E : Phosphorylation of Chk1 is not impaired in Ubc9 D100A cells . Stable U2OS cells were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 2 . 5 mM hydroxyurea . At the indicated times , cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies .

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E: Phosphorylation of Chk1 is not impaired in Ubc9 D100A cells. Stable U2OS cells were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 2.5 mM hydroxyurea. At the indicated times, cells were lysed in Laemmli buffer and analysed by immunoblotting with the indicated antibodies.


A : Impaired y - H2AX and 53BP1 accumulation in Ubc9 D100A cells . Stable U2OS cells were transfected with siRNA against endogenous Ubc9 for 72 h . Cells were treated with 125 µM H2O2 and immunofluorescence microscopy was performed . Left panel : representative immunofluorescence images . Right panel : quantification of cells with γ - H2AX and 53BP1 foci . Error bars represent S . E . M , n = 3 independent experiments * p < 0 . 05 .

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A: Impaired y-H2AX and 53BP1 accumulation in Ubc9 D100A cells. Stable U2OS cells were transfected with siRNA against endogenous Ubc9 for 72 h. Cells were treated with 125 µM H2O2 and immunofluorescence microscopy was performed. Left panel: representative immunofluorescence images. Right panel: quantification of cells with γ-H2AX and 53BP1 foci. Error bars represent S.E.M, n=3 independent experiments * p < 0.05.


B : H2O2 exposure leads to prolonged DNA damage in Ubc9 wt and Ubc9 D100A cells . Cells were treated as described in A . Stable U2OS cells were transfected with siRNA against endogenous Ubc9 for 72 h . Cells were treated with 125 µM H2O2 and immunofluorescence microscopy was performed . An alkaline comet assay was performed to evaluate the DNA damage . Representative images are shown . Error intervals represent S . E . M , n = 4 independent experiments .

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B: H2O2 exposure leads to prolonged DNA damage in Ubc9 wt and Ubc9 D100A cells. Cells were treated as described in A. Stable U2OS cells were transfected with siRNA against endogenous Ubc9 for 72 h. Cells were treated with 125 µM H2O2 and immunofluorescence microscopy was performed. An alkaline comet assay was performed to evaluate the DNA damage. Representative images are shown. Error intervals represent S.E.M, n=4 independent experiments.


A : Diamide is inducing the SUMO E1 ~ E2 disulfide but not ATM phosphorylation . U2OS stable cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 500 µM diamide or 500 µM H2O2 . Cells were subsequently washed in PBS with 20 mM NEM and lysed in Laemmli buffer . The lysates were resolved by SDS - PAGE .

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A: Diamide is inducing the SUMO E1~E2 disulfide but not ATM phosphorylation. U2OS stable cell populations were depleted from endogenous Ubc9 by siRNA for 72 h before addition of 500 µM diamide or 500 µM H2O2. Cells were subsequently washed in PBS with 20 mM NEM and lysed in Laemmli buffer. The lysates were resolved by SDS-PAGE.


B : Left panel : The PP2A inhibitor okadaic acid rescues the y - H2AX defect of the " Ubc9 DA " cells . " Ubc9 wt " or " Ubc9 DA " cells were treated with 0 . 25 µM okadaic acid for 30 min . After addition of 250 µM H2O2 in fresh medium for 3 hours , immunofluorescence microscopy was performed as described in Fig . 7A . Error bars represent S . E . M , n = 3 independent experiments . Right panel : Representative immunofluorescence images .

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B: Left panel: The PP2A inhibitor okadaic acid rescues the y-H2AX defect of the "Ubc9 DA" cells. "Ubc9 wt" or "Ubc9 DA" cells were treated with 0.25 µM okadaic acid for 30 min. After addition of 250 µM H2O2 in fresh medium for 3 hours, immunofluorescence microscopy was performed as described in Fig. 7A. Error bars represent S.E.M, n=3 independent experiments. Right panel: Representative immunofluorescence images.


C : Left panel : Ubc9 D100A enhances sensitivity to Ara - C or etoposide . Cell survival assays were performed as described in Fig . 5C . Cells were exposed to Ara - C or etoposide ( VP16 ) for 1 h and evaluated 10 days later . Right panel : N - acetylcysteine treatment rescues the higher toxicity of Ara - C or etoposide on " Ubc9 DA " cells . Cells were treated with 0 . 5 mM NAC 16 h prior to addition of etoposide or Ara - C . Graphs represent means of three to four independent experiments , error bars represent S . E . M . * p < 0 . 05 .

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C: Left panel: Ubc9 D100A enhances sensitivity to Ara-C or etoposide. Cell survival assays were performed as described in Fig. 5C. Cells were exposed to Ara-C or etoposide (VP16) for 1 h and evaluated 10 days later. Right panel: N-acetylcysteine treatment rescues the higher toxicity of Ara-C or etoposide on "Ubc9 DA" cells. Cells were treated with 0.5 mM NAC 16 h prior to addition of etoposide or Ara-C. Graphs represent means of three to four independent experiments, error bars represent S.E.M. * p < 0.05.


( b ) IRGMC mRNA was significantly enriched in miR - 196B complexes . Extracts of cells expressing FLAG - tagged - AGO1 and transfected with biotinylated miR - 196B or control miR - 20 as indicated and then with IRGMC or IRGMT plasmids were submitted to tandem affinity purification ( immunoprecipitation with FLAG antibodies followed by affinity purification on streptavidin beads ) . IRGM mRNA variants were quantified using quantitative RT - PCR ; results are presented as the ratio between miR - 196B and miR - 20 ( non - relevant miRNA ) pull - downs and the mean of three independent experiments ± standard deviation ( s . d . ) . IP strep , immunoprecipitation straptavidine .

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(b) IRGMC mRNA was significantly enriched in miR-196B complexes. Extracts of cells expressing FLAG-tagged-AGO1 and transfected with biotinylated miR-196B or control miR-20 as indicated and then with IRGMC or IRGMT plasmids were submitted to tandem affinity purification (immunoprecipitation with FLAG antibodies followed by affinity purification on streptavidin beads). IRGM mRNA variants were quantified using quantitative RT-PCR; results are presented as the ratio between miR-196B and miR-20 (non-relevant miRNA) pull-downs and the mean of three independent experiments ± standard deviation (s.d.). IP strep, immunoprecipitation straptavidine.


( c ) HEK293 cells ( IRGMC / C ) were transfected with either FLAG - tagged IRGMC or FLAG - tagged IRGMT plasmids and co - transfected with miR - 196B . Immunoblotting with an IRGM antibody revealed the specificity of the downregulation effect mediated by the miRNA IRGM mRNA interaction . Quantification of the immunoblot signals are presented as IRGM expression relative to actin ( mean of at least three independent experiments ± s . d . ) .

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(c) HEK293 cells (IRGMC/C) were transfected with either FLAG-tagged IRGMC or FLAG-tagged IRGMT plasmids and co-transfected with miR-196B. Immunoblotting with an IRGM antibody revealed the specificity of the downregulation effect mediated by the miRNA IRGM mRNA interaction. Quantification of the immunoblot signals are presented as IRGM expression relative to actin (mean of at least three independent experiments ± s.d.).


( a ) Representative in situ hybridization of frozen sections obtained from colon biopsies of genotyped healthy controls ( n = 40 ) or individuals with Crohn ' s disease ( CD ) ( n = 67 ) without or with active inflammation and labeled for miR - 196A . ( L , lumen ; LP , lamina propria ) . Scale bars in the upper panel , 25 μm ; scale bars in the lower panel , 10 μm .

(a) Representative in situ hybridization of frozen sections obtained from colon biopsies of genotyped healthy controls (n = 40) or individuals with Crohn's disease (CD) (n = 67) without or with active inflammation and labeled for miR-196A. (L, lumen; LP, lamina propria). Scale bars in the upper panel, 25 μm; scale bars in the lower panel, 10 μm.


( b ) Epithelial or laminal fractions were captured from sections of biopsies of healthy controls ( n = 8 ) or individuals with Crohn ' s disease with no inflammation ( n = 16 ) , quiescent ( defined as low - grade inflammation ) ( n = 8 ) or acute inflammation ( n = 8 ) using laser capture microdissection . After RNA extraction , miR - 196A ( black bars ) and miR - 196B ( white bars ) relative expression was analyzed using RNU19 , 44 and U6 . To overcome possible inter - individual bias , the lamina propria fraction value was used for relative quantification . Due to high differences in expression between healthy and inflamed tissues , the results are presented as a log2 - fold ratio . Error bars indicate the s . d . of the ΔΔCt values .

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(b) Epithelial or laminal fractions were captured from sections of biopsies of healthy controls (n = 8) or individuals with Crohn's disease with no inflammation (n = 16), quiescent (defined as low-grade inflammation) (n = 8) or acute inflammation (n = 8) using laser capture microdissection. After RNA extraction, miR-196A (black bars) and miR-196B (white bars) relative expression was analyzed using RNU19, 44 and U6. To overcome possible inter-individual bias, the lamina propria fraction value was used for relative quantification. Due to high differences in expression between healthy and inflamed tissues, the results are presented as a log2-fold ratio. Error bars indicate the s.d. of the ΔΔCt values.


( c ) Representative in situ staining for IRGM of TMAs from colon biopsies of healthy individuals or individuals with Crohn ' s disease with a defined genotype in a healthy non - inflamed or an acute inflamed phase . Scale bars , 32 μm . ( d ) Mean ( black line ) , s . e . m . ( white box ) and 95 % CI of the mean of the IRGM expression level for 40 healthy subjects ( 32 individuals with C / C and 8 individuals with C / T ) and 67 individuals with Crohn ' s disease ( 45 with C / C and 22 with C / T ) with quiescent or inflamed colon mucosa . We performed statistical analysis using ANOVA ( P = 0 . 0015 ) and an unpaired Student ' s t - test ( the one tail P value is indicated on the figure ) .

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(c) Representative in situ staining for IRGM of TMAs from colon biopsies of healthy individuals or individuals with Crohn's disease with a defined genotype in a healthy non-inflamed or an acute inflamed phase. Scale bars, 32 μm. (d) Mean (black line), s.e.m. (white box) and 95% CI of the mean of the IRGM expression level for 40 healthy subjects (32 individuals with C/C and 8 individuals with C/T) and 67 individuals with Crohn's disease (45 with C/C and 22 with C/T) with quiescent or inflamed colon mucosa. We performed statistical analysis using ANOVA (P = 0.0015) and an unpaired Student's t-test (the one tail P value is indicated on the figure).


( a ) The basal flux of autophagy is affected by IRGM expression level . HEK293 cells transfected with an IRGM - expressing plasmid , miR - 196 or siIRGM were treated with bafilomycin A1 for 2 h and processed for immunoblotting with anti - LC3B . ( b ) Quantification of LC3 - II relative to actin ( mean of three independent experiments ± s . d . ) .

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(a) The basal flux of autophagy is affected by IRGM expression level. HEK293 cells transfected with an IRGM-expressing plasmid, miR-196 or siIRGM were treated with bafilomycin A1 for 2 h and processed for immunoblotting with anti-LC3B. (b) Quantification of LC3-II relative to actin (mean of three independent experiments ± s.d.).


( c ) Downregulation of IRGM expression by miR - 196 abrogates AIEC - mediated autophagy in cells treated with autophagic inhibitors ( Inh ) or transfected with miR - 196B and infected for 4 h with AIEC LF82 ( mean of three independent experiments ± s . d . ) .

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(c) Downregulation of IRGM expression by miR-196 abrogates AIEC-mediated autophagy in cells treated with autophagic inhibitors (Inh) or transfected with miR-196B and infected for 4 h with AIEC LF82 (mean of three independent experiments ± s.d.).


( d ) Confocal microscopic examination of LC3 revealed a significant decrease in the percentage of LC3 - associated ( red ) LF82 bacteria ( green ) in miR - 196 transfected cells compared to control cells ( mean ± s . d . ) .

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(d) Confocal microscopic examination of LC3 revealed a significant decrease in the percentage of LC3-associated (red) LF82 bacteria (green) in miR-196 transfected cells compared to control cells (mean ± s.d.).


( e ) miR - 196 transfection leads to increased intracellular LF82 replication . Results are expressed as a fold increase ± s . e . m . of intracellular bacteria .

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(e) miR-196 transfection leads to increased intracellular LF82 replication. Results are expressed as a fold increase ± s.e.m. of intracellular bacteria.


( f ) IRGM overexpression did not inhibit autophagic flux and it increased LC3 - II accumulation slightly in response to AIEC infection . HEK293 cells were transfected with IRGM - expressing plasmid , treated with autophagic inhibitors and infected with AIEC bacteria for 4 h ( mean of three independent experiments ± s . d . ) .

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(f) IRGM overexpression did not inhibit autophagic flux and it increased LC3-II accumulation slightly in response to AIEC infection. HEK293 cells were transfected with IRGM-expressing plasmid, treated with autophagic inhibitors and infected with AIEC bacteria for 4 h (mean of three independent experiments ± s.d.).


( g ) Confocal microscopic examination showed an increased percentage of LC3 - associated AIEC bacteria in IRGM cells compared to control cells ( mean ± s . d . ) . ( h ) IRGM overexpression led to a high rate of intracellular replication of LF82 bacteria . ( i ) Most of the bacteria reside in non - acidic vacuoles , as shown with lysotracker at 8 h post infection ( means of three independent experiments ± s . d . ) .

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(g) Confocal microscopic examination showed an increased percentage of LC3-associated AIEC bacteria in IRGM cells compared to control cells (mean ± s.d.). (h) IRGM overexpression led to a high rate of intracellular replication of LF82 bacteria. (i) Most of the bacteria reside in non-acidic vacuoles, as shown with lysotracker at 8 h post infection (means of three independent experiments ± s.d.).


( a ) Kinetics of the appearance of P14 cells in spleens of C57BL / 6 mice given adoptive transfer of 1 × 104 P14 cells , monitored after infection of recipients with LCMV Armstrong strain .

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(a) Kinetics of the appearance of P14 cells in spleens of C57BL/6 mice given adoptive transfer of 1 × 104 P14 cells, monitored after infection of recipients with LCMV Armstrong strain.


( b ) Flow cytometry of cell size at various stages of P14 differentiation , assessed by forward scatter ( FSC ) .

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(b) Flow cytometry of cell size at various stages of P14 differentiation, assessed by forward scatter (FSC).


( c ) Immunoblot analysis of LC3b and p62 at various stages of P14 differentiation . Data are representative of two independent experiments with samples pooled from 5 - 20 mice for each time point .

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(c) Immunoblot analysis of LC3b and p62 at various stages of P14 differentiation. Data are representative of two independent experiments with samples pooled from 5-20 mice for each time point.


( b ) Flow cytometry of P14 cells from the spleens of recipient mice given adoptive transfer of P14 cells transduced with retrovirus as in a , assessed on days 5 , 8 and 30 after infection of recipients with LCMV . Numbers in blue outlined areas indicate percent GFP − cells among transduced ( Thy - 1 . 1 + ) P14 cells . ( c ) Summary of results in b , showing autophagy activity as percent GFP − cells . * P ≤ 0 . 0005 ( unpaired t - test ) .

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(b) Flow cytometry of P14 cells from the spleens of recipient mice given adoptive transfer of P14 cells transduced with retrovirus as in a, assessed on days 5, 8 and 30 after infection of recipients with LCMV. Numbers in blue outlined areas indicate percent GFP− cells among transduced (Thy-1.1+) P14 cells. (c) Summary of results in b, showing autophagy activity as percent GFP− cells. *P ≤ 0.0005 (unpaired t-test).


( d ) Multispectral - imaging flow cytometry of P14 cells from the spleens of recipient mice given adoptive transfer of P14 cells transduced with retrovirus as in a , assessed on day 8 after infection , showing GFP signals from GFP + transduced ( CD45 . 1 + Thy - 1 . 1 + ) P14 cells . Original magnification , × 60 . ( e ) Summary of results in d , presented as the frequency of cells with more than one GFP + punctum among vector transduced Thy - 1 . 1 + P14 cells . Each symbol represents an individual mouse ; small horizontal lines indicate the mean ( ± s . e . m . ) . Data are representative of three independent experiments ( b , d ) or three independent experiments with three or more mice per group ( e ; error bars , s . e . m . ) or six mice per group ( c ; error bars , s . e . m . ) .

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(d) Multispectral-imaging flow cytometry of P14 cells from the spleens of recipient mice given adoptive transfer of P14 cells transduced with retrovirus as in a, assessed on day 8 after infection, showing GFP signals from GFP+ transduced (CD45.1+ Thy-1.1+) P14 cells. Original magnification, ×60. (e) Summary of results in d, presented as the frequency of cells with more than one GFP+ punctum among vector transduced Thy-1.1+ P14 cells. Each symbol represents an individual mouse; small horizontal lines indicate the mean (±s.e.m.). Data are representative of three independent experiments (b,d) or three independent experiments with three or more mice per group (e; error bars, s.e.m.) or six mice per group (c; error bars, s.e.m.).


( b ) Flow cytometry of splenocytes from recipient mice ( n = 6 ) given adoptive transfer of P14 cells transduced with retrovirus as in a , assessed on days 5 and 8 after infection of recipients with LCMV ( left ) , and quantification of P14 cells per spleen ( right ) . Numbers adjacent to outlined areas ( left ) indicate percent P14 ( CD45 . 1 + ) cells . * P ≤ 0 . 0005 ( unpaired t - test ) .

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(b) Flow cytometry of splenocytes from recipient mice (n = 6) given adoptive transfer of P14 cells transduced with retrovirus as in a, assessed on days 5 and 8 after infection of recipients with LCMV (left), and quantification of P14 cells per spleen (right). Numbers adjacent to outlined areas (left) indicate percent P14 (CD45.1+) cells. *P ≤ 0.0005 (unpaired t-test).


( c ) Flow cytometry of the size of P14 cells transduced with wild - type mCherry - GFP - LC3b retrovirus ( assessed as in Fig . 1b ) in mice treated as in b , assessed at days 5 and 8 after infection .

(c) Flow cytometry of the size of P14 cells transduced with wild-type mCherry-GFP-LC3b retrovirus (assessed as in Fig. 1b) in mice treated as in b, assessed at days 5 and 8 after infection.


( d ) Efficiency of retroviral transduction ( left ) and expression of GFP and mCherry ( right ) in retrovirus - transduced P14 cells , assessed by flow cytometry gated on CD45 . 1 + P14 cells ( left ) or CD45 . 1 + Thy - 1 . 1 + P14 cells ( right ) . Numbers in quadrants indicate percent CD45 . 1 + Thy - 1 . 1 − ( untransduced ) P14 cells ( top left ) or CD45 . 1 + Thy - 1 . 1 + ( transduced ) P14 cells ( top right ; magenta ) ( left ) or percent cells in each ( right ) . ( e ) Ratio of the geometric mean fluorescence intensity ( gMFI ) of GFP to that of mCherry ( GFP / mCherry ) for the cells in d ; each symbol represents an individual mouse ; numbers above lines connecting groups indicate difference in ratio . Data are representative of two independent experiments with three or more mice per group ( error bars ( b ) , s . e . m . ) .

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(d) Efficiency of retroviral transduction (left) and expression of GFP and mCherry (right) in retrovirus-transduced P14 cells, assessed by flow cytometry gated on CD45.1+ P14 cells (left) or CD45.1+Thy-1.1+ P14 cells (right). Numbers in quadrants indicate percent CD45.1+Thy-1.1− (untransduced) P14 cells (top left) or CD45.1+Thy-1.1+ (transduced) P14 cells (top right; magenta) (left) or percent cells in each (right). (e) Ratio of the geometric mean fluorescence intensity (gMFI) of GFP to that of mCherry (GFP/mCherry) for the cells in d; each symbol represents an individual mouse; numbers above lines connecting groups indicate difference in ratio. Data are representative of two independent experiments with three or more mice per group (error bars (b), s.e.m.).


( a ) Genomic deletion of Atg7 in H - 2Db - gp33 + T cells and in total activated ( CD44hiCD62Llo ) CD8 + T cells from Atg7fl / fl mice ( Cre − ) and Atg7fl / flGzmb - Cremice ( Cre + ) at day 8 after infection with LCMV Armstrong strain ; results are presented relative to those of Atg7fl / fl mice , set as 100 % . Each symbol represents an individual mouse ; small horizontal lines indicate the mean ( ± s . e . m . ) .

(a) Genomic deletion of Atg7 in H-2Db-gp33+ T cells and in total activated (CD44hiCD62Llo) CD8+ T cells from Atg7fl/fl mice (Cre −) and Atg7fl/flGzmb-Cremice (Cre +) at day 8 after infection with LCMV Armstrong strain; results are presented relative to those of Atg7fl/fl mice, set as 100%. Each symbol represents an individual mouse; small horizontal lines indicate the mean (±s.e.m.).


( b ) Immunoblot analysis of Atg7 and the products of its enzymatic activity ( left margin ) in CD44hiCD62Llo CD8 + T cells isolated at day 8 after infection as in a ; β - actin serves as a loading control .

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(b) Immunoblot analysis of Atg7 and the products of its enzymatic activity (left margin) in CD44hiCD62Llo CD8+ T cells isolated at day 8 after infection as in a; β-actin serves as a loading control.


( c ) Flow cytometry of T cells in the peripheral blood at day 8 after infection as in a , gated on CD8 + T cells ( left ) , and of the H - 2Db - gp33 + T cells identified at left , identifying terminal effector ( KLRG1hiCD127lo ) and memory precursor ( KLRG1loCD127hi ) populations ( right ) . Numbers adjacent to outlined areas ( left ) indicate percent H - 2Db - gp33 + T cells ; numbers in quadrants ( right ) indicate percent cells in each .

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(c) Flow cytometry of T cells in the peripheral blood at day 8 after infection as in a, gated on CD8+ T cells (left), and of the H-2Db-gp33+ T cells identified at left, identifying terminal effector (KLRG1hiCD127lo) and memory precursor (KLRG1loCD127hi) populations (right). Numbers adjacent to outlined areas (left) indicate percent H-2Db-gp33+ T cells; numbers in quadrants (right) indicate percent cells in each.


( d ) Total antigen - specific CD8 + T cells in the spleen , liver and lungs at day 8 after infection as in a .

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(d) Total antigen-specific CD8+ T cells in the spleen, liver and lungs at day 8 after infection as in a.


( e ) Production of IFN - γ and TNF in cells obtained 8 d after infection and stimulated ex vivo with various peptides ( above plots ) , gated on total CD8 + T cells . Numbers in quadrants indicate percent cells in each .

(e) Production of IFN-γ and TNF in cells obtained 8 d after infection and stimulated ex vivo with various peptides (above plots), gated on total CD8+ T cells. Numbers in quadrants indicate percent cells in each.


( f ) Flow cytometry of CD8 + T cells in the peripheral blood of mice at day 15 after infection as in a ( numbers in plots as in c , left ) .

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(f) Flow cytometry of CD8+ T cells in the peripheral blood of mice at day 15 after infection as in a (numbers in plots as in c, left).


( g ) Quantification of tetramer - positive cells in spleens at day 15 after infection as in a .

(g) Quantification of tetramer-positive cells in spleens at day 15 after infection as in a.


( h ) Kinetics of the appearance H - 2Db - gp33 + CD8 + T cells among peripheral blood mononuclear cells ( PBMCs ) after infection as in a ( left ) ; each line represents one mouse . Right , frequency of H - 2Db - gp33 + CD8 + T cells remaining on day 15 relative to that on day 8 , set as 100 % .

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(h) Kinetics of the appearance H-2Db-gp33+ CD8+ T cells among peripheral blood mononuclear cells (PBMCs) after infection as in a (left); each line represents one mouse. Right, frequency of H-2Db-gp33+ CD8+ T cells remaining on day 15 relative to that on day 8, set as 100%.


( i ) Quantification of tetramer - positive cells at day 30 after infection as in a . Horizontal dashed lines ( g , h ) indicate the limit of detection . * P ≤ 0 . 05 , * * P ≤ 0 . 005 and * * * P ≤ 0 . 0005 ( unpaired t - test ) . Data were pooled from two independent experiments ( a ) or at least two independent experiments ( c - i ) with three to seven mice per group ( error bars , s . e . m . ) or are from one experiment with samples obtained from two independent experiments ( b ) .

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

(i) Quantification of tetramer-positive cells at day 30 after infection as in a. Horizontal dashed lines (g,h) indicate the limit of detection. *P ≤ 0.05, **P ≤ 0.005 and ***P ≤ 0.0005 (unpaired t-test). Data were pooled from two independent experiments (a) or at least two independent experiments (c-i) with three to seven mice per group (error bars, s.e.m.) or are from one experiment with samples obtained from two independent experiments (b).


( a ) Flow cytometry of circulating LCMV - specific CD8 + T cells among peripheral blood mononuclear cells from Atg5fl / fl and Atg5fl / flGzmb - Cremice on days 8 , 15 and 50 after infection with LCMV Armstrong strain . Numbers adjacent to outlined areas indicate percent H - 2Db - gp33 + T cells , gated on CD8 + T cells . ( b ) Longitudinal kinetics of the appearance of H - 2Db - gp33 + T cells in the peripheral blood of mice as in a ( left ) , and percent H - 2Db - gp33 + CD8 + T cells remaining on day 15 ( right ) ( all presented as in Fig . 4h ) .

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(a) Flow cytometry of circulating LCMV-specific CD8+ T cells among peripheral blood mononuclear cells from Atg5fl/fl and Atg5fl/flGzmb-Cremice on days 8, 15 and 50 after infection with LCMV Armstrong strain. Numbers adjacent to outlined areas indicate percent H-2Db-gp33+ T cells, gated on CD8+ T cells. (b) Longitudinal kinetics of the appearance of H-2Db-gp33+ T cells in the peripheral blood of mice as in a (left), and percent H-2Db-gp33+ CD8+ T cells remaining on day 15 (right) (all presented as in Fig. 4h).


( c ) Flow cytometry of T cells in tissues on day 279 after infection as in a ( numbers in plots as in a , among total gated CD8 + T cells ) . ( d ) Total T cells specific for H - 2Db - gp33 ( left ) or H - 2Db - NP396 ( right ) in the spleen , liver and lungs of mice as in a . * P ≤ 0 . 05 , * * P ≤ 0 . 005 and * * * P ≤ 0 . 0005 ( unpaired t - test ) . Data are representative of two independent experiments with three to seven mice per group ( error bars ( d ) , s . e . m . ) .

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(c) Flow cytometry of T cells in tissues on day 279 after infection as in a (numbers in plots as in a, among total gated CD8+ T cells). (d) Total T cells specific for H-2Db-gp33 (left) or H-2Db-NP396 (right) in the spleen, liver and lungs of mice as in a. *P ≤ 0.05, **P ≤ 0.005 and ***P ≤ 0.0005 (unpaired t-test). Data are representative of two independent experiments with three to seven mice per group (error bars (d), s.e.m.).


( a , b ) Flow cytometry of CD8 + T cell obtained from chimeras generated ( as in Supplementary Fig . 5a ) by reconstitution of wild - type mice with a mixture of bone marrow cells from Atg7fl / fl C57BL / 6 and wild - type C57BL / 6C57BL / 6 ( Atg7fl / fl vs C57BL / 6 ) or from Atg7fl / flGzmb - Cremice and wild - type C57BL / 6mice and ( Atg7fl / flGzmb - Cre vs C57BL / 6 ) , gated ( far left ) as populations specific for H - 2Db - gp33 ( a ) or H - 2Db - NP396 ( b ) and then assessed , on days 8 , 15 and 30 after infection of recipients with LCMV Armstrong infection , as CD45 . 2 + ( Atg7fl / fl or Atg7fl / flGzmb - Cre ) donor cells or CD45 . 1 + ( C57BL / 6 ) donor cells ( middle ) . Far right , appearance of tetramer - positive T cells ( key , donor source ) in the peripheral blood of chimeras from day 8 to day 30 after infection , presented relative to that at day 8 after infection , set as 100 % .

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(a,b) Flow cytometry of CD8+ T cell obtained from chimeras generated (as in Supplementary Fig. 5a) by reconstitution of wild-type mice with a mixture of bone marrow cells from Atg7fl/fl C57BL/6 and wild-type C57BL/6C57BL/6 (Atg7fl/fl vs C57BL/6) or from Atg7fl/flGzmb-Cremice and wild-type C57BL/6mice and (Atg7fl/flGzmb-Cre vs C57BL/6), gated (far left) as populations specific for H-2Db-gp33 (a) or H-2Db-NP396 (b) and then assessed, on days 8, 15 and 30 after infection of recipients with LCMV Armstrong infection, as CD45.2+ (Atg7fl/fl or Atg7fl/flGzmb-Cre) donor cells or CD45.1+ (C57BL/6) donor cells (middle). Far right, appearance of tetramer-positive T cells (key, donor source) in the peripheral blood of chimeras from day 8 to day 30 after infection, presented relative to that at day 8 after infection, set as 100%.


( c ) Frequency of CD45 . 2 + antigen - specific CD8 + T cells in the spleen , liver and lungs on day 30 after infectionwith LCMV Armstrongas in a , b . Data are representative of two independent experiments with three to four mice per group ( error bars , s . e . m . ) .

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(c) Frequency of CD45.2+ antigen-specific CD8+ T cells in the spleen, liver and lungs on day 30 after infectionwith LCMV Armstrongas in a,b. Data are representative of two independent experiments with three to four mice per group (error bars, s.e.m.).


( a ) Kinetics of the appearance of H - 2Db - gp276 + T cells in the peripheral blood of Atg7fl / fl and Atg7fl / flGzmb - Cre mice at various times ( horizontal axis ) after infection with LCMV clone 13 .

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(a) Kinetics of the appearance of H-2Db-gp276+ T cells in the peripheral blood of Atg7fl/fl and Atg7fl/flGzmb-Cre mice at various times (horizontal axis) after infection with LCMV clone 13.


( b ) Total IFN - γ - secreting cells in spleens obtained from mice at day 8 or day 15 after infection as in a , assessed after 5 h of stimulation ex vivo with various peptides ( horizontal axes ) .

(b) Total IFN-γ-secreting cells in spleens obtained from mice at day 8 or day 15 after infection as in a, assessed after 5 h of stimulation ex vivo with various peptides (horizontal axes).


( c ) Viral titers in serum of mice at various times ( horizontal axis ) after infection with LCMV clone 13 as in a . Each symbol represents an individual mouse ; small horizontal lines indicate the mean ( ± s . e . m . ) . * P ≤ 0 . 05 , * * P ≤ 0 . 005 and * * * P ≤ 0 . 0005 ( unpaired t - test ) . Data are representative of two independent experiments with three to five mice per group ( error bars , s . e . m . ) .

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(c) Viral titers in serum of mice at various times (horizontal axis) after infection with LCMV clone 13 as in a. Each symbol represents an individual mouse; small horizontal lines indicate the mean (±s.e.m.). *P ≤ 0.05, **P ≤ 0.005 and ***P ≤ 0.0005 (unpaired t-test). Data are representative of two independent experiments with three to five mice per group (error bars, s.e.m.).


B : Single - cell growth of drug - treated cells as in A as a function of time since MreB - motion stop according to panel A . Left and middle : Relative increase ( left ) and single - cell rates ( middle ) of volume , mass , surface , and length . Gray asterisks indicate single - cell mass at lysis . Right : Relative changes of dry - mass density , surface - to - mass ratio , surface - to - volume ratio , and width .

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B: Single-cell growth of drug-treated cells as in A as a function of time since MreB-motion stop according to panel A. Left and middle: Relative increase (left) and single-cell rates (middle) of volume, mass, surface, and length. Gray asterisks indicate single-cell mass at lysis. Right: Relative changes of dry-mass density, surface-to-mass ratio, surface-to-volume ratio, and width.


D : Complex nutrient shift of non - dividing cells ( S382 ) growing on agarose pad ( MM + 0 . 02 % glucose ) in the presence of vancomycin ( 100 μg / mL ) . Same quantities as in B , as a function of time since MreB - motion stop . Cells were transiently starved for glucose ( through addition of 0 . 5 % ( w / v ) αMG and 0 . 25 % ( w / v ) 2DG ) , followed by addition of excess glucose ( 1 % ) . Different from A - C , all compounds were added to the agarose pad as droplets and reached cells by diffusion .

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D: Complex nutrient shift of non-dividing cells (S382) growing on agarose pad (MM+0.02% glucose) in the presence of vancomycin (100 μg/mL). Same quantities as in B, as a function of time since MreB-motion stop. Cells were transiently starved for glucose (through addition of 0.5% (w/v) αMG and 0.25% (w/v) 2DG), followed by addition of excess glucose (1%). Different from A-C, all compounds were added to the agarose pad as droplets and reached cells by diffusion.


A : Single - cell growth of non - dividing b42 cells ( ΔmepS / pBAD30 - mepS ) grown on LB agarose pads as a function of time after addition of arabinose ( 0 . 2 % ) in form of a droplet . Left : Relative increase of volume , mass , surface , and length . Middle : Elongation rate and surface - growth rate . Right : Relative changes of surface - to - mass ratio and width .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Single-cell growth of non-dividing b42 cells (ΔmepS/pBAD30-mepS) grown on LB agarose pads as a function of time after addition of arabinose (0.2%) in form of a droplet. Left: Relative increase of volume, mass, surface, and length. Middle: Elongation rate and surface-growth rate. Right: Relative changes of surface-to-mass ratio and width.


B - C : Cell bending : B : Time - lapses of bending filamentous cells ( S290 ) in donut microchambers ( 3 % agarose ; RDM + glu ) and grown without ( top ) or with ( bottom ) D - cycloserine ( 1 mM ) as a function of time since D - cycloserine treatment , starting > 10 min after D - cycloserine treatment ( the time of MreB - motion stop D - cycloserine - treated cells were osmotically shocked ( NaCl ) after 33 min of treatment , which releases a fraction of cells C : Change of cell centerline curvature as a function of cell length between 10 min after start of D - cycloserine treatment and release of cells through osmotic

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

B-C: Cell bending: B: Time-lapses of bending filamentous cells (S290) in donut microchambers (3% agarose; RDM+glu) and grown without (top) or with (bottom) D-cycloserine (1 mM) as a function of time since D-cycloserine treatment, starting >10 min after D-cycloserine treatment (the time of MreB-motion stop D-cycloserine-treated cells were osmotically shocked (NaCl) after 33 min of treatment, which releases a fraction of cells C: Change of cell centerline curvature as a function of cell length between 10 min after start of D-cycloserine treatment and release of cells through osmotic shock


D - E : Cell straightening . D : Time - lapses of straightening filamentous cells ( S290 ) previously bent in donuts : control ( top ) and D - cycloserine treated cells ( bottom ) as a function of time since simultaneous release from donuts and beginning of drug treatment . E : Change of centerline curvature as a function of relative length from 10 min after D - cycloserine treatment ( time of MreB - motion stop ) .

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

D-E: Cell straightening. D: Time-lapses of straightening filamentous cells (S290) previously bent in donuts: control (top) and D-cycloserine treated cells (bottom) as a function of time since simultaneous release from donuts and beginning of drug treatment. E: Change of centerline curvature as a function of relative length from 10 min after D-cycloserine treatment (time of MreB-motion stop).


C : Relative change of cell width of non - dividing cells ( S257 ) grown in LB during combined D - cycloserine treatment ( 5 mM ) and a hypo - osmotic ramp as in A ( DCS + Hypo ramp ) , as compared to cells exposed to the hypo - osmotic ramp only ( Hypo ramp ) or treated with D - cycloserine only ( DCS ) . D - cycloserine was contained in both agarose pads , and treatment started 15 min prior to the shock .

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

C: Relative change of cell width of non-dividing cells (S257) grown in LB during combined D-cycloserine treatment (5 mM) and a hypo-osmotic ramp as in A (DCS + Hypo ramp), as compared to cells exposed to the hypo-osmotic ramp only (Hypo ramp) or treated with D-cycloserine only (DCS). D-cycloserine was contained in both agarose pads, and treatment started 15 min prior to the shock.


Comparable mean SV number per ribbon in wild - type at the distal ( Ds . , H ) and the proximal ( Pr . , I ) part of the ribbon . More SVs are observed at inhibitory OtofPga / Pga ribbons compared to inhibitory wild - type synapses at both ribbon parts , and further at OtofPga / Pga synapses under rest in the Pr . part . Data are presented as mean ± SEM ; the red dotted line highlights the wild - type mean SV number per ribbon under rest as a reference point ;

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Comparable mean SV number per ribbon in wild-type at the distal (Ds., H) and the proximal (Pr., I) part of the ribbon. More SVs are observed at inhibitory OtofPga/Pga ribbons compared to inhibitory wild-type synapses at both ribbon parts, and further at OtofPga/Pga synapses under rest in the Pr. part. Data are presented as mean ± SEM; the red dotted line highlights the wild-type mean SV number per ribbon under rest as a reference point;


Representative virtual sections of wild - type ( J ) and OtofPga / Pga ( K ) ribbon synapses under inhibition . Counted RA - SVs are marked with white asterisks . Magnification , 12 , 000x ; scale bar , 100 nm .

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Representative virtual sections of wild-type (J) and OtofPga/Pga (K) ribbon synapses under inhibition. Counted RA-SVs are marked with white asterisks. Magnification, 12,000x; scale bar, 100 nm.


Exemplary virtual sections of wild - type and OtofPga / Pga under inhib ( L , M ) , rest ( L ' , M ' ) and stim ( L ' ' , M ' ' ) . At OtofPga / Pga SVs are densely packed . Blue arrowheads indicate proteinaceous filaments between wild - type SVs . Magnification 12 , 000x ; scale bar , 100 nm .

Exemplary virtual sections of wild-type and OtofPga/Pga under inhib (L,M), rest (L',M') and stim (L'',M''). At OtofPga/Pga SVs are densely packed. Blue arrowheads indicate proteinaceous filaments between wild-type SVs. Magnification 12,000x; scale bar, 100 nm.


Illustrations show the two major SVs associated with filament / s in the wild - type RA - SV pool at rest ; synaptic ribbon ( R , red ) , SVs ( dark green ) with filament / s ( blue ) . Not drawn to scale . ( A , B ) Ri - attached SVs ( Ri . ) and ( C , D ) interconnected SVs ( In . ) harbor single ( A , C ) or multiple ( B , D ) filaments . Example virtual section from resting wild - type ; single Ri . ( A ' , A ' ' ) and In . ( C ' , C ' ' ) , multiple Ri . ( B ' , B ' ' ) and In . ( D ' , D ' ' ) . Magnification 12 , 000x ; scale bar , 40 nm . Structures are color - coded similarly to the illustrations , the number of ribbon - attached filaments per SV are indicated with red arrowhead / s and interconnectors per SV with purple arrowhead / s in A ' ' , B ' ' , C ' ' and D ' ' . Magnification 12 , 000x ; scale bar , 40 nm .

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

Illustrations show the two major SVs associated with filament/s in the wild-type RA-SV pool at rest; synaptic ribbon (R, red), SVs (dark green) with filament/s (blue). Not drawn to scale. (A,B) Ri-attached SVs (Ri.) and (C,D) interconnected SVs (In.) harbor single (A,C) or multiple (B,D) filaments. Example virtual section from resting wild-type; single Ri. (A',A'') and In. (C',C''), multiple Ri. (B',B'') and In. (D',D''). Magnification 12,000x; scale bar, 40 nm. Structures are color-coded similarly to the illustrations, the number of ribbon-attached filaments per SV are indicated with red arrowhead/s and interconnectors per SV with purple arrowhead/s in A'', B'', C'' and D''. Magnification 12,000x; scale bar, 40 nm.


The graph is representing the percentage of RA - SVs around the whole ribbon at wild - type under inhib , rest and stim . Upon stimulation , the percentage of SVs without filaments and ribbon - attached SVs are significantly reduced , while interconnected SVs increased significantly .

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The graph is representing the percentage of RA-SVs around the whole ribbon at wild-type under inhib, rest and stim. Upon stimulation, the percentage of SVs without filaments and ribbon-attached SVs are significantly reduced, while interconnected SVs increased significantly.


Depicted are the fractions of SVs without filaments , ribbon - attached SVs ( Ri . ) , interconnected SVs ( In . ) and SVs with Ri . + In . per ribbon , at ( A ) the distal ( Ds . ) and ( B ) the proximal ( Pr . ) part of the ribbon . Data are represented as mean ± SEM of wild - type and OtofPga / Pga IHCs under inhib , rest and stim . ( A ) Upon stimulation , in the Ds . part at wild - type , the fraction of SVs without filaments reduces , but of SVs with Ri . + In . increases significantly . At OtofPga / Pga IHCs the fraction of SVs without filaments is reduced when comparing stim vs . inhib . The fraction of interconnected SVs increases with stimulation compared to inhibition and rest . In the mutant ( inhib ) , the fraction of interconnected SVs decreases compared to wild - type ( inhib ) , and Ri . + In . SVs reduce vs . wild - type ( stim ) . ( B ) The fraction of SVs without filaments increased at inhibited OtofPga / Pga synapses . The red dotted lines in ( A ) and ( B ) represent the mean fraction of RA - SVs / ribbon part of resting wild - type for each defined sub - pool .

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

Depicted are the fractions of SVs without filaments, ribbon-attached SVs (Ri.), interconnected SVs (In.) and SVs with Ri. + In. per ribbon, at (A) the distal (Ds.) and (B) the proximal (Pr.) part of the ribbon. Data are represented as mean ± SEM of wild-type and OtofPga/Pga IHCs under inhib, rest and stim. (A) Upon stimulation, in the Ds. part at wild-type, the fraction of SVs without filaments reduces, but of SVs with Ri. + In. increases significantly. At OtofPga/Pga IHCs the fraction of SVs without filaments is reduced when comparing stim vs. inhib. The fraction of interconnected SVs increases with stimulation compared to inhibition and rest. In the mutant (inhib), the fraction of interconnected SVs decreases compared to wild-type (inhib), and Ri. + In. SVs reduce vs. wild-type (stim). (B) The fraction of SVs without filaments increased at inhibited OtofPga/Pga synapses. The red dotted lines in (A) and (B) represent the mean fraction of RA-SVs/ribbon part of resting wild-type for each defined sub-pool.


Representative wild - type virtual section under rest depicts SVs with r - attached fil ( Ra . fil . , red arrowheads ) and one interconnector ( Intercon . , purple arrowhead ) , around the ribbon ( R ) . Magnification 12 , 000x ; scale bar , 40 nm . Example 3D reconstruction of the RA - SVs with filaments ( green ) in resting wild - type . Larger average length is found for ribbon - attached filaments ( Ra . fil . , red ) vs . SV interconnectors ( Intercon . , purple ) . For clear visualization , the ribbon ( R ) is transparent and the same SV pair is shown in a front ( A ' ) , side ( A ' ' ) and top view ( A ' ' ' ) . Scale bar , 50 nm .

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

Representative wild-type virtual section under rest depicts SVs with r-attached fil (Ra.fil., red arrowheads) and one interconnector (Intercon., purple arrowhead), around the ribbon (R). Magnification 12,000x; scale bar, 40 nm. Example 3D reconstruction of the RA-SVs with filaments (green) in resting wild-type. Larger average length is found for ribbon-attached filaments (Ra.fil., red) vs. SV interconnectors (Intercon., purple). For clear visualization, the ribbon (R) is transparent and the same SV pair is shown in a front (A'), side (A'') and top view (A'''). Scale bar, 50 nm.


Increased mean length of ribbon - attached filaments vs . SV interconnectors in all conditions for both genotypes . The red dotted lines represent the mean RA - filament length in resting wild - type for each sub - pool .

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

Increased mean length of ribbon-attached filaments vs. SV interconnectors in all conditions for both genotypes. The red dotted lines represent the mean RA-filament length in resting wild-type for each sub-pool.


The cumulative probability distribution of RA - SVs plotted against the individual filament length . The graph illustrates shorter filaments in wild - type and OtofPga / Pga ribbon synapses upon stimulation .

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

The cumulative probability distribution of RA-SVs plotted against the individual filament length. The graph illustrates shorter filaments in wild-type and OtofPga/Pga ribbon synapses upon stimulation.


Decreased mean filament length upon stimulation in wild - type and OtofPga / Pga ribbon synapses . Resting OtofPga / Pga exhibit the shortest mean filament length . The red dotted lines represent the mean RA - filament length in resting wild - type for each sub - pool .

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

Decreased mean filament length upon stimulation in wild-type and OtofPga/Pga ribbon synapses. Resting OtofPga/Pga exhibit the shortest mean filament length. The red dotted lines represent the mean RA-filament length in resting wild-type for each sub-pool.


Representative tomographic virtual sections from resting wild - type . Five different types of MP - SVs are observed that we classify as sub - pools : non - tethered SVs ( A ' , A ' ' ; yellow ) , single presynaptic density - attached SVs ( B ' , B ' ' ; Pd . a , orange , pink arrowhead ) or single m - attached SVs ( C ' , C ' ' ; Ma . , orange , orange arrowhead ) , or multiple - tethered SVs ( D ' , D ' ' ; orange , brown arrowheads ) and docked SVs ( E ' , E ' ' ; light orange ) . Ribbon ( R ) , presynaptic density ( PD , pink ) and AZ - membrane ( blue ) . For clarity , a simplified illustration is provided for each sub - pool . Magnification : 12 , 000x ; scale bar , 40 nm . The graph is showing the percentage of the MP sub - pools shown in A - E of all MP - SVs at inhibitory , resting and stimulatory wild - type synapses .

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

Representative tomographic virtual sections from resting wild-type. Five different types of MP-SVs are observed that we classify as sub-pools: non-tethered SVs (A',A''; yellow), single presynaptic density-attached SVs (B',B''; Pd.a, orange, pink arrowhead) or single m-attached SVs (C',C''; Ma., orange, orange arrowhead), or multiple-tethered SVs (D',D''; orange, brown arrowheads) and docked SVs (E',E''; light orange). Ribbon (R), presynaptic density (PD, pink) and AZ-membrane (blue). For clarity, a simplified illustration is provided for each sub-pool. Magnification: 12,000x; scale bar, 40 nm. The graph is showing the percentage of the MP sub-pools shown in A-E of all MP-SVs at inhibitory, resting and stimulatory wild-type synapses.


Depicts the fraction of non - tethered and single - tethered MP - SVs per ribbon . Data are presented in mean ± SEM . The fraction of non - tethered SVs reduces in wild - type and OtofPga / Pga after stimulation and the fraction of single membrane - attached tethers increases in wild - type upon stimulation . In resting wild - type , most single - tethered SVs are attached to the presynaptic density .

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

Depicts the fraction of non-tethered and single-tethered MP-SVs per ribbon. Data are presented in mean ± SEM. The fraction of non-tethered SVs reduces in wild-type and OtofPga/Pga after stimulation and the fraction of single membrane-attached tethers increases in wild-type upon stimulation. In resting wild-type, most single-tethered SVs are attached to the presynaptic density.


Example tomographic virtual sections from inhibitory : wild - type ( A ) and OtofPga / Pga ( B ) ; stimulatory : wild - type ( C ) and OtofPga / Pga ( D ) . Tethered SVs are highlighted in orange with orange tether ( s ) ; single - tethers ( with orange arrowhead ) and multiple - tethers ( with brown arrowhead ) . The number of tethers per SV is indicated . Magnification : 12 , 000x ; scale bar , 40 nm . Virtual sections from inhibitory : wild - type ( E ) and OtofPga / Pga ( F ) ; stimulatory : wild - type ( G ) and OtofPga / Pga ( H ) . Docked SVs at the AZ - membrane ( blue line ) are highlighted ( orange asterisk ) . Some docked SVs are localized close to the presynaptic density ( PD , pink outline ) , anchoring the ribbon ( R ) . Magnification : 12 , 000x ; scale bar , 100 nm .

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

Example tomographic virtual sections from inhibitory: wild-type (A) and OtofPga/Pga (B); stimulatory: wild-type (C) and OtofPga/Pga (D). Tethered SVs are highlighted in orange with orange tether(s); single-tethers (with orange arrowhead) and multiple-tethers (with brown arrowhead). The number of tethers per SV is indicated. Magnification: 12,000x; scale bar, 40 nm. Virtual sections from inhibitory: wild-type (E) and OtofPga/Pga (F); stimulatory: wild-type (G) and OtofPga/Pga (H). Docked SVs at the AZ-membrane (blue line) are highlighted (orange asterisk). Some docked SVs are localized close to the presynaptic density (PD, pink outline), anchoring the ribbon (R). Magnification: 12,000x; scale bar, 100 nm.


Percentage of multiple - tethered MP - SVs per ribbon of the total MP - SV pool show more multiple - tethered SVs in OtofPga / Pga IHCs . Percentage of docked MP - SVs per ribbon of the total MP - pool . The number of docked SVs is significantly increased at OtofPga / Pga inhibited and specifically at stimulated AZs . P - values for ( I ) and ( J ) are calculated by Fisher ' s exact Chi - square test , wild - type : inhib , n = 102 SVs ; rest , n = 82 SVs ; stim , n = 99 SVs ; OtofPga / Pga : inhib , n = 123 SVs ; rest , n = 96 SVs ; stim , n = 111 SVs ; ns p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 .

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

Percentage of multiple-tethered MP-SVs per ribbon of the total MP-SV pool show more multiple-tethered SVs in OtofPga/Pga IHCs. Percentage of docked MP-SVs per ribbon of the total MP-pool. The number of docked SVs is significantly increased at OtofPga/Pga inhibited and specifically at stimulated AZs. P-values for (I) and (J) are calculated by Fisher's exact Chi-square test, wild-type: inhib, n = 102 SVs; rest, n = 82 SVs; stim, n = 99 SVs; OtofPga/Pga: inhib, n = 123 SVs; rest, n = 96 SVs; stim, n = 111 SVs; ns p > 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001


Bar - plots show the distance of multiple - tethered ( O ) and docked ( P ) SVs from presynaptic density ( PD ) , 0 - 100 nm : MP - SV pool ( dark blue ) ; 100 - 500 nm : peripheral SV pool ( light blue ) . Upon stimulation , multiple - tethered and docked SVs are localized significantly closer to the PD in OtofPga / Pga . P - values for ( O ) and ( P ) are calculated by Wilcoxon Rank test , * p < 0 . 05 , * * * p < 0 . 001 , ns p > 0 . 05 and Ø inconclusive statistics because occurrence of docked SVs in wild - type resting condition is extremely rare . Multiple - tethered SVs , wild - type : inhib , n = 3 SVs ; rest , n = 13 SVs ; stim , n = 3 SVs ; OtofPga / Pga : inhib , n = 21 SVs ; rest , n = 34 SVs ; stim , n = 16 SVs ; docked SVs , wild - type : inhib , n = 2 SVs ; rest , n = 1 SVs ; stim , n = 7 SVs ; OtofPga / Pga : inhib , n = 20 SVs ; rest , n = 4 SVs ; stim , n = 18 SVs .

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

Bar-plots show the distance of multiple-tethered (O) and docked (P) SVs from presynaptic density (PD), 0-100 nm: MP-SV pool (dark blue); 100-500 nm: peripheral SV pool (light blue). Upon stimulation, multiple-tethered and docked SVs are localized significantly closer to the PD in OtofPga/Pga. P-values for (O) and (P) are calculated by Wilcoxon Rank test, * p < 0.05, *** p < 0.001, ns p > 0.05 and Ø inconclusive statistics because occurrence of docked SVs in wild-type resting condition is extremely rare. Multiple-tethered SVs, wild-type: inhib, n = 3 SVs; rest, n = 13 SVs; stim, n = 3 SVs; OtofPga/Pga: inhib, n = 21 SVs; rest, n = 34 SVs; stim, n = 16 SVs; docked SVs, wild-type: inhib, n = 2 SVs; rest, n = 1 SVs; stim, n = 7 SVs; OtofPga/Pga: inhib, n = 20 SVs; rest, n = 4 SVs; stim, n = 18 SVs.


Example virtual sections depict SVs with single membrane - ( orange arrowhead ) and presynaptic density ( pink arrowhead ) - attached tethers in resting wild - type . Magnification 12 , 000x ; scale bar , 40 nm . 3D reconstructions of same tethered SVs ( orange ) as shown in ( A ) and ( B ) ; membrane - attached ( A ' , yellow tether ) and presynaptic density ( PD ) - attached ( B ' , pink tether ) . Individual tether lengths are depicted . For clarity , ribbon ( R ) and AZ - membrane are shown in transparent . Scale bar , 20 nm .

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

Example virtual sections depict SVs with single membrane- (orange arrowhead) and presynaptic density (pink arrowhead) -attached tethers in resting wild-type. Magnification 12,000x; scale bar, 40 nm. 3D reconstructions of same tethered SVs (orange) as shown in (A) and (B); membrane-attached (A', yellow tether) and presynaptic density (PD) -attached (B', pink tether). Individual tether lengths are depicted. For clarity, ribbon (R) and AZ-membrane are shown in transparent. Scale bar, 20 nm.


The graph depicts the mean length of single membrane - , presynaptic density - attached and multiple - tethers . Data are presented as mean ± SEM . Stimulated OtofPga / Pga synapses exhibit a shorter average tether length compared to stimulated wild - type .

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

The graph depicts the mean length of single membrane-, presynaptic density-attached and multiple-tethers. Data are presented as mean ± SEM. Stimulated OtofPga/Pga synapses exhibit a shorter average tether length compared to stimulated wild-type.


The cumulative probability distribution of MP - SVs plotted against the tether length . The red dotted lines represent the mean MP tether length in resting wild - type for each sub - pool .

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

The cumulative probability distribution of MP-SVs plotted against the tether length. The red dotted lines represent the mean MP tether length in resting wild-type for each sub-pool.


Total mean tether length in the MP - SV pool . Data are presented as mean ± SEM . Longer tethers are present upon stimulation in wild - type . Resting OtofPga / Pga ribbon synapses harbor the longest tethers , and tether where shorter at stimulated OtofPga / Pga synapses compared to wild - type . The red dotted lines represent the mean MP tether length in resting wild - type for each sub - pool .

Total mean tether length in the MP-SV pool. Data are presented as mean ± SEM. Longer tethers are present upon stimulation in wild-type. Resting OtofPga/Pga ribbon synapses harbor the longest tethers, and tether where shorter at stimulated OtofPga/Pga synapses compared to wild-type. The red dotted lines represent the mean MP tether length in resting wild-type for each sub-pool.


One - cell stage embryos were injected animally with mRNA encoding dkk1 ( 35 / 45 pg ) protein . Total RNA was isolated from ten control embryos at neurula stage 17 ( lane 2 ) and ten embryos from each dkk1 injected group ( lanes 3 - 4 ) . Various neural AP markers were examined by sqRT - PCR : cement gland and forebrain ( xag1 , xanf1 , otx2 ) , hindbrain ( krox20 , hoxb3 ) , hindbrain / spinal cord border ( hoxb4 ) , and spinal cord ( hoxb9 , cdx1 - 2 - 4 , hoxd10 ) . Ef1α serves as a control for quantitating RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 1 ) .

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

One-cell stage embryos were injected animally with mRNA encoding dkk1 (35/45 pg) protein. Total RNA was isolated from ten control embryos at neurula stage 17 (lane 2) and ten embryos from each dkk1 injected group (lanes 3-4). Various neural AP markers were examined by sqRT-PCR: cement gland and forebrain (xag1, xanf1, otx2), hindbrain (krox20, hoxb3), hindbrain/spinal cord border (hoxb4), and spinal cord (hoxb9, cdx1-2-4, hoxd10). Ef1α serves as a control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


One - cell stage embryos were injected animally with the zygotic - expressing Nxfz8 / pCS2 encoding plasmid ( 100 / 200 pg ) . Total RNA was isolated from ten control embryos at neurula stage 17 ( lane 2 ) and ten embryos from each Nxfz8 injected group ( lanes 3 - 4 ) . Various neural AP markers were examined by sqRT - PCR : cement gland and forebrain ( xag1 , xanf1 , otx2 ) , hindbrain ( krox20 , hoxb3 ) , hindbrain / spinal cord border ( hoxb4 ) , and spinal cord ( hoxb9 , cdx1 - 2 - 4 , hoxd10 ) . Ef1α serves as a control for quantitating RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 1 ) .

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

One-cell stage embryos were injected animally with the zygotic-expressing Nxfz8/pCS2 encoding plasmid (100/200 pg). Total RNA was isolated from ten control embryos at neurula stage 17 (lane 2) and ten embryos from each Nxfz8 injected group (lanes 3-4). Various neural AP markers were examined by sqRT-PCR: cement gland and forebrain (xag1, xanf1, otx2), hindbrain (krox20, hoxb3), hindbrain/spinal cord border (hoxb4), and spinal cord (hoxb9, cdx1-2-4, hoxd10). Ef1α serves as a control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


Embryos from the same experiment as in ( A ) underwent in situ hybridization . Expression of the hindbrain marker krox20 ( upper two panels ) was reduced by Dkk1 ( 45pg ) in 83 % of the embryos ( n = 18 ) . Expression of spinal cord markers ( hoxb9 , cdx4 , hox10 ) was normal , being unchanged in 89 % of the embryos ( n = 18 for each marker ) . Control embryos , ( CE ) .

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

Embryos from the same experiment as in (A) underwent in situ hybridization. Expression of the hindbrain marker krox20 (upper two panels) was reduced by Dkk1 (45pg) in 83% of the embryos (n=18). Expression of spinal cord markers (hoxb9, cdx4, hox10) was normal, being unchanged in 89% of the embryos (n=18 for each marker). Control embryos, (CE).


One - cell stage embryos were injected animally with the Zic1 - MO ( 25 / 40 ng ) . Total RNA was isolated from ten control embryos at neurula stage 18 ( lane 2 ) and ten embryos from each Zic1 - MO injected group ( lanes 3 - 4 ) . Neural AP markers were examined by sqRT - PCR : forebrain ( xanf1 , otx2 ) , hindbrain ( krox20 , hoxb1 , hoxb3 ) , spinal cord ( hoxb9 , cdx1 - 4 ) and panneural ( soxd ) . Ef1α is the control for quantitating RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 1 ) .

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

One-cell stage embryos were injected animally with the Zic1-MO (25/40 ng). Total RNA was isolated from ten control embryos at neurula stage 18 (lane 2) and ten embryos from each Zic1-MO injected group (lanes 3-4). Neural AP markers were examined by sqRT-PCR: forebrain (xanf1, otx2), hindbrain (krox20, hoxb1, hoxb3), spinal cord (hoxb9, cdx1-4) and panneural (soxd). Ef1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


One - cell stage embryos were injected animally with mRNA encoding Zic5 dominant - negative ( 0 . 5 ng ) protein . Total RNA was isolated from five control embryos at neurula stage 18 ( lane 1 ) and five embryos from the injected group ( lanes 2 ) . Neural AP markers were examined by sqRT - PCR : hindbrain ( krox20 ) and spinal cord ( cdx1 - 4 ) . Ef1α is the control for quantitating RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 3 ) .

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

One-cell stage embryos were injected animally with mRNA encoding Zic5 dominant-negative (0.5 ng) protein. Total RNA was isolated from five control embryos at neurula stage 18 (lane 1) and five embryos from the injected group (lanes 2). Neural AP markers were examined by sqRT-PCR: hindbrain (krox20) and spinal cord (cdx1-4). Ef1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 3).


One - cell stage embryos were injected animally with the Zic1 - MO ( 25 ng ) . Embryos underwent in situ hybridization . Expression of the panneural sox2 marker was normal , the forebrain marker xanf1 was expanded in 80 % of the embryos , the hindbrain representative marker krox20 was reduced in 80 % of the embryos and the representative spinal cord marker cdx4 was expanded in 90 % of the embryos . N = 23 embryos were per marker .

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

One-cell stage embryos were injected animally with the Zic1-MO (25 ng). Embryos underwent in situ hybridization. Expression of the panneural sox2 marker was normal, the forebrain marker xanf1 was expanded in 80% of the embryos, the hindbrain representative marker krox20 was reduced in 80% of the embryos and the representative spinal cord marker cdx4 was expanded in 90% of the embryos. N = 23 embryos were per marker.


One - cell stage embryos were injected animally with mRNAs encoding the noggin ( 5 / 20 pg ) and / or meis3 ( 0 . 5 ng ) proteins . AC explants were removed from control and injected embryos at blastula stage 9 , and explants were cultured to neurula stage 17 . Total RNA was isolated from seven control embryos ( lane 2 ) , and from eighteen ACs from each group ( lanes 3 - 8 ) . Neural AP markers were examined by sqRT - PCR : forebrain ( xanf1 , otx2 ) , hindbrain ( krox20 , hoxb1 , hoxb3 ) , spinal cord ( hoxb9 , cdx1 - 4 ) and panneural ( soxd ) . Ef1α is the control for quantitating RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 1 ) .

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

One-cell stage embryos were injected animally with mRNAs encoding the noggin (5/20 pg) and/or meis3 (0.5 ng) proteins. AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 17. Total RNA was isolated from seven control embryos (lane 2), and from eighteen ACs from each group (lanes 3-8). Neural AP markers were examined by sqRT-PCR: forebrain (xanf1, otx2), hindbrain (krox20, hoxb1, hoxb3), spinal cord (hoxb9, cdx1-4) and panneural (soxd). Ef1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


One - cell stage embryos were injected animally with mRNA encoding bmp4 ( 0 . 2 ng ) protein . Total RNA was isolated from five control embryos at neurula stage 17 ( lane 1 ) and five embryos from the injected group ( lanes 2 ) . Neural and mesodermal markers were examined by sqRT - PCR : forebrain ( otx2 ) , hindbrain ( krox20 , hoxb3 ) , spinal cord ( hoxb9 , hoxc10 , cdx4 ) , panneural ( nrp1 , ncam ) and mesodermal ( muscle actin - MA ) . ODC is the control for quantitating RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 1 ) .

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

One-cell stage embryos were injected animally with mRNA encoding bmp4 (0.2 ng) protein. Total RNA was isolated from five control embryos at neurula stage 17 (lane 1) and five embryos from the injected group (lanes 2). Neural and mesodermal markers were examined by sqRT-PCR: forebrain (otx2), hindbrain (krox20, hoxb3), spinal cord (hoxb9, hoxc10, cdx4), panneural (nrp1, ncam) and mesodermal (muscle actin - MA). ODC is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


One - cell stage embryos were injected animally with mRNA encoding bmp4 ( 0 . 2 ng ) protein Embryos underwent in situ hybridization . Expression of the hindbrain marker hoxb3 was reduced / eliminated by bmp4 ( 0 . 2 ng ) in 68 % of the embryos ( n = 19 ) . Expression of the spinal cord marker cdx4 was slightly increased / normal , in 100 % of the embryos ( n = 19 ) .

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

One-cell stage embryos were injected animally with mRNA encoding bmp4 (0.2 ng) protein Embryos underwent in situ hybridization. Expression of the hindbrain marker hoxb3 was reduced/eliminated by bmp4 (0.2 ng) in 68% of the embryos (n=19). Expression of the spinal cord marker cdx4 was slightly increased/normal, in 100% of the embryos (n=19).


One - cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA ( 5 - 50 pg ) . AC explants were removed from control and injected embryos at blastula stage 9 , and explants were cultured to neurula stage 16 . Total RNA was isolated from five control embryos ( lane 2 ) , and from eighteen ACs from each group ( lanes 3 - 7 ) . Neural , epidermal and mesodermal markers were examined by sqRT - PCR : hindbrain ( krox20 ) , spinal cord ( hoxb9 , hoxc10 / d10 , cdx1 - 2 - 4 ) , epidermal cytokeratin ( E13 ) , lateral mesoderm ( osr1 - 2 ) and dorsal - lateral mesoderm muscle actin ( MA ) . ODC is the control for quantitating the RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 1 ) .

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

One-cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA (5-50 pg). AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 16. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-7). Neural, epidermal and mesodermal markers were examined by sqRT-PCR: hindbrain (krox20), spinal cord (hoxb9, hoxc10/d10, cdx1-2-4), epidermal cytokeratin (E13), lateral mesoderm (osr1-2) and dorsal-lateral mesoderm muscle actin (MA). ODC is the control for quantitating the RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


One - cell stage embryos were injected animally with mRNAs encoding the bmp4 ( 50 / 150 pg ) and / or meis3 ( 0 . 7 ng ) proteins . AC explants were removed from control and injected embryos at blastula stage 9 , and explants were cultured to neurula stage 16 . Total RNA was isolated from five control embryos ( lane 2 ) , and from eighteen ACs from each group ( lanes 3 - 8 ) . Neural and mesodermal markers were examined by sqRT - PCR : hindbrain ( krox20 ) , spinal cord ( hoxb9 , hoxc10 , cdx2 ) , and lateral mesoderm ( osr1 ) . Ef1α is the control for quantitating the RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 1 ) .

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

One-cell stage embryos were injected animally with mRNAs encoding the bmp4 (50/150 pg) and/or meis3 (0.7 ng) proteins. AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 16. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-8). Neural and mesodermal markers were examined by sqRT-PCR: hindbrain (krox20), spinal cord (hoxb9, hoxc10, cdx2), and lateral mesoderm (osr1). Ef1α is the control for quantitating the RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


One - cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA ( 75 - 200 pg ) . AC explants were removed from control and injected embryos at blastula stage 9 , and explants were cultured to gastrula stage 11 - 11 . 5 . Total RNA was isolated from five control embryos ( lane 2 ) , and from eighteen ACs from each group ( lanes 3 - 6 ) . Marker expression was determined by sqRT - PCR . Xenopus brachyury ( xbra ) expression was induced by BMP4 in a dose - dependent manner . Marker expression was determined by sqRT - PCR . Neither dorsal ( chd , goosecoid - gsc ) or ventral - lateral ( osr1 ) mesoderm markers were induced by BMP4 . Sox2 / 3 neural markers are expressed in the ACs and are unchanged by BMP expression . EF1α is the control for quantitating RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 1 ) .

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

One-cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA (75-200 pg). AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to gastrula stage 11-11.5. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-6). Marker expression was determined by sqRT-PCR. Xenopus brachyury (xbra) expression was induced by BMP4 in a dose-dependent manner. Marker expression was determined by sqRT-PCR. Neither dorsal (chd, goosecoid-gsc) or ventral-lateral (osr1) mesoderm markers were induced by BMP4. Sox2/3 neural markers are expressed in the ACs and are unchanged by BMP expression. EF1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


One - cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA ( 5 - 50 pg ) . AC explants were removed from control and injected embryos at blastula stage 9 , and explants were cultured to neurula stage 16 . Total RNA was isolated from five control embryos ( lane 2 ) , and from eighteen ACs from each group ( lanes 3 - 6 ) . FGF3 - 4 - 8 expression was determined by sqRT - PCR . ODC is the control for quantitating RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 1 ) .

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

One-cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA (5-50 pg). AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 16. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-6). FGF3-4-8 expression was determined by sqRT-PCR. ODC is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


One - cell stage embryos were injected animally with mRNAs encoding the bmp4 ( 50 pg ) and / or FGF dominant - negative receptor ( FGF - DNR , 1 . 5 ng ) proteins . AC explants were removed from control and injected embryos at blastula stage 9 , and explants were cultured to neurula stage 16 . Total RNA was isolated from five control embryos ( lane 2 ) , and from eighteen ACs from each group ( lanes 3 - 6 ) . In addition to fgf3 , neural , epidermal and mesodermal markers were examined by sqRT - PCR : hindbrain ( krox20 , hoxb3 ) , spinal cord ( hoxb9 , hoxc10 , cdx4 ) , lateral mesoderm ( osr1 ) , dorsal - lateral mesoderm muscle actin ( MA ) , and epidermal cytokeratin ( E13 ) . ODC is the control for quantitating RNA levels . - RT - PCR was performed on total RNA isolated from control embryos ( lane 1 ) .

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

One-cell stage embryos were injected animally with mRNAs encoding the bmp4 (50 pg) and/or FGF dominant-negative receptor (FGF-DNR, 1.5 ng) proteins. AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 16. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-6). In addition to fgf3, neural, epidermal and mesodermal markers were examined by sqRT-PCR: hindbrain (krox20, hoxb3), spinal cord (hoxb9, hoxc10, cdx4), lateral mesoderm (osr1), dorsal-lateral mesoderm muscle actin (MA), and epidermal cytokeratin (E13). ODC is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


A . Representative immunoblots and quantifications ( below ) of the indicated proteins in lysates of ARPE - 19 cells treated with 1 mM DFP for the indicated time ( h , hours ) .

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

A. Representative immunoblots and quantifications (below) of the indicated proteins in lysates of ARPE-19 cells treated with 1 mM DFP for the indicated time (h, hours).


B - C . Representative confocal images of ARPE - 19 cells stably expressing the mito - QC reporter ( B ) , or the pexo - QC reporter ( C ) treated with 1 mM DFP for the indicated time ( h , hours ) . Enlarged images of the area outlined in white are shown in the lower corners . The ' red - only ' mask is generated during images analysis and highlights red - only puncta ( autolysosomes ) based on the mCherry / GFP ratio and thresholding criteria . Nuclei were stained in blue ( Hoechst ) . Scale bar : 10μm . At right , quantification of total red - only punctate per cell ( mitolysosomes or pexolysosomes ) .

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

B-C. Representative confocal images of ARPE-19 cells stably expressing the mito-QC reporter (B), or the pexo-QC reporter (C) treated with 1 mM DFP for the indicated time (h, hours). Enlarged images of the area outlined in white are shown in the lower corners. The 'red-only' mask is generated during images analysis and highlights red-only puncta (autolysosomes) based on the mCherry/GFP ratio and thresholding criteria. Nuclei were stained in blue (Hoechst). Scale bar: 10μm. At right, quantification of total red-only punctate per cell (mitolysosomes or pexolysosomes).


D . ARPE - 19 cells were transfected with 50 pmol of non - targeting siRNA ( siNT ) or 50 pmol of siRNA targeting ATG13 or ULK1 . 48 hours post knockdown , cells were treated with 1 mM DFP for an additional 48 hours . Cell lysates were subject to immunoblotting with the indicated antibodies . Quantification is shown on the right .

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

D. ARPE-19 cells were transfected with 50 pmol of non-targeting siRNA (siNT) or 50 pmol of siRNA targeting ATG13 or ULK1. 48 hours post knockdown, cells were treated with 1 mM DFP for an additional 48 hours. Cell lysates were subject to immunoblotting with the indicated antibodies. Quantification is shown on the right.


A . Representative immunoblots of the indicated proteins in lysates of WT or HIF1α KO U2OS cells , treated with 1mM DFP for 48 hours .

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

A. Representative immunoblots of the indicated proteins in lysates of WT or HIF1α KO U2OS cells, treated with 1mM DFP for 48 hours.


B . Representative confocal images of WT or HIF1α KO U2OS cells stably expressing the pexo - QC reporter and treated with 1 mM DFP for 48 hours ( left panel ) and flow cytometry analysis of the mCherry / GFP ratio ( right panel ) .

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

B. Representative confocal images of WT or HIF1α KO U2OS cells stably expressing the pexo-QC reporter and treated with 1 mM DFP for 48 hours (left panel) and flow cytometry analysis of the mCherry/GFP ratio (right panel).


C . Representative immunoblots ( left panel ) and quantification ( right panel ) of the indicated proteins in lysates of WT or two NIX KO ARPE - 19 clones ( Cl ) , treated with 1 mM DFP for the indicated time ( h , hours ) .

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

C. Representative immunoblots (left panel) and quantification (right panel) of the indicated proteins in lysates of WT or two NIX KO ARPE-19 clones (Cl), treated with 1 mM DFP for the indicated time (h, hours).


D - E . Representative confocal images of WT or NIX KO ARPE - 19 cells stably expressing the mito - QC reporter ( D ) or the pexo - QC reporter ( E ) treated with 1 mM DFP for 48 hours ( left panel ) and flow cytometry analysis of the mCherry / GFP ratio ( right panel ) .

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

D-E. Representative confocal images of WT or NIX KO ARPE-19 cells stably expressing the mito-QC reporter (D) or the pexo-QC reporter (E) treated with 1 mM DFP for 48 hours (left panel) and flow cytometry analysis of the mCherry/GFP ratio (right panel).


F . Representative immunoblots ( left panel ) and quantification ( right panel ) of the indicated proteins . NIX KO ARPE - 19 cells ( Cl 31 ) stably expressing a pBabe empty vector ( ∅ ) or a pBabe NIX vector , were treated with 1 mM DFP for 48 hours prior lysis . Two biological replicates are shown in the immunoblot .

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

F. Representative immunoblots (left panel) and quantification (right panel) of the indicated proteins. NIX KO ARPE-19 cells (Cl 31) stably expressing a pBabe empty vector (∅) or a pBabe NIX vector, were treated with 1 mM DFP for 48 hours prior lysis. Two biological replicates are shown in the immunoblot.


A - B . Representative confocal images of untreated ( CTRL ) and DFP - treated ARPE - 19 cells ( 1mM , 48 hours ) , stably expressing GFP - WT NIX ( green ) and stained with anti - catalase antibody ( A , red ) or anti - PMP70 antibody ( B , red ) . At right , quantification of Pearson correlation coefficients between NIX and the peroxisomal markers catalase and PMP70 , with or without DFP treatment for 48 hours . Peroxisomes enriched for GFP - NIX or having minimal signal for GFP - NIX are depicted with white arrowheads or white arrows , respectively .

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

A-B. Representative confocal images of untreated (CTRL) and DFP-treated ARPE-19 cells (1mM, 48 hours), stably expressing GFP-WT NIX (green) and stained with anti-catalase antibody (A, red) or anti-PMP70 antibody (B, red). At right, quantification of Pearson correlation coefficients between NIX and the peroxisomal markers catalase and PMP70, with or without DFP treatment for 48 hours. Peroxisomes enriched for GFP-NIX or having minimal signal for GFP-NIX are depicted with white arrowheads or white arrows, respectively.


C . Representative confocal images of ARPE - 19 cells stably expressing GFP - WT NIX , GFP - NIXΔTM or GFP - NIX TM only ( green ) , treated with DFP ( 1mM ) for 48 hours and immunostained with anti - catalase antibody ( red ) . At right , quantification of Pearson correlation coefficients between the green and the red signal .

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

C. Representative confocal images of ARPE-19 cells stably expressing GFP-WT NIX, GFP-NIXΔTM or GFP-NIX TM only (green), treated with DFP (1mM) for 48 hours and immunostained with anti-catalase antibody (red). At right, quantification of Pearson correlation coefficients between the green and the red signal.


B . Representative immunoblots of the indicated proteins in lysates of WT or HA - Parkin ( PRKN ) - ARPE - 19 cells treated as in A .

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

B. Representative immunoblots of the indicated proteins in lysates of WT or HA-Parkin(PRKN)- ARPE-19 cells treated as in A.


B . Representative immunoblots of the indicated proteins in lysates of NIX KO ARPE - 19 cells ( Cl 31 ) stably expressing a pBabe Flag vector ( ∅ ) , a pBabe Flag - NIX vector ( NIX ) , a pBabe Flag - NIX W36A \ L39A vector ( W36A \ L39A ) or a pBabe Flag - NIX S34E \ S35E vector ( S34E \ S35E ) , and treated with 1 mM DFP for 48 hours prior lysis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative immunoblots of the indicated proteins in lysates of NIX KO ARPE-19 cells (Cl 31) stably expressing a pBabe Flag vector (∅), a pBabe Flag-NIX vector (NIX), a pBabe Flag-NIX W36A\L39A vector (W36A\L39A) or a pBabe Flag-NIX S34E\S35E vector (S34E\S35E), and treated with 1 mM DFP for 48 hours prior lysis.


C - D . Representative confocal images of cells as in B , stably expressing the mito - QC reporter ( C ) or the pexo - QC reporter ( D ) and treated with 1 mM DFP for 24 hours ( C ) or 48 hours ( D ) ( left panel ) and flow cytometry analysis of the mCherry / GFP ratio ( right panel ) . Enlarged images of the area outlined in white are shown in the lower corners . Nuclei were stained in blue ( Hoechst )

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

C-D. Representative confocal images of cells as in B, stably expressing the mito-QC reporter (C) or the pexo-QC reporter (D) and treated with 1 mM DFP for 24 hours (C) or 48 hours (D) (left panel) and flow cytometry analysis of the mCherry/GFP ratio (right panel). Enlarged images of the area outlined in white are shown in the lower corners. Nuclei were stained in blue (Hoechst)


A . Representative confocal images of optical section from WT and NIX KO mouse retina immunostained with peroxisomal markers ( PMP70 and catalase ) or a lysosomal marker LAMP1 . Nuclei are coloured in white ( DAPI ) . RPE , retinal pigment epithelium ; ONL , outer nuclear layer ; OPL , outer plexiform layer ; INL ; inner nuclear layer ; IPL , inner plexiform layer ; RGC , retinal ganglion cells . B . Quantification of signal area in the ONL and INL from images as A . Each data point represents a single mouse ( n = 3 - 4 per group ) .

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

A. Representative confocal images of optical section from WT and NIX KO mouse retina immunostained with peroxisomal markers (PMP70 and catalase) or a lysosomal marker LAMP1. Nuclei are coloured in white (DAPI). RPE, retinal pigment epithelium; ONL, outer nuclear layer; OPL, outer plexiform layer; INL; inner nuclear layer; IPL, inner plexiform layer; RGC, retinal ganglion cells. B. Quantification of signal area in the ONL and INL from images as A. Each data point represents a single mouse (n=3-4 per group).


GST - Mif2 _ 1 - 41 was immobilized on GSH Sepharose beads and incubated with Mtw1 - Nnf1 complex ( MN ) in the presence of increasing concentrations of Ame1 _ 1 - 20 peptide ( lanes 4 - 9 ) . GST - Ame1 _ 1 - 30 served as control ( lane 2 and 3 ) . After washing , bound complexes were eluted by adding SDS sample buffer . Protein bands in Coomassie stained gel are highlighted in the indicated color . Integrated density of Mtw1 bands was quantified using ImageJ and correlated to GST - Mif2 amounts in the same lane .

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

GST-Mif2_1-41 was immobilized on GSH Sepharose beads and incubated with Mtw1-Nnf1 complex (MN) in the presence of increasing concentrations of Ame1_1-20 peptide (lanes 4-9). GST-Ame1_1-30 served as control (lane 2 and 3). After washing, bound complexes were eluted by adding SDS sample buffer. Protein bands in Coomassie stained gel are highlighted in the indicated color. Integrated density of Mtw1 bands was quantified using ImageJ and correlated to GST-Mif2 amounts in the same lane.


Binding of FITC - Mif21 - 41 to MN was directly determined in a fluorescence polarization ( FP ) assay . This interaction at EC50 was than competed with increasing concentration of Ame11 - 20 unlabeled peptide . Both experiments were performed in triplicates of which the mean and standard deviation is shown .

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

Binding of FITC-Mif21-41 to MN was directly determined in a fluorescence polarization (FP) assay. This interaction at EC50 was than competed with increasing concentration of Ame11-20 unlabeled peptide. Both experiments were performed in triplicates of which the mean and standard deviation is shown.