMd . Proteins were eluted from the beads and immunoblotted . Two lanes on the left indicate immunoblotting of samples used to incubate beads .

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(f) Agarose beads (control beads) and cardiolipin-bound agarose beads (CL beads) were incubated with GST or GST-IRGMd. Proteins were eluted from the beads and immunoblotted. Two lanes on the left indicate immunoblotting of samples used to incubate beads.


( g ) Nitrocellulose filters spotted with the indicated lipids ( defined in e ) were incubated with wild - type IRGMd , IRGMdS47N , and the IRGM isoform IRGMb at the indicated concentrations . GST control is shown in Supplementary Information , Figure S1d . Membranes in e - g were probed with anti - GST antibody .

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(g) Nitrocellulose filters spotted with the indicated lipids (defined in e) were incubated with wild-type IRGMd, IRGMdS47N, and the IRGM isoform IRGMb at the indicated concentrations. GST control is shown in Supplementary Information, Figure S1d. Membranes in e-g were probed with anti-GST antibody.


( a ) HeLa cells transfected with plasmids encoding GFP or GFP - IRGMd were stained with MTR and imaged by live - cell confocal microscopy . Scale bars , 5 μm . ( b - d ) Quantification of mitochondrial morphologies in cells transfected with IRGMd , IRGMdS47N or IRGMb GFP fusions ( control cells express GFP only ) .

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(a) HeLa cells transfected with plasmids encoding GFP or GFP-IRGMd were stained with MTR and imaged by live-cell confocal microscopy. Scale bars, 5 μm. (b-d) Quantification of mitochondrial morphologies in cells transfected with IRGMd, IRGMdS47N or IRGMb GFP fusions (control cells express GFP only).


( e ) HeLa were transfected with the indicated siRNAs and vector encoding GFP - IRGMd , and labelled with MTR . The percentage of cells with mitochondrial morphologies ranging from normal , punctiform ( dots ) and elongated were quantified .

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(e)HeLa were transfected with the indicated siRNAs and vector encoding GFP-IRGMd, and labelled with MTR. The percentage of cells with mitochondrial morphologies ranging from normal, punctiform (dots) and elongated were quantified.


( f ) HeLa were transfected with the indicated siRNAs and vector encoding GFP - IRGMd ( or vector encoding GFP only , as a control ) and labelled with MTR . Percentages of GFP + cells with MTR staining at 48 h were quantified .

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(f) HeLa were transfected with the indicated siRNAs and vector encoding GFP-IRGMd (or vector encoding GFP only, as a control) and labelled with MTR. Percentages of GFP+ cells with MTR staining at 48 h were quantified.


( g ) Confocal microscopy images of HeLa cells transfected with vector encoding GFP - DRP1 ( A ) or GFP - IRGMd ( B - G ) and labelled with MTR ( A - D ) or antibodies against COX - IV ( F ) . A , steady - state DRP1 distribution ; B - D , translocation of IRGMd from the cytosol to mitochondria at indicated times post - transfection ; E - G , co - localization of IRGMd and COX - IV ( G is merged from E and F ) . Scale bars , 5 μm .

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(g) Confocal microscopy images of HeLa cells transfected with vector encoding GFP-DRP1 (A) or GFP-IRGMd (B-G) and labelled with MTR (A-D) or antibodies against COX-IV (F). A, steady-state DRP1 distribution; B-D, translocation of IRGMd from the cytosol to mitochondria at indicated times post-transfection; E-G, co-localization of IRGMd and COX-IV (G is merged from E and F). Scale bars, 5 μm.


( h ) HeLa cells , co - transfected with GFP - IRGMd , and control , BECN1 or ATG7 siRNA , were labelled with MTR 48 h post - transfection , imaged by live - cell microscopy , and percentages of GFP + cells that were also MTR + were quantified .

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(h) HeLa cells, co-transfected with GFP-IRGMd, and control, BECN1 or ATG7 siRNA, were labelled with MTR 48 h post-transfection, imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified.


( i ) Cells from Atg7 wild - type ( WT ) or Atg7 − / − MEFs transfected with vectors encoding GFP - IRGMd for 48 h were stained with MTR , imaged , and percentage of GFP + cells that were also MTR + cells was quantified .

(i) Cells from Atg7 wild-type (WT) or Atg7−/− MEFs transfected with vectors encoding GFP-IRGMd for 48 h were stained with MTR, imaged, and percentage of GFP+ cells that were also MTR+ cells was quantified.


( j ) Wild - type W2 ( Bax / Bak + / + ) or mutant D3 ( Bax / Bak − / − ) BMK cells were transfected with vectors encoding GFP - IRGMd and after 48 h were labelled with MTR . Cells were imaged by live - cell microscopy , and percentages of GFP + cells that were also MTR + were quantified .

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(j) Wild-type W2 (Bax/Bak+/+) or mutant D3 (Bax/Bak−/−) BMK cells were transfected with vectors encoding GFP-IRGMd and after 48 h were labelled with MTR. Cells were imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified.


( k ) HeLa cells were transfected with vectors encoding GFP - IRGMd , treated with z - VAD as indicated , and after 48 h stained with MTR . Cells were imaged by live - cell microscopy , and percentages of GFP + cells that were also MTR + were quantified . Data are means ± s . e . m . ( n = 3 ) . Dagger indicates P ≥ 0 . 05 , asterisk indicates P 0 . 05 and double asterisks indicate P 0 . 01 ( t - test ) for the indicated data .

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(k) HeLa cells were transfected with vectors encoding GFP-IRGMd, treated with z-VAD as indicated, and after 48 h stained with MTR. Cells were imaged by live-cell microscopy, and percentages of GFP+ cells that were also MTR+ were quantified. Data are means ± s.e.m. (n = 3). Dagger indicates P ≥ 0.05, asterisk indicates P 0.05 and double asterisks indicate P 0.01 (t-test) for the indicated data.


( a ) Percentage of HeLa cells transfected with vectors encoding GFP or GFP - IRGMd that were rounded 48 h post - transfection .

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(a) Percentage of HeLa cells transfected with vectors encoding GFP or GFP-IRGMd that were rounded 48 h post-transfection.


( b ) HeLa cells transfected with plasmids encoding GFP or GFP - IRGMd were stained with 7 - AAD after 48 h and percentages of GFP + cells that were also 7 - AAD + were quantified .

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(b) HeLa cells transfected with plasmids encoding GFP or GFP-IRGMd were stained with 7-AAD after 48 h and percentages of GFP+ cells that were also 7-AAD+ were quantified.


( c ) HeLa cells transfected with vectors encoding GFP - IRGMd versus GFP - IRGMdS47N and YFP - IRGMb ( vector encoding GFP only was transfected as a control ) were stained with 7 - AAD after 48 h , and percentages of GFP + ( for IRGMd and IRGMS47N ) or YFP + ( for IRGMb ) cells that were also 7 - AAD + were quantified .

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(c) HeLa cells transfected with vectors encoding GFP-IRGMd versus GFP-IRGMdS47N and YFP-IRGMb (vector encoding GFP only was transfected as a control) were stained with 7-AAD after 48 h, and percentages of GFP+ (for IRGMd and IRGMS47N) or YFP+ (for IRGMb) cells that were also 7-AAD+ were quantified.


( d ) HeLa cells were transfected with plamsids encoding GFP - IRGMd , GFP - IRGMDS47N or YFP - IRGMb ( vector encoding GFP only was transfected as a control ) , and after 48 h were stained with propidium iodide ( PI ) . Percentages of GFP + or YFP + cells that were also PI + were quantified .

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(d) HeLa cells were transfected with plamsids encoding GFP-IRGMd, GFP-IRGMDS47N or YFP-IRGMb (vector encoding GFP only was transfected as a control), and after 48 h were stained with propidium iodide (PI). Percentages of GFP+ or YFP+ cells that were also PI+ were quantified.


( e ) Images of HeLa cells transfected with vectors encoding GFP - IRGMd ( bottom ) were stained for active caspases 3 and 7 using FLICA dye . Staurosporine ( STS ; middle images ) was used as a positive control . Cells were transfected with vectors encoding GFP only as a control ( top ) .

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(e) Images of HeLa cells transfected with vectors encoding GFP-IRGMd (bottom) were stained for active caspases 3 and 7 using FLICA dye. Staurosporine (STS; middle images) was used as a positive control. Cells were transfected with vectors encoding GFP only as a control (top).


( f ) HeLa cells were transfected with plasmids encoding GFP - IRGMd and were stained with antibodies against HMGB1 ( absence of nuclear HMGB1 stain is a marker of HMGB1 release ) or DAPI . Top : images indicate GFP - IRGMd ( left ) , HMGB1 ( middle ) and DAPI ( right ) localization . Bottom : phase - contrast microscopy image of cells ( left ) and image merged from other panels ( right ) . Scale bars , 5 μm .

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(f) HeLa cells were transfected with plasmids encoding GFP-IRGMd and were stained with antibodies against HMGB1 (absence of nuclear HMGB1 stain is a marker of HMGB1 release) or DAPI. Top: images indicate GFP-IRGMd (left), HMGB1 (middle) and DAPI (right) localization. Bottom: phase-contrast microscopy image of cells (left) and image merged from other panels (right). Scale bars, 5 μm.


( g - i ) Cells were transfected with plasmids encoding GFP or GFP - IRGMd , and control or DRP1 siRNA , as indicated . After 48 h , ( g ) cells were stained with 7 - AAD and percentages of GFP + cells that were also 7 - AAD + were quantified , ( h ) rounded cells were quantified , or ( i ) cells were stained with HMGB1 and HMGB1 + nuclei were quantified . Data are means ± s . e . m . ( n = 3 ) . Asterisk indicates P 0 . 05 and double asterisk indicate P 0 . 01 ( t - test ) .

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(g-i) Cells were transfected with plasmids encoding GFP or GFP-IRGMd, and control or DRP1 siRNA, as indicated. After 48 h, (g) cells were stained with 7-AAD and percentages of GFP+ cells that were also 7-AAD+ were quantified, (h) rounded cells were quantified, or (i) cells were stained with HMGB1 and HMGB1+ nuclei were quantified. Data are means ± s.e.m. (n = 3). Asterisk indicates P 0.05 and double asterisk indicate P 0.01 (t-test).


( a , b ) HeLa cells were transfected with plasmids encoding GFP , CFP - IRGMa , GFP - IRGMc or GFP - IRGMd , and after 24 h ( a ) or 48 h ( b ) , stained with MTR . Percentages of positively transfected cells that were also MTR + were then quantified . ( c , d ) Cells were transfected as in a , b , and after 24 h ( c ) or 48 h ( d ) the percentages of positively transfected cells that were also rounded by morphology were quantified .

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(a, b) HeLa cells were transfected with plasmids encoding GFP, CFP-IRGMa, GFP-IRGMc or GFP-IRGMd, and after 24 h (a) or 48 h (b), stained with MTR. Percentages of positively transfected cells that were also MTR+ were then quantified. (c, d) Cells were transfected as in a, b, and after 24 h (c) or 48 h (d) the percentages of positively transfected cells that were also rounded by morphology were quantified.


B Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis from animals of the indicated timepoint ( in hours ) after the L4 stage . Animals have been exposed to the indicated knockdown RNAi bacterial strains or a strain that only contains the empty RNAi vector . * P < 0 . 001 , Student t - test corrected for multiple comparisons at each time point using the Holm - Sidek method . N = 20 animals per genotype and timepoint . Error bars indicate SEM .

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B Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from animals of the indicated timepoint (in hours) after the L4 stage. Animals have been exposed to the indicated knockdown RNAi bacterial strains or a strain that only contains the empty RNAi vector. *P<0.001, Student t-test corrected for multiple comparisons at each time point using the Holm-Sidek method. N=20 animals per genotype and timepoint. Error bars indicate SEM.


C , D Co - expression via the col - 19 promoter of UbG76V - GFP ( green ) and mRFP ( red ) in C . elegans hypodermis from a single integrated transgene at L4 + 48 hours . Wild - type animals are shown . Bar , 50 microns . UbG76V - GFP fluorescence is barely detectable . E , F Co - expression via the sur - 5 promoter of UbG76V - GFP ( green ) and mCherry ( red ) in C . elegans from two separate integrated transgenes ( with focus on the intestine ) at L4 + 48 hours . Wild - type animals are shown . Bar , 50 microns . UbG76V - GFP fluorescence is barely detectable . G , H Co - expression of UbG76V - GFP ( green ) and mRFP ( red ) in C . elegans hypodermis at L4 + 48 hours in dop - 1 ( vs100 ) mutants . Bar , 50 microns . Abundant , stable UbG76V - GFP fluorescence is observed . I , J Co - expression of UbG76V - GFP ( green ) and mCherry ( red ) in C . elegans from the sur - 5 promoter at L4 + 48 hours in dop - 1 ( vs100 ) mutants . Bar , 50 microns . Abundant , stable UbG76V - GFP fluorescence is observed , particularly in the intestine .

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C,D Co-expression via the col-19 promoter of UbG76V-GFP (green) and mRFP (red) in C. elegans hypodermis from a single integrated transgene at L4+48 hours. Wild-type animals are shown. Bar, 50 microns. UbG76V-GFP fluorescence is barely detectable.E,F Co-expression via the sur-5 promoter of UbG76V-GFP (green) and mCherry (red) in C. elegans from two separate integrated transgenes (with focus on the intestine) at L4+48 hours. Wild-type animals are shown. Bar, 50 microns. UbG76V-GFP fluorescence is barely detectable.G,H Co-expression of UbG76V-GFP (green) and mRFP (red) in C. elegans hypodermis at L4+48 hours in dop-1(vs100) mutants. Bar, 50 microns. Abundant, stable UbG76V-GFP fluorescence is observed.I,J Co-expression of UbG76V-GFP (green) and mCherry (red) in C. elegans from the sur-5 promoter at L4+48 hours in dop-1(vs100) mutants. Bar, 50 microns. Abundant, stable UbG76V-GFP fluorescence is observed, particularly in the intestine.


K Quantified fluorescence of UbG76V - GFP normalized to mRFP in the hypodermis from animals of the indicated timepoint ( in hours ) after the L4 stage and of the indicated genotype . * P < 0 . 001 , Student t - test corrected for multiple comparisons at each time point using the Holm - Sidek method . N = 20 animals per genotype and timepoint . Error bars indicate SEM .

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K Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis from animals of the indicated timepoint (in hours) after the L4 stage and of the indicated genotype. *P<0.001, Student t-test corrected for multiple comparisons at each time point using the Holm-Sidek method. N=20 animals per genotype and timepoint. Error bars indicate SEM.