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 .

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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.


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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 ) .

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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 .

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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 .

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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 .

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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 .

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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.


( A ) Tumor volume at the indicated time points after CT26 cell transplant in mice treated or not with LPS n ( number of animals per group ) = 6 .

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(A) Tumor volume at the indicated time points after CT26 cell transplant in mice treated or not with LPS n (number of animals per group)=6.


( B ) Tumor volume in mice depleted of DCs or NK cells before tumor cell transplant and LPS administration n ( number of animals per group ) = 5 .

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(B) Tumor volume in mice depleted of DCs or NK cells before tumor cell transplant and LPS administration n (number of animals per group)=5.


( C ) Tumor volume in SCIDmice treated or not with LPS n ( number of animals per group ) = 5 .

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(C) Tumor volume in SCIDmice treated or not with LPS n (number of animals per group)=5.


( D ) Tumor volume at the indicated time point after repetitive LPS injections as depicted in the bottom scheme n ( number of animals per group ) = 6 .

(D) Tumor volume at the indicated time point after repetitive LPS injections as depicted in the bottom scheme n (number of animals per group)=6.


( E ) Percent of NK cells within the tumor in mice treated or not with LPS and depleted or not of DCs n ( number of animals per group ) ≥ 4 . NT , control ; DT , diphtheria toxin treated animals to deplete DCs ; LPS , mice treated with LPS . Error bars depict SEM . Statistical significance was determined with a two - tail t test . NT , untreated .

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(E) Percent of NK cells within the tumor in mice treated or not with LPS and depleted or not of DCs n (number of animals per group) ≥ 4. NT, control; DT, diphtheria toxin treated animals to deplete DCs; LPS, mice treated with LPS. Error bars depict SEM. Statistical significance was determined with a two-tail t test. NT, untreated.


( A ) Explanted tumors at Day 12 after repetitive injections of LPS as depicted in the scheme at the bottom of the panel .

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(A) Explanted tumors at Day 12 after repetitive injections of LPS as depicted in the scheme at the bottom of the panel.


( B ) Detection of blood vessels by immunohistochemistry using anti - CD31 antibody ( brown staining ) in frozen sections of tumors from mice treated or not with with LPS as in the scheme in ( A ) . Nuclei are stained with hematoxylin . Two representative sections are shown , magnification 20X . Left panel scale bars , 500m ; right panel scale bars 100 m . Quantification of vessel mean dimensions and area coverage by vessels is also shown . Three sections from three tumors have been analyzed . Error bars depict SEM . Statistical significance was determined with a two - tail t test . NT , untreated .

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(B) Detection of blood vessels by immunohistochemistry using anti-CD31 antibody (brown staining) in frozen sections of tumors from mice treated or not with with LPS as in the scheme in (A). Nuclei are stained with hematoxylin. Two representative sections are shown, magnification 20X. Left panel scale bars, 500m; right panel scale bars 100 m. Quantification of vessel mean dimensions and area coverage by vessels is also shown. Three sections from three tumors have been analyzed. Error bars depict SEM. Statistical significance was determined with a two-tail t test. NT, untreated.


( A , B ) Absolute numbers of IFN - γ + and total NK cells in draining lymph nodes at the indicated time points before and after LPS administration in mice treated or not with FTY720 ( 25g / mouse ) to reduce the ingress of lymphocytes in the efferent lymphatics .

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(A,B) Absolute numbers of IFN-γ+ and total NK cells in draining lymph nodes at the indicated time points before and after LPS administration in mice treated or not with FTY720 (25g/mouse) to reduce the ingress of lymphocytes in the efferent lymphatics.


( C ) Tumor volume in mice treated or not with LPS and / or FTY720 according to the scheme .

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(C) Tumor volume in mice treated or not with LPS and/or FTY720 according to the scheme.


( D ) Percent of IFN - γ + NK cells within the tumor in mice treated or not with LPS and with FTY720 . n ( number of animals per group ) = 4 . Error bars depict SEM . FTY : FTY720 . Statistical significance was determined with a two - tail t test . NT , untreated .

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(D) Percent of IFN-γ+ NK cells within the tumor in mice treated or not with LPS and with FTY720. n (number of animals per group)=4. Error bars depict SEM. FTY: FTY720. Statistical significance was determined with a two-tail t test. NT, untreated.


( A ) Absolute numbers of NK cells in one of the 4 draining lymph nodes in mice treated or not with the anti - CD62L antibody to inhibit NK cel ingress in the lymph node from blood . Where indicated mice were challenged with LPS .

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(A) Absolute numbers of NK cells in one of the 4 draining lymph nodes in mice treated or not with the anti-CD62L antibody to inhibit NK cel ingress in the lymph node from blood. Where indicated mice were challenged with LPS.


( B ) Absolute numbers of IFN - γ + NK cells in one of the 4 draining lymph nodes before and after LPS administration in mice in which NK cell entry in the lymph node was blocked or not by anti - CD62L treatment .

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(B) Absolute numbers of IFN-γ+ NK cells in one of the 4 draining lymph nodes before and after LPS administration in mice in which NK cell entry in the lymph node was blocked or not by anti-CD62L treatment.


( C ) ( left panel ) Absolute numbers of endogenous and adoptively transferred ( CFSE + ) NK cells in the draining and contralateral lymph node 5 hours after LPS administration ; ( right panel ) percent of endogenous and adoptively transferred NK cells on total lymphocytes in the draining and contralateral lymph node 5 hours after LPS administration .

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(C) (left panel) Absolute numbers of endogenous and adoptively transferred (CFSE+) NK cells in the draining and contralateral lymph node 5 hours after LPS administration; (right panel) percent of endogenous and adoptively transferred NK cells on total lymphocytes in the draining and contralateral lymph node 5 hours after LPS administration.


( D ) Flow cytometry analysis of LPS draining and contralateral lymph node from mice that received CFSE labeled cells at the moment of LPS administration . Lymph node cells were stained with anti - CD3 , anti - DX5 and anti - IFN - γ antibodies and the analysis performed on gated CD3 - DX5high cells . One representative of 4 independent experiments is shown .

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(D) Flow cytometry analysis of LPS draining and contralateral lymph node from mice that received CFSE labeled cells at the moment of LPS administration. Lymph node cells were stained with anti-CD3, anti-DX5 and anti-IFN-γ antibodies and the analysis performed on gated CD3-DX5high cells. One representative of 4 independent experiments is shown.


( E ) Tumor volume in mice treated or not with LPS , where indicated mice received anti - CD62L antibody 12h before LPS treatment .

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(E) Tumor volume in mice treated or not with LPS, where indicated mice received anti-CD62L antibody 12h before LPS treatment.


( F ) Percent of IFN - γ + NK cells within the tumor in mice treated or not with LPS and with the anti - CD62L antibody .

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(F) Percent of IFN-γ+ NK cells within the tumor in mice treated or not with LPS and with the anti-CD62L antibody.


( A ) Representative image of an NK cell ( red ) interacting with four DCs ( green ) . ( B , C , D ) Plot of the confinement ratio ( CR , black squares ) , the normalized NK speed Vthr ( ti ) ( red circles ) and the digitalized NK - DC distance , DistD ( ti ) ( green triangles ) as a function of time for the NK cell reported in the image . The B , C and D panels correspond respectively to the interaction with the DC1 , DC2 and DC4 displayed in ( A ) . For reference also the normalized distance Dist ( ti ) is reported in the plots ( blue down triangles ) . The time periods in which all the three conditions discussed in the text are fulfilled are marked with red striped rectangles in the plots . The green up - triangles indicate the parameter TonToff defined as TonToff = - 1 if Dist ( ti ) < d = 25 µm and TonToff = - 1 if Dist ( ti ) d and indicate the putative interactions according to a more simplified algorithm based on the DC - NK cell distance alone .

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(A) Representative image of an NK cell (red) interacting with four DCs (green). (B, C, D) Plot of the confinement ratio (CR, black squares), the normalized NK speed Vthr(ti) (red circles) and the digitalized NK-DC distance, DistD(ti) (green triangles) as a function of time for the NK cell reported in the image. The B, C and D panels correspond respectively to the interaction with the DC1, DC2 and DC4 displayed in (A). For reference also the normalized distance Dist(ti) is reported in the plots (blue down triangles). The time periods in which all the three conditions discussed in the text are fulfilled are marked with red striped rectangles in the plots. The green up-triangles indicate the parameter TonToff defined as TonToff = -1 if Dist(ti) < d = 25 µm and TonToff = -1 if Dist(ti) d and indicate the putative interactions according to a more simplified algorithm based on the DC-NK cell distance alone.


( A ) NK and DC trajectories as they are tracked by Volocity software on the 4D volumes collected in lymph nodes on the TPE microscope . The sequence of images shows the interaction between an NK cell ( red ) and a DC ( green ) . In the top panels the NK cell moves according to a directional random motion while in the lower left and middle panels the NK cell seems to recognize the DC and to go back . In the right lower panel the stable contact DC - NK cell is clearly visible . ( B ) Time duration of the DC - NK contacts in steady state ( - LPS ) and inflammatory ( + LPS ) conditions . Bars indicate the mean of the distribution . The percent of cells showing long interaction times ( ≥ 15 min ) are shown . ( C ) NK cell 3D velocities . 3D velocities of NK cells taking contacts with DCs before ( black ) and 5 h ( green ) after LPS treatment .

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(A) NK and DC trajectories as they are tracked by Volocity software on the 4D volumes collected in lymph nodes on the TPE microscope. The sequence of images shows the interaction between an NK cell (red) and a DC (green). In the top panels the NK cell moves according to a directional random motion while in the lower left and middle panels the NK cell seems to recognize the DC and to go back. In the right lower panel the stable contact DC-NK cell is clearly visible. (B) Time duration of the DC-NK contacts in steady state (-LPS) and inflammatory (+LPS) conditions. Bars indicate the mean of the distribution. The percent of cells showing long interaction times (≥ 15 min) are shown. (C) NK cell 3D velocities. 3D velocities of NK cells taking contacts with DCs before (black) and 5 h (green) after LPS treatment.


( A ) Differential interference contrast images ( left panels ) and confocal microscopy analysis of IL - 18 ( right panels ) in a DC / NK cell conjugate after 2 hours of coculture in the presence of LPS and in unstimulated cells ( NT ) . Representative images from 3 experiments are shown , magnification 63X . Scale bars , 4 . 36 m .

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(A) Differential interference contrast images (left panels) and confocal microscopy analysis of IL-18 (right panels) in a DC/NK cell conjugate after 2 hours of coculture in the presence of LPS and in unstimulated cells (NT). Representative images from 3 experiments are shown, magnification 63X. Scale bars, 4.36 m.


( B ) IFN - γ release by NK cells cultured in the presence of supernatants recovered from untreated DCs ( untreated ) or DCs treated O / N with LPS ( LPS ) . Where indicated IL - 18 was added to the cultures .

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(B) IFN-γ release by NK cells cultured in the presence of supernatants recovered from untreated DCs (untreated) or DCs treated O/N with LPS (LPS). Where indicated IL-18 was added to the cultures.


( C ) Cell - contact dependent activation of NK cells by DCs depends on IL - 18 . Unstimulated or LPS - activated DCs and NK cells were cocultured in the same wells ( DC + NK + LPS ) or separated by a porous membrane ( ( DC + LPS ) / NK ) . Where indicated IL - 18 was added to the transwells ( ( DCs + LPS ) / ( NK + IL - 18 ) ) . NK cells alone were also cultured in the presence of the three selected cytokines ; IL - 2 and IFN - β in the upper chamber and NK cells and IL - 18 in the lower chamber . IFN - γ in the supernatant was then measured by ELISA after 8 hours of coculture . ( B , C ) . n ( number of independent experiments ) = 3 . Error bars depict SEM .

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(C) Cell-contact dependent activation of NK cells by DCs depends on IL-18. Unstimulated or LPS-activated DCs and NK cells were cocultured in the same wells (DC + NK + LPS) or separated by a porous membrane ((DC+LPS)/NK). Where indicated IL-18 was added to the transwells ((DCs+LPS)/(NK+IL-18)). NK cells alone were also cultured in the presence of the three selected cytokines; IL-2 and IFN-β in the upper chamber and NK cells and IL-18 in the lower chamber. IFN-γ in the supernatant was then measured by ELISA after 8 hours of coculture. (B, C). n (number of independent experiments)=3. Error bars depict SEM.


A . IFT22 nucleotide - binding experiments . Fluorescence measurements using increasing amounts of TbIFT22 and TbIFT22 / 74342 - 401 / 81397 - 450 core complex incubated with mant - labeled GDP ( mant - GDP ) or non - hydrolysable GTP / ATP analogs ( mant - GMPPNP / mant - AMPPNP ) . The fluorescence intensity is plotted as a function of protein concentration . Data were fitted to a single - site binding equation for determination of the dissociation constant ( Kd ) . Kd values and standard deviations are calculated from three independent experiments .

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A. IFT22 nucleotide-binding experiments. Fluorescence measurements using increasing amounts of TbIFT22 and TbIFT22/74342-401/81397-450 core complex incubated with mant-labeled GDP (mant-GDP) or non-hydrolysable GTP / ATP analogs (mant-GMPPNP / mant-AMPPNP). The fluorescence intensity is plotted as a function of protein concentration. Data were fitted to a single-site binding equation for determination of the dissociation constant (Kd). Kd values and standard deviations are calculated from three independent experiments.


D . Top : Cartoon representation of IFT22 ( grey ) with positions of two nucleotide - binding mutants highlighted . GTP is shown in stick representation and Mg2 + as a ball . D175 ( blue ) is the unusual residue binding the guanine base while S19 ( pink ) is a conserved residue required for coordination of the Mg2 + cation and is commonly mutated to an asparagine to prevent nucleotide binding . Bottom : Nucleotide - binding experiments of IFT22 nucleotide - binding mutants D175E , D175A and S19N with fluorescently labeled nucleotides . Only the S19N mutation ( light pink ) abolished IFT22 nucleotide - binding ability completely .

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D. Top: Cartoon representation of IFT22 (grey) with positions of two nucleotide-binding mutants highlighted. GTP is shown in stick representation and Mg2+ as a ball. D175 (blue) is the unusual residue binding the guanine base while S19 (pink) is a conserved residue required for coordination of the Mg2+ cation and is commonly mutated to an asparagine to prevent nucleotide binding. Bottom: Nucleotide-binding experiments of IFT22 nucleotide-binding mutants D175E, D175A and S19N with fluorescently labeled nucleotides. Only the S19N mutation (light pink) abolished IFT22 nucleotide-binding ability completely.


C . SDS - PAGE gel of a Ni2 + - NTA pulldown using His - tagged TbIFT22 ( WT and mutants ) and untagged CrIFT25 / 27 / 74 / 81 . WT TbIFT22 is able to pull down the Cr tetrameric complex , thus forming a chimeric IFT - B1 pentamer , while both the A86R and S19N mutant fail to bind the complex .

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C. SDS-PAGE gel of a Ni2+-NTA pulldown using His-tagged TbIFT22 (WT and mutants) and untagged CrIFT25/27/74/81. WT TbIFT22 is able to pull down the Cr tetrameric complex, thus forming a chimeric IFT-B1 pentamer, while both the A86R and S19N mutant fail to bind the complex.


A . SDS - PAGE gel of a Ni2 + - NTA pulldown using His - tagged IFT74342 - 401 / 81397 - 450 and untagged IFT22 ( WT and mutants ) . Pulldowns were done from cell lysates of co - expressed proteins . Lanes 1 - 4 show similar total expression levels of the different co - expressed constructs ( input samples ) . Lanes 5 - 8 show pulldown elutions . The IFT22S19N ( inactive GTPase mutant ) and the IFT22A86R mutant ( IFT74 / 81 - binding mutant ) did not interact with the IFT74342 - 401 / 81397 - 450 complex in the pull - down experiment .

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A. SDS-PAGE gel of a Ni2+-NTA pulldown using His-tagged IFT74342-401/81397-450 and untagged IFT22 (WT and mutants). Pulldowns were done from cell lysates of co-expressed proteins. Lanes 1-4 show similar total expression levels of the different co-expressed constructs (input samples). Lanes 5-8 show pulldown elutions. The IFT22S19N (inactive GTPase mutant) and the IFT22A86R mutant (IFT74/81-binding mutant) did not interact with the IFT74342-401/81397-450 complex in the pull-down experiment.


B . SDS - PAGE gel of a Ni2 + - NTA pulldown using the His - tagged IFT7479 - 401 / 811 - 450 and untagged IFT22 ( WT and mutants ) . Pulldowns were done from cell lysates of co - expressed proteins . Lanes 1 - 4 show similar total expression levels of the different co - expressed constructs ( input samples ) . Lanes 5 - 8 show pulldown elutions . The IFT22S19N ( inactive GTPase mutant ) and the IFT22A86R mutant ( IFT74 / 81 - binding mutant ) did not interact with the IFT7479 - 401 / 811 - 450 complex in the pull - down experiment .

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B. SDS-PAGE gel of a Ni2+-NTA pulldown using the His-tagged IFT7479-401/811-450 and untagged IFT22 (WT and mutants). Pulldowns were done from cell lysates of co-expressed proteins. Lanes 1-4 show similar total expression levels of the different co-expressed constructs (input samples). Lanes 5-8 show pulldown elutions. The IFT22S19N (inactive GTPase mutant) and the IFT22A86R mutant (IFT74/81-binding mutant) did not interact with the IFT7479-401/811-450 complex in the pull-down experiment.


C . SDS - PAGE gel of a Ni2 + - NTA pull - down of full - length IFT25 / 27 / 74 / 81 complex with His - tagged IFT22 ( WT and S19N mutant ) . The nucleotide - binding deficient mutant IFT22S19N interacts with IFT25 / 27 / 74 / 81 in the pull - down experiment .

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C. SDS-PAGE gel of a Ni2+-NTA pull-down of full-length IFT25/27/74/81 complex with His-tagged IFT22 (WT and S19N mutant). The nucleotide-binding deficient mutant IFT22S19N interacts with IFT25/27/74/81 in the pull-down experiment.


D . SDS - PAGE gel of a Ni2 + - NTA pull - down of full - length IFT25 / 27 / 74 / 81 complex with His - tagged IFT22 ( WT and A86R mutant ) . The IFT74 / 81 - binding mutant IFT22A86R fails to pull down the full - length tetrameric complex .

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D. SDS-PAGE gel of a Ni2+-NTA pull-down of full-length IFT25/27/74/81 complex with His-tagged IFT22 (WT and A86R mutant). The IFT74/81-binding mutant IFT22A86R fails to pull down the full-length tetrameric complex.


A . Western blot analysis of the IFT22RNAi + GFP : : IFT22S19N cell line probed with the anti - IFT22 antibody ( bottom ) and with an anti - paraflagellar rod ( PFR ) as loading control ( top ) .

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A. Western blot analysis of the IFT22RNAi+GFP::IFT22S19N cell line probed with the anti-IFT22 antibody (bottom) and with an anti-paraflagellar rod (PFR) as loading control (top).


B . IFA of the indicated trypanosome cell lines using the mAb25 ( marker for the axoneme , left panels ) and an anti - IFT172 antibody ( marker for IFT , right panels ) . The top panels show the phase contrast images merged with DAPI ( cyan ) that stains nuclear and mitochondrial DNA . Scale bars correspond to 5 µm .

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B. IFA of the indicated trypanosome cell lines using the mAb25 (marker for the axoneme, left panels) and an anti-IFT172 antibody (marker for IFT, right panels). The top panels show the phase contrast images merged with DAPI (cyan) that stains nuclear and mitochondrial DNA. Scale bars correspond to 5 µm.


C . Kymographs showing the movement of the GFP : : IFT22S19N in the presence ( left ) or the absence ( right ) of the IFT22 endogenous protein . Note the improved signal - to - noise ratio in the absence of endogenous IFT22 . P and D mark the proximal and distal ends of the flagellum , respectively . Scale bars are 5 seconds ( time , vertical ) and 5 µm ( length , horizontal )

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C. Kymographs showing the movement of the GFP::IFT22S19N in the presence (left) or the absence (right) of the IFT22 endogenous protein. Note the improved signal-to-noise ratio in the absence of endogenous IFT22. P and D mark the proximal and distal ends of the flagellum, respectively. Scale bars are 5 seconds (time, vertical) and 5 µm (length, horizontal)


D . Dot plot representation of flagellum length in the indicated cell lines and conditions . For each condition , 100 flagella were measured and statistical analysis was performed with the ANOVA test . Significant differences are indicated with a star ( P < 0 .

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D. Dot plot representation of flagellum length in the indicated cell lines and conditions. For each condition, 100 flagella were measured and statistical analysis was performed with the ANOVA test. Significant differences are indicated with a star (P<0.0001


A . Western blot analysis of the IFT22RNAi + GFP : : IFT22A86R cell line probed with the anti - IFT22 antibody ( bottom ) and with an anti - BiP as loading control ( top ) .

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A. Western blot analysis of the IFT22RNAi+GFP::IFT22A86R cell line probed with the anti-IFT22 antibody (bottom) and with an anti-BiP as loading control (top).


B . IFA with the indicated cell lines using the mAb25 ( marker for the axoneme , central panels ) and an anti - IFT172 antibody ( marker for IFT , bottom panels ) . The top panels show the phase contrast images merged with DAPI ( cyan ) that stains nuclear and mitochondrial DNA . The arrowheads indicate the presence of short flagella stained with the Mab25 antibody in the IFT22RNAi cells . Scale bars correspond to 5 µm .

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B. IFA with the indicated cell lines using the mAb25 (marker for the axoneme, central panels) and an anti-IFT172 antibody (marker for IFT, bottom panels). The top panels show the phase contrast images merged with DAPI (cyan) that stains nuclear and mitochondrial DNA. The arrowheads indicate the presence of short flagella stained with the Mab25 antibody in the IFT22RNAi cells. Scale bars correspond to 5 µm.


C . Sections of IFT22RNAi + GFP : : IFT22rescue ( top panels ) or IFT22RNAi + GFP : : IFT22A86R cells ( bottom panels ) were analysed by transmission electron microscopy . Sections through the flagellar pocket , the transition zone and the flagellum are shown . Scale bars are 500 nm ( flagellar pocket sections ) or 200 nm ( transition zone and flagellum sections ) . The white arrow indicates an endocytic vesicle budding off the flagellar pocket whereas the black one points at an IFT train . ( Axo = axoneme , TZ = transition zone , BB = basal body )

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C. Sections of IFT22RNAi+GFP::IFT22rescue (top panels) or IFT22RNAi+GFP::IFT22A86R cells (bottom panels) were analysed by transmission electron microscopy. Sections through the flagellar pocket, the transition zone and the flagellum are shown. Scale bars are 500 nm (flagellar pocket sections) or 200 nm (transition zone and flagellum sections). The white arrow indicates an endocytic vesicle budding off the flagellar pocket whereas the black one points at an IFT train. (Axo = axoneme, TZ = transition zone, BB = basal body)


E . Naïve MCF7 cells and MCF7aTT cells were exposed to the indicated stress conditions for 3 h , and cell viability was assessed by staining dead cells with trypan blue . The bar graphs represent the percentage of trypan blue excluding cells . ( means + / - SD , n = 3 , numbers above bars indicate p values calculated with the two - stage linear step - up procedure of Benjamini , Krieger and Yekutieli ) .

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E. Naïve MCF7 cells and MCF7aTT cells were exposed to the indicated stress conditions for 3 h, and cell viability was assessed by staining dead cells with trypan blue. The bar graphs represent the percentage of trypan blue excluding cells. (means +/- SD, n = 3, numbers above bars indicate p values calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli).


A . Whole lysates of MCF7 and MCF7aTT cells exposed to heat shock and / or proteasome ( MG132 , 20 μM , 1h ) and HSP70 ( VER - 155008 , 50 μM , 1h ) inhibitors were prepared as described in Materials and Methods . Total lysate , soluble fractions and insoluble fractions were stained by SYPRO Ruby ( left ) and quantified ( right ) . ( means + / - SD , n = 3 , numbers above the bars indicate p values calculated with the two - stage linear step - up procedure of Benjamini , Krieger and Yekutieli ) .

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A. Whole lysates of MCF7 and MCF7aTT cells exposed to heat shock and/or proteasome (MG132, 20 μM, 1h) and HSP70 (VER-155008, 50 μM, 1h) inhibitors were prepared as described in Materials and Methods. Total lysate, soluble fractions and insoluble fractions were stained by SYPRO Ruby (left) and quantified (right). (means +/- SD, n = 3, numbers above the bars indicate p values calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli).


C . The soluble and insoluble fractions shown in ( A ) were assayed for the level of K48 - linked polyubiquitin , and polyubiquitylated proteins were quantified ( blue line graph , means + / - SEM , n = 4 , numbers above the bars indicate p values ) . The data for bulk protein obtained in ( A ) were overlaid onto the graph for comparison ( red line , means + / - SEM , n = 3 ) .

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C. The soluble and insoluble fractions shown in (A) were assayed for the level of K48-linked polyubiquitin, and polyubiquitylated proteins were quantified (blue line graph, means +/- SEM, n = 4, numbers above the bars indicate p values). The data for bulk protein obtained in (A) were overlaid onto the graph for comparison (red line, means +/- SEM, n = 3).


B . The same experiment as described in ( A ) but with MCF7 cells exposed to MG132 ( 20 μM , 1 h ) or puromycin ( 100 μg / ml , 10 min ) . Means + / - SEM , n = 2 . C . Lysate from MCF7 cells exposed to MG132 ( 20 μM , 1 h ) or puromycin ( 100 μg / ml , 10 min ) as indicated were subjected to immunoprecipitation with anti - puromycin antibodies , followed by assaying for co - precipitation of K48 - ubiquitylated proteins . Actin is shown as a reference .

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B. The same experiment as described in (A) but with MCF7 cells exposed to MG132 (20 μM, 1 h) or puromycin (100 μg/ml, 10 min). Means +/- SEM, n = 2. C. Lysate from MCF7 cells exposed to MG132 (20 μM, 1 h) or puromycin (100 μg/ml, 10 min) as indicated were subjected to immunoprecipitation with anti-puromycin antibodies, followed by assaying for co-precipitation of K48-ubiquitylated proteins. Actin is shown as a reference.


B . Same experiment as in ( A ) with thermotolerant MCF7aTT cells incubated with the HSP70 inhibitor VER - 155008 ( 50 μM , 1h ) or MG132 ( 20 μM , 1h ) . The signal obtained for ribosomal protein RPS19 and RPL7 are shown for reference , documenting residual polysomes despite the flat UV trace . Right panels are quantifications of K48 - ub ( means + / - SEM , n = 2 ) , black vertical lines indicate splicing of lanes that were run on different gels due to limitations in lane capacity . The bar graphs represent the P / M ratios ( means + / - SD , n = 3 , numbers above bars indicate p values calculated with the two - stage linear step - up procedure of Benjamini , Krieger and Yekutieli ) . C . Sucrose density gradient fractions described in ( B ) were assayed for partitioning of HSPA1 and HSPH1 by immunoblotting . HSPA1 data were quantified in a line graph ( means + / - SEM , n = 4 ) . D

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B. Same experiment as in (A) with thermotolerant MCF7aTT cells incubated with the HSP70 inhibitor VER-155008 (50 μM, 1h) or MG132 (20 μM, 1h). The signal obtained for ribosomal protein RPS19 and RPL7 are shown for reference, documenting residual polysomes despite the flat UV trace. Right panels are quantifications of K48-ub (means +/- SEM, n = 2), black vertical lines indicate splicing of lanes that were run on different gels due to limitations in lane capacity. The bar graphs represent the P/M ratios (means +/- SD, n = 3, numbers above bars indicate p values calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli). C. Sucrose density gradient fractions described in (B) were assayed for partitioning of HSPA1 and HSPH1 by immunoblotting. HSPA1 data were quantified in a line graph (means +/- SEM, n = 4). D


D . Cell lysate was spun through a 30 % sucrose cushion to pellet polysomes . Nascent proteins were released from polysomes by dissociating 80S ribosomes with EDTA ( see Appendix Figures S3 ) . Soluble and insoluble fractions were prepared by centrifugation and assayed for the level of K48 - linked polyubiquitin . The levels of K48 - linked polyubiquitin were quantified ( means + / - SD , n = 3 ) . Immunoblots within black squares were run on the same gel but irrelevant lanes were removed as indicated by white lines .

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D. Cell lysate was spun through a 30% sucrose cushion to pellet polysomes. Nascent proteins were released from polysomes by dissociating 80S ribosomes with EDTA (see Appendix Figures S3). Soluble and insoluble fractions were prepared by centrifugation and assayed for the level of K48-linked polyubiquitin. The levels of K48-linked polyubiquitin were quantified (means +/- SD, n = 3). Immunoblots within black squares were run on the same gel but irrelevant lanes were removed as indicated by white lines.


D . Total cell lysates were prepared from naïve MCF7 and thermoresistant MCF7aTT cells , and puromycin labeled peptides were pull down as illustrated in the scheme . Precipitates were assayed for the presence of K48 - linked polyubiquitin , chaperones , and proteasome subunits by immunoblotting .

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D. Total cell lysates were prepared from naïve MCF7 and thermoresistant MCF7aTT cells, and puromycin labeled peptides were pull down as illustrated in the scheme. Precipitates were assayed for the presence of K48-linked polyubiquitin, chaperones, and proteasome subunits by immunoblotting.


E . Whole lysates of cells KYSE150 and KYSE150HSPH1 - / - cells exposed to the indicated temperature conditions ( aTT scheme and heat shock ) were fractionated into soluble and insoluble fractions as described in Materials and Methods . Insoluble fractions were assayed for the level of K48 - linked polyubiquitin ( left panel ) . Total levels of HSPA1 and HSPH1 are shown for reference . K48 ubiquitin signals were quantified and displayed in a bar graph ( means + / - SD , n = 4 ) .

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E. Whole lysates of cells KYSE150 and KYSE150HSPH1-/- cells exposed to the indicated temperature conditions (aTT scheme and heat shock) were fractionated into soluble and insoluble fractions as described in Materials and Methods. Insoluble fractions were assayed for the level of K48-linked polyubiquitin (left panel). Total levels of HSPA1 and HSPH1 are shown for reference. K48 ubiquitin signals were quantified and displayed in a bar graph (means +/- SD, n = 4).


F . Lysates of cells KYSE150 and KYSE150HSPH1 - / - cells exposed to the indicated temperature conditions ( aTT scheme and heat shock ) were obtained and polysomes were isolated by centrifugation through a 30 % sucrose cushion . Whole cell lysates ( left panel ) and the polysomal pellets ( right panel ) were assayed by immunoblotting for the levels of HSPH1 , HSPA1 and proteasome subunits .

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F. Lysates of cells KYSE150 and KYSE150HSPH1-/- cells exposed to the indicated temperature conditions (aTT scheme and heat shock) were obtained and polysomes were isolated by centrifugation through a 30% sucrose cushion. Whole cell lysates (left panel) and the polysomal pellets (right panel) were assayed by immunoblotting for the levels of HSPH1, HSPA1 and proteasome subunits.


A . Representative micrographs of tissue sections derived from oral squamous cell carcinomas ( OSCC ) and esophageal squamous cell carcinomas ( ESCC ) stained with the indicated antibodies by immunohistochemistry ( IHC ) . Top panels show routine H & E staining . Black squares point to regions magnified in the adjacent panels to the right .

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A. Representative micrographs of tissue sections derived from oral squamous cell carcinomas (OSCC) and esophageal squamous cell carcinomas (ESCC) stained with the indicated antibodies by immunohistochemistry (IHC). Top panels show routine H&E staining. Black squares point to regions magnified in the adjacent panels to the right.


C . Kaplan - Meier plots showing the correlation between HSPH1 mRNA expression level and the survival of patients with ESCC . The expression data was obtained from and visualized with KM Plotter ( www . kmplot . com , ( Nagy et al , 2018 ) using the Pan Cancer algorithm at default settings .

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C. Kaplan-Meier plots showing the correlation between HSPH1 mRNA expression level and the survival of patients with ESCC. The expression data was obtained from and visualized with KM Plotter (www.kmplot.com, (Nagy et al, 2018) using the Pan Cancer algorithm at default settings.


D . KYSE150 and KYSE150HSPH1 - / - cells were injected into nude mice , and xenograft tumor growth was monitored over time ( left panel ; mean tumor volume + / - SEM , n = 4 with 5 animals per n ) . Mean tumor weights at the end of the experiments were determined ( means + / - SD , n = 4 with 5 animals per n , number above the bar graph represent p values , * p < 0 . 05 , * * p < 0 . 01 ( calculated with the two - stage linear step - up procedure of Benjamini , Krieger and Yekutieli ) ) .

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D. KYSE150 and KYSE150HSPH1-/- cells were injected into nude mice, and xenograft tumor growth was monitored over time (left panel; mean tumor volume +/- SEM, n = 4 with 5 animals per n). Mean tumor weights at the end of the experiments were determined (means +/- SD, n = 4 with 5 animals per n, number above the bar graph represent p values, * p<0.05, **p<0.01 (calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli)).


( A ) Representative images of WT , Mfn1 KO and Mfn2 KO MEFs transfected for 48 h with a plasmid encoding mitochondria - targeted RFP ( mt - RFP ) . Scale bar : 5 μm .

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(A) Representative images of WT, Mfn1 KO and Mfn2 KO MEFs transfected for 48 h with a plasmid encoding mitochondria-targeted RFP (mt-RFP). Scale bar: 5 μm.


( A ) Representative images of WT and Mfn2 KO MEFs co - transfected with plasmids encoding mitochondria - targeted RFP and ER - targeted GFP . Scale bar : 5 μm .

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(A) Representative images of WT and Mfn2 KO MEFs co-transfected with plasmids encoding mitochondria-targeted RFP and ER-targeted GFP. Scale bar: 5 μm.


( B ) After 48 h , the cells were fixed and the degree of colocalization between ER and mitochondria based on red and green fluorescent pixel overlap was analyzed using Mander ' s coefficient ( n = 30 cells analyzed in 3 independent experiments ) . Data are presented as mean ± SEM . * p < 0 . 05 , two - tailed Student ' s t - test .

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(B) After 48 h, the cells were fixed and the degree of colocalization between ER and mitochondria based on red and green fluorescent pixel overlap was analyzed using Mander's coefficient (n= 30 cells analyzed in 3 independent experiments). Data are presented as mean ± SEM. *p<0.05, two-tailed Student's t-test.


I - K ) Mitochondrial Ca2 + was determined in WT and Mfn2 KO MEFs loaded with Rhod2 . The effect of ( I ) caffeine - induced ( 20 mM ) , ( J ) IP3R activation with ATP - induced ( 100 μM ) and ( K ) histamine - induced ( 100 μM ) ER Ca2 + release on mitochondrial Ca2 + was measured ( n = 30 cells from 3 independent experiments ) . Data are presented as mean ± SEM .

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I-K) Mitochondrial Ca2+ was determined in WT and Mfn2 KO MEFs loaded with Rhod2. The effect of (I) caffeine-induced (20 mM), (J) IP3R activation with ATP-induced (100 μM) and (K) histamine-induced (100 μM) ER Ca2+ release on mitochondrial Ca2+ was measured (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM.


WT and Mfn2 KO cells were transfected with plasmids encoding : GFP and the indicated combination of non - targeting siRNA ( siCT ) , siRNA targeting MCU ( siMCU ) , ChiMERA or control ( indicated as WT or Mfn2 alone ) plasmids . ( R ) MMP was analyzed ( n = 90 cells analyzed in 3 independent experiments ) .

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WT and Mfn2 KO cells were transfected with plasmids encoding: GFP and the indicated combination of non-targeting siRNA (siCT), siRNA targeting MCU (siMCU), ChiMERA or control (indicated as WT or Mfn2 alone) plasmids. (R) MMP was analyzed (n= 90 cells analyzed in 3 independent experiments).


WT and Mfn2 KO cells were transfected with plasmids encoding GFP and the indicated combination of non - targeting siRNA ( siCT ) , siRNA targeting MCU ( siMCU ) , ChiMERA or control ( indicated as WT or Mfn2 alone ) plasmids . ( S ) ATP levels of GFP + cells was analyzed after 24 h treatment with 2DG ( 10 mM ) ( n = 3 independent experiments ) .

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WT and Mfn2 KO cells were transfected with plasmids encoding GFP and the indicated combination of non-targeting siRNA (siCT), siRNA targeting MCU (siMCU), ChiMERA or control (indicated as WT or Mfn2 alone) plasmids. (S) ATP levels of GFP+ cells was analyzed after 24 h treatment with 2DG (10 mM) (n= 3 independent experiments).


WT and Mfn2 KO MEFs were co - transfected with plasmids encoding : mt - RFP and ER - Mfn2 or control ( CT ) plasmid . After 48 h , the cells were fixed . ( C ) Representative images . Scale bar = 5 μm .

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WT and Mfn2 KO MEFs were co-transfected with plasmids encoding: mt-RFP and ER-Mfn2 or control (CT) plasmid. After 48 h, the cells were fixed. (C) Representative images. Scale bar= 5 μm.


WT and Mfn2 KO MEFs were co - transfected with plasmids encoding : J - N ) GFP and ER - Mfn2 or control ( CT ) plasmid . ( J ) MMP was determined . The values were normalized to surrounding untransfected cells . ( n = 90 cells analyzed in 3 independent experiments ) . ( K ) After 24 h , the transfected cells were sorted and plated for another 24 h , after which cells were treated with 2 - DG ( 10 mM ) for 6 h , and ATP levels were measured ( n = 3 independent experiments ) . ( L ) Oxygen consumption was measured and ( M ) RCR and ( N ) SRC was calculated ( n = 4 independent experiments ) . Data are presented as mean ± SEM . Data information : * p < 0 . 05 , one - way ANOVA followed by Tukey ' s post hoc test . Note that experiments were performed at the same time , so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity .

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WT and Mfn2 KO MEFs were co-transfected with plasmids encoding: J-N) GFP and ER-Mfn2 or control (CT) plasmid. (J) MMP was determined. The values were normalized to surrounding untransfected cells. (n= 90 cells analyzed in 3 independent experiments). (K) After 24 h, the transfected cells were sorted and plated for another 24 h, after which cells were treated with 2-DG (10 mM) for 6 h, and ATP levels were measured (n= 3 independent experiments). (L) Oxygen consumption was measured and (M) RCR and (N) SRC was calculated (n= 4 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.


WT and Mfn1 / 2 double KO were transfected with : A ) mt - RFP , ER - GFP and plasmids expressing the indicated proteins . Mitochondria - ER colocalization was analyzed using Mander ' s coefficient ( n = 15 cells analyzed in 3 independent experiments ) . Data are presented as mean ± SEM . B ) GFP and ERMITO - RLuc and indicated expression vectors . After 24 h , the transfected cells were sorted and plated for another 24 h , after which RLuc was analyzed ( n = 3 independent experiments ) . Data are presented as mean ± SEM . C ) Data information : The red dashed line indicates the WT cells results . * p < 0 . 05 versus Mfn1 / 2 DKO cells , one - way ANOVA followed by Tukey ' s post hoc test . Note that experiments were performed at the same time , so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity .

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WT and Mfn1/2 double KO were transfected with: A) mt-RFP, ER-GFP and plasmids expressing the indicated proteins. Mitochondria-ER colocalization was analyzed using Mander's coefficient (n= 15 cells analyzed in 3 independent experiments). Data are presented as mean ± SEM. B) GFP and ERMITO-RLuc and indicated expression vectors. After 24 h, the transfected cells were sorted and plated for another 24 h, after which RLuc was analyzed (n= 3 independent experiments). Data are presented as mean ± SEM. C) Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.


WT and Mfn1 / 2 double KO were transfected with : C ) GFP and the indicated plasmids . After 48 h , mitochondrial Ca2 + was determined ( n = 30 cells from 3 independent experiments ) . Data are presented as mean ± SEM . Data information : The red dashed line indicates the WT cells results . * p < 0 . 05 versus Mfn1 / 2 DKO cells , one - way ANOVA followed by Tukey ' s post hoc test . Note that experiments were performed at the same time , so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity .

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WT and Mfn1/2 double KO were transfected with: C) GFP and the indicated plasmids. After 48 h, mitochondrial Ca2+ was determined (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM. Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.


WT and Mfn1 / 2 double KO were transfected with : D ) LAR - GECO1 and the indicated plasmids . After 48 h ER Ca2 + was released with caffeine ( 20 mM ) as indicated ( n = 25 - 27 cells from 3 independent experiments ) . Data are presented as mean ± SEM . Data information : The red dashed line indicates the WT cells results . * p < 0 . 05 versus Mfn1 / 2 DKO cells , one - way ANOVA followed by Tukey ' s post hoc test . Note that experiments were performed at the same time , so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity .

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WT and Mfn1/2 double KO were transfected with: D) LAR-GECO1 and the indicated plasmids. After 48 h ER Ca2+ was released with caffeine (20 mM) as indicated (n= 25-27 cells from 3 independent experiments). Data are presented as mean ± SEM. Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.


WT and Mfn1 / 2 double KO were transfected with : E ) GFP and the indicated plasmid . After 48 h , the effect of caffeine - induced ( 20 mM ) ER Ca2 + release on mitochondrial Ca2 + was measured ( n = 25 cells from 3 independent experiments ) . Data are presented as mean ± SEM .

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WT and Mfn1/2 double KO were transfected with: E) GFP and the indicated plasmid. After 48 h, the effect of caffeine-induced (20 mM) ER Ca2+ release on mitochondrial Ca2+ was measured (n= 25 cells from 3 independent experiments). Data are presented as mean ± SEM.


B ) Representative Western blots of the indicated proteins during the neuronal maturation in vitro ( n = 3 independent experiments ) . C ) Representative Western blots of the indicated proteins of WT and Mfn2 KO neurons at DIV7 ( n = 3 independent experiments ) . D

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B) Representative Western blots of the indicated proteins during the neuronal maturation in vitro (n= 3 independent experiments). C) Representative Western blots of the indicated proteins of WT and Mfn2 KO neurons at DIV7 (n= 3 independent experiments). D


D ) Primary neurons of tamoxifen - inducible Mfn2 KO mice were transfected at DIV3 with ER - GFP , mt - RFP plus the indicated plasmids . Tamoxifen - treated ( for 72 h ) and vehicle - treated neurons ( WT ; red dashed line ) were fixed and mitochondria - ER co - localization was analyzed using Mander ' s coefficient ( n = 15 neurons analyzed in 3 independent experiments ) . Data are presented as mean ± SEM . Data information : * p < 0 . 05 versus WT ; # p < 0 . 05 versus Mfn2 KO neurons , one - way ANOVA followed by Tukey ' s post - hoc test . Scale bar : 500 μm .

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D) Primary neurons of tamoxifen-inducible Mfn2 KO mice were transfected at DIV3 with ER-GFP, mt-RFP plus the indicated plasmids. Tamoxifen-treated (for 72 h) and vehicle-treated neurons (WT; red dashed line) were fixed and mitochondria-ER co-localization was analyzed using Mander's coefficient (n= 15 neurons analyzed in 3 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05 versus WT; #p<0.05 versus Mfn2 KO neurons, one-way ANOVA followed by Tukey's post-hoc test. Scale bar: 500 μm.