( e ) The heatmap shows changes of the indicated cell type signatures in published bulk RNA - seq data ( GEO GSE124685 ) across different histopathological stages that represent increasing extent of fibrosis from stage 1 - 3 , as determined by quantitative micro - CT imaging and tissue histology Samples used in this study were 10 IPF and 6 control patients .

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(e) The heatmap shows changes of the indicated cell type signatures in published bulk RNA-seq data (GEO GSE124685) across different histopathological stages that represent increasing extent of fibrosis from stage 1-3, as determined by quantitative micro-CT imaging and tissue histology Samples used in this study were 10 IPF and 6 control patients.


( f ) The heatmaps show z - scores for the individual marker genes of the indicated cell types across IPF stages and controls .

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(f) The heatmaps show z-scores for the individual marker genes of the indicated cell types across IPF stages and controls.


( b ) The boxplots show the number of proteins quantified ( y - axis ) across various diagnoses ( x - axis ) . The mean and 10 - 90 percentiles are shown ; the dotted line marks 1000 proteins ( IPF n = 16 , HP / EAA n = 8 , COP n = 11 , NSIP n = 10 , RB - IL , n = 3 , Sarcoidosis n = 22 , other ILDs n = 25 , non - IL , n = 29 ) .

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(b) The boxplots show the number of proteins quantified (y-axis) across various diagnoses (x-axis). The mean and 10-90 percentiles are shown; the dotted line marks 1000 proteins (IPF n=16, HP/EAA n=8, COP n=11, NSIP n=10, RB-IL, n=3, Sarcoidosis n=22, other ILDs n=25, non-IL, n=29).


( c Empirical cumulative density plots depict the distribution of correlation coefficients for ( c ) Myofibroblast markers ( red points ) with DLCO in comparison to all background proteins ( black line ) .

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(c Empirical cumulative density plots depict the distribution of correlation coefficients for (c) Myofibroblast markers (red points) with DLCO in comparison to all background proteins (black line).


d ) Empirical cumulative density plots depict the distribution of correlation coefficients for ( d ) Plasma cell markers ( red points ) with % alveolar macrophages in BAL in comparison to all background proteins ( black line ) .

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d) Empirical cumulative density plots depict the distribution of correlation coefficients for (d) Plasma cell markers (red points) with % alveolar macrophages in BAL in comparison to all background proteins (black line).


( e ) Barplot displays coefficients derived from correlating lung function associations at the protein level with cell type specific ILD associations at the RNA level .

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(e) Barplot displays coefficients derived from correlating lung function associations at the protein level with cell type specific ILD associations at the RNA level.


( f ) The heatmap illustrates gene expression changes associated with lung fibrosis across indicated cell types ( columns ) for selected BALF protein biomarkers ( rows ) . The dotplot visualizes the frequency changes of the indicated cell types inferred from the deconvolution of bulk mRNA data of ILD samples compared to controls ( GSE47460 ) . Samples used in this study from the LTRC n = 254 ILD patients and n = 108 controls .

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(f) The heatmap illustrates gene expression changes associated with lung fibrosis across indicated cell types (columns) for selected BALF protein biomarkers (rows). The dotplot visualizes the frequency changes of the indicated cell types inferred from the deconvolution of bulk mRNA data of ILD samples compared to controls (GSE47460). Samples used in this study from the LTRC n = 254 ILD patients and n = 108 controls.


( a , b ) Scatter plots stratify genes based on the protein lung function associations ( y - axis ) and cell type specific ILD associations ( x - axis ) . The size of the dots represents the detection level of each gene in the corresponding cell type . Colors highlight genes with marginal associations at the protein and RNA levels .

O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O 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) Scatter plots stratify genes based on the protein lung function associations (y-axis) and cell type specific ILD associations (x-axis). The size of the dots represents the detection level of each gene in the corresponding cell type. Colors highlight genes with marginal associations at the protein and RNA levels.


( c , d ) The box plots illustrate differences in mRNA detection for the indicated genes between tissues from end stage lung fibrosis patients in ( c ) basal cells , and ( d ) alveolar epithelial cells when compared to controls . The boxes represent the interquartile range , the horizontal line in the box is the median , and the whiskers represent 1 . 5 times the interquartile range ( Chicago cohort : ILD n = 9 , controls n = 8 ; Nashville cohort : ILD n = 20 , controls n = 10 ; Munich cohort : ILD n = 3 , controls n = 11 ) .

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(c, d) The box plots illustrate differences in mRNA detection for the indicated genes between tissues from end stage lung fibrosis patients in (c) basal cells, and (d) alveolar epithelial cells when compared to controls. The boxes represent the interquartile range, the horizontal line in the box is the median, and the whiskers represent 1.5 times the interquartile range (Chicago cohort: ILD n = 9, controls n = 8; Nashville cohort: ILD n = 20, controls n = 10; Munich cohort: ILD n = 3, controls n = 11).


( e , f ) The scatter plots show the positive correlation of the indicated proteins in BALF ( MS - intensity , x - axis ) with meta lung function ( y - axis ) .

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(e, f) The scatter plots show the positive correlation of the indicated proteins in BALF (MS-intensity, x-axis) with meta lung function (y-axis).


( g - j ) Immunofluorescence analysis of the indicated proteins and cell type markers in IPF ( n = 3 ) and control samples ( n = 3 ) .

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(g-j) Immunofluorescence analysis of the indicated proteins and cell type markers in IPF (n=3) and control samples (n=3).


( a ) The scatter plots show the positive correlation of CFHR1 in BALF ( MS - intensity , x - axis ) with meta lung function ( y - axis ) .

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(a) The scatter plots show the positive correlation of CFHR1 in BALF (MS-intensity, x-axis) with meta lung function (y-axis).


( c ) The volcano plot depicts the fold changes ( x - axis ) and the - log10 p - values ( y - axis ) derived from the differential gene expression analysis using diffxpy between ILD and controls within the subset of PDGFRB + pericytes ( outlier values truncated ) .

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(c) The volcano plot depicts the fold changes (x-axis) and the -log10 p-values (y-axis) derived from the differential gene expression analysis using diffxpy between ILD and controls within the subset of PDGFRB+ pericytes (outlier values truncated).


( e ) The heatmap shows the expression of genes most strongly associated with SSTR2 expression within the pericytes ( left color bar indicates significant correlation or anti - correlation ) .

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(e) The heatmap shows the expression of genes most strongly associated with SSTR2 expression within the pericytes (left color bar indicates significant correlation or anti-correlation).


( f ) Immunofluorescence analysis of SSTR2 , the pericyte cell type marker PDGFRB and a marker for smooth muscle cells DESMIN in IPF ( n = 3 ) and control samples ( n = 3 ) .

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(f) Immunofluorescence analysis of SSTR2, the pericyte cell type marker PDGFRB and a marker for smooth muscle cells DESMIN in IPF (n=3) and control samples (n=3).


( g ) Representative images of immunohistochemistry analysis of SSTR2 protein expression in tissue regions ( n = 474 ) from ILD patients ( n = 53 ) and control patients ( n = 26 ) .

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(g) Representative images of immunohistochemistry analysis of SSTR2 protein expression in tissue regions (n = 474) from ILD patients (n = 53) and control patients (n = 26).


( h ) Correlation of immunohistochemistry signal of SSTR2 with Ashcroft scores .

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(h) Correlation of immunohistochemistry signal of SSTR2 with Ashcroft scores.


( i ) The bar graph shows the genes most strongly correlated with SSTR2 belonging to the GO category " transcription regulators " . The dotted line marks a correlation coefficient of zero .

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(i) The bar graph shows the genes most strongly correlated with SSTR2 belonging to the GO category "transcription regulators". The dotted line marks a correlation coefficient of zero.


( c ) Relative expression level of CRTAC1 across human organs .

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(c) Relative expression level of CRTAC1 across human organs.


( d ) The box plots illustrate differences in mRNA detection for CRTAC1 in alveolar epithelial cells from fibrosis patients compared to control samples across the three indicated patient cohorts ( Chicago cohort : ILD n = 9 , controls n = 8 ; Nashville cohort : ILD n = 20 , controls n = 10 ; Munich cohort : ILD n = 3 , controls n = 11 ) .

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(d) The box plots illustrate differences in mRNA detection for CRTAC1 in alveolar epithelial cells from fibrosis patients compared to control samples across the three indicated patient cohorts (Chicago cohort: ILD n = 9, controls n = 8; Nashville cohort: ILD n = 20, controls n = 10; Munich cohort: ILD n = 3, controls n = 11).


( e ) Relative gene expression levels of CRTAC1 in GSE47460 . Dots represent average expression in the tissue of individual patients . The line represents the mean , error bars show SD . CRTAC1 is significantly downregulated in ILD but not COPD

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(e) Relative gene expression levels of CRTAC1 in GSE47460. Dots represent average expression in the tissue of individual patients. The line represents the mean, error bars show SD. CRTAC1 is significantly downregulated in ILD but not COPD patients


( f ) For each single cell cohort , the gene - gene correlations with CRTAC1 within the SFTPC + AT - 2 cells were calculated . The indicated genes were selected based on their common direction of correlation across cohorts .

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(f) For each single cell cohort, the gene-gene correlations with CRTAC1 within the SFTPC+ AT-2 cells were calculated. The indicated genes were selected based on their common direction of correlation across cohorts.


( h ) The bar graph shows the gene categories most strongly correlated with CRTAC1 belonging to the GO category of " transcription regulators " . The dotted line marks a correlation coefficient of zero .

O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

(h) The bar graph shows the gene categories most strongly correlated with CRTAC1 belonging to the GO category of "transcription regulators". The dotted line marks a correlation coefficient of zero.


( n ) Immunofluorescence analysis of SPRR1A , KRT8 as well as SFTPC in IPF ( n = 3 ) and control samples ( n = 2 ) .

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(n) Immunofluorescence analysis of SPRR1A, KRT8 as well as SFTPC in IPF (n=3) and control samples (n=2).


( o ) A high throughput experimental workflow for plasma proteomics ( Niu allowed for profiling of two independent cohorts of ILD patients ( Munich , n = 30 and Hannover , n = 81 ; healthy age matched controls , n = 30 ) . All proteins quantified in plasma are shown , ranked by their abundance measured by mass spectrometry ( MS - intensity ) .

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(o) A high throughput experimental workflow for plasma proteomics(Niu allowed for profiling of two independent cohorts of ILD patients (Munich, n=30 and Hannover, n=81; healthy age matched controls, n=30). All proteins quantified in plasma are shown, ranked by their abundance measured by mass spectrometry (MS-intensity).


( p ) The indicated proteins from the plasma analysis were selected based on their common direction of correlation with patient lung function in two independent patient cohorts with distinct clinical characteristics .

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(p) The indicated proteins from the plasma analysis were selected based on their common direction of correlation with patient lung function in two independent patient cohorts with distinct clinical characteristics.


( q ) The heatmap shows the predicted relative contribution of lung cell types to the association of protein biomarker signatures in plasma with lung function ( forced vital capacity - FVC ) . Patients were split in two groups , one with a mild decline in lung function [ FVC 60 - 100 % ] and one with severe loss of lung function [ FVC 20 - 60 % ] and compared to healthy age matched controls .

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(q) The heatmap shows the predicted relative contribution of lung cell types to the association of protein biomarker signatures in plasma with lung function (forced vital capacity - FVC). Patients were split in two groups, one with a mild decline in lung function [FVC 60-100%] and one with severe loss of lung function [FVC 20-60%] and compared to healthy age matched controls.


CSTB gene expression analysis in hCOs , starting from day ( d ) 16 until d140 . For every time point , at least 3 different samples were analyzed ; each sample was made by a pool of 3 to 4 hCOs . Data are represented as mean ± SEM . Unpaired t - test ( * P < 0 . 05 , * * P < 0 . 01 ) .

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CSTB gene expression analysis in hCOs, starting from day (d) 16 until d140. For every time point, at least 3 different samples were analyzed; each sample was made by a pool of 3 to 4 hCOs. Data are represented as mean ± SEM. Unpaired t-test (*P < 0.05, **P < 0.01).


Micrographs of sections of control hCOs immunostained with CSTB antibody at different time points in culture . The dashed lines represent the apical surface of the cavities / ventricles ( V - like ) . The cortical plate like side is indicated ( CP - like ) . Nuclei ( blue ) are stained with DAPI .

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Micrographs of sections of control hCOs immunostained with CSTB antibody at different time points in culture. The dashed lines represent the apical surface of the cavities/ventricles (V-like). The cortical plate like side is indicated (CP-like). Nuclei (blue) are stained with DAPI.


Real time qPCR results of PAX6 , NEUN , NESTIN , CSTB gene expression levels in PAX6 - and NEUN - FACS sorted nuclei from f - CTRL d135 hCOs . Data are represented as mean ± SEM . Statistical significance was based on T - student test ( * P < 0 . 05 , * * P < 0 . 01 ) . For PAX6 , 10 samples were analyzed as biological replicates : 5 for Pax6 + sorted Nuclei and 5 for Neun + sorted Nuclei ; for NEUN , NESTIN and CSTB , 9 samples were analyzed as biological replicates : 5 for Pax6 + sorted Nuclei and 4 for Neun + sorted Nuclei .

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Real time qPCR results of PAX6, NEUN, NESTIN, CSTB gene expression levels in PAX6- and NEUN- FACS sorted nuclei from f-CTRL d135 hCOs. Data are represented as mean ± SEM. Statistical significance was based on T-student test (*P < 0.05, **P < 0.01). For PAX6, 10 samples were analyzed as biological replicates: 5 for Pax6+sorted Nuclei and 5 for Neun+sorted Nuclei; for NEUN, NESTIN and CSTB, 9 samples were analyzed as biological replicates: 5 for Pax6+sorted Nuclei and 4 for Neun+sorted Nuclei.


Western blot analysis and relative quantifications ( arbitrary units , a . u . ) on protein extracts from cortical ( Cx ) and ganglionic eminence ( GE ) tissues for Cstb using Actb as a loading control . Cx and GE tissues from mouse embryos at different time of development as indicated in figure .

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Western blot analysis and relative quantifications (arbitrary units, a.u.) on protein extracts from cortical (Cx) and ganglionic eminence (GE) tissues for Cstb using Actb as a loading control. Cx and GE tissues from mouse embryos at different time of development as indicated in figure.


Micrograph of coronal sections of E16 mouse cerebral cortices immunostained for Cstb and acetylated tubulin ( acTub ) as marker for stabilized microtubules . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Nuclei ( blue ) are stained with DAPI .

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Micrograph of coronal sections of E16 mouse cerebral cortices immunostained for Cstb and acetylated tubulin (acTub) as marker for stabilized microtubules. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Nuclei (blue) are stained with DAPI.


Micrograph sections of d40 hCOs electroporated with GFP - empty vector control or GFP - CSTB and analyzed 5 dpe . Sections were then immunostained for GFP and KI67 . The dashed lines represent the apical surface of the cavities / ventricles ( V - like ) . The cortical plate like side is indicated ( CP - like ) . Quantification of the total number of proliferating KI67 + cells / area ( µm2 ) of V - like structures transfected with GFP - empty vector control or GFP - CSTB in ( A ) .

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Micrograph sections of d40 hCOs electroporated with GFP-empty vector control or GFP-CSTB and analyzed 5 dpe. Sections were then immunostained for GFP and KI67. The dashed lines represent the apical surface of the cavities/ventricles (V-like). The cortical plate like side is indicated (CP-like). Quantification of the total number of proliferating KI67+ cells/area (µm2) of V-like structures transfected with GFP-empty vector control or GFP-CSTB in (A).


Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP - empty vector or GFP - Cstb and analyzed 2 dpe and immunostained with GFP , Ki67 and BrdU 30 minutes after BrdU injection . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Quantification of the total number of proliferating Ki67 + cells / area ( µm2 ) ( D ) and Ki67 + Brdu + cells / area ( µm2 ) ( E ) of ventricles transfected with GFP - empty vector or GFP - Cstb in ( C ) .

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Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector or GFP-Cstb and analyzed 2 dpe and immunostained with GFP,Ki67 and BrdU 30 minutes after BrdU injection. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of proliferating Ki67+ cells/area(µm2)(D) and Ki67+Brdu+ cells/area(µm2)(E) of ventricles transfected with GFP-empty vector or GFP-Cstb in (C).


Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP - empty vector or GFP - Cstb , analyzed 2 dpe and immunostained with GFP and Phospho - Histone H3 ( PH3 ) ( F ) and with GFP , Pax6 and Tbr2 ( G ) . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles .

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Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector or GFP-Cstb, analyzed 2 dpe and immunostained with GFP and Phospho-Histone H3 (PH3)(F) and with GFP, Pax6 and Tbr2 (G). Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles.


Distribution of proliferating Ki67 + / BrdU + cells ( H ) and mitotic progenitor PH3 + cells ( I ) in the mouse cortex . The cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3 + cells or Ki67 + / BrdU + cells calculated in each Bin .

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Distribution of proliferating Ki67+/BrdU+cells (H) and mitotic progenitor PH3+ cells (I) in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3+ cells or Ki67+/BrdU+ cells calculated in each Bin.


Distribution of Tbr2 + intermediate progenitors ( J ) and Pax6 + Tbr2 + cells ( K ) in the mouse cortex . The cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side and Bin5 to pial side of the cortex and Tbr2 + cells or Pax6 + Tbr2 + cells calculated in each Bin .

O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-TISSUE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O

Distribution of Tbr2+ intermediate progenitors (J) and Pax6+Tbr2+ cells (K) in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and Tbr2+ cells or Pax6+Tbr2+ cells calculated in each Bin.


Western blot analysis for CSTB in the CSF from 2 different E14 mouse embryos ( A ) , in the CM from E14 cortical cells in culture for 4

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Western blot analysis for CSTB in the CSF from 2 different E14 mouse embryos (A), in the CM from E14 cortical cells in culture for 4 days


Western blot analysis for CSTB in the CM from hCOs after 4 days in culture ( C ) . The CM was collected from hCOs at different time points in culture ( d19 , d38 , d60 , d80 ) . Protein extract ( PE ) from 60 days hCOs was used as a positive control . GAPDH immunostaining was used as a negative control on hCOs CM . Ponceau red staining is shown to indicate the presence of proteins at comparable levels in the CM

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O

Western blot analysis for CSTB in the CM from hCOs after 4 days in culture (C). The CM was collected from hCOs at different time points in culture (d19, d38, d60, d80). Protein extract (PE) from 60 days hCOs was used as a positive control. GAPDH immunostaining was used as a negative control on hCOs CM. Ponceau red staining is shown to indicate the presence of proteins at comparable levels in the CM samples


Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co - electroporated with mCherry expressing vector and HA - empty vector or - Cstb , and analyzed 3 dpe . Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65 - GFP transgenic mouse line . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Distribution of GFP + interneurons in the 5 equal Bins of the mouse developing cortex - Bin1 corresponded to apical side and Bin5 to pial side . Ventricle ( V ) is indicated . The dashed lines represent the apical surface of the ventricles .

O O O O O O B-ORGANISM B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O B-EXP_ASSAY B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O B-TISSUE O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O O B-TISSUE O O O B-GENEPROD O B-CELL_TYPE O O O O O O O B-ORGANISM O B-TISSUE O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O B-TISSUE O

Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co-electroporated with mCherry expressing vector and HA-empty vector or -Cstb, and analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65-GFP transgenic mouse line. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of GFP+ interneurons in the 5 equal Bins of the mouse developing cortex-Bin1 corresponded to apical side and Bin5 to pial side. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles.


Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co - electroporated with mCherry and HA - Cstb expressing vector , analyzed 3 dpe . Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD67 - GFP transgenic mouse line . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Distribution of GFP + interneurons in the 5 equal Bins of the mouse developing cortex comparing electroporated sides with the contralateral ones in the same section to avoid different distribution of interneurons rostro - caudally .

O O O O O O B-ORGANISM B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O B-EXP_ASSAY B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O B-TISSUE O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O O B-TISSUE O O O B-GENEPROD O B-CELL_TYPE O O O O O O O B-ORGANISM O B-TISSUE O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O

Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co-electroporated with mCherry and HA-Cstb expressing vector, analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD67-GFP transgenic mouse line. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of GFP+ interneurons in the 5 equal Bins of the mouse developing cortex comparing electroporated sides with the contralateral ones in the same section to avoid different distribution of interneurons rostro-caudally.


Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP - miR - neg vector ( CTRL ) and GFP - miRNA - Cstb ( KD ) , analyzed 3 dpe and immunostained with GFP . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Distribution of electroporated GFP + cells in the mouse cortex . The cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side and Bin5 to pial side of the cortex and GFP + cells calculated in each Bin . Nuclei ( blue ) are stained with DAPI .

O O O O O O B-ORGANISM B-TISSUE I-TISSUE O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD B-TISSUE O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O O B-TISSUE O O O O B-GENEPROD O O O O B-ORGANISM B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-TISSUE O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O

Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP-miR-neg vector (CTRL) and GFP-miRNA-Cstb (KD), analyzed 3 dpe and immunostained with GFP. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of electroporated GFP+ cells in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and GFP+ cells calculated in each Bin. Nuclei (blue) are stained with DAPI.


Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP - empty vector control and GFP - miRNA - Cstb , analyzed 3 dpe and immunostained with GFP and Ki67 . Ventricle ( V ) is indicated . The dashed lines represent the apical surface of the ventricles . Quantification of the total number of proliferating Ki67 + cells / area ( µm2 ) of ventricular structures transfected with GFP - miR - neg vector or GFP - miRNA - Cstb in ( C ) . Nuclei ( blue ) are stained with DAPI .

O O O O O O B-ORGANISM B-TISSUE I-TISSUE O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O O O O O O O O O O O O B-TISSUE O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O B-TISSUE O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O

Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 with GFP-empty vector control and GFP-miRNA-Cstb, analyzed 3 dpe and immunostained with GFP and Ki67. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of proliferating Ki67+ cells/area (µm2) of ventricular structures transfected with GFP-miR-neg vector or GFP-miRNA- Cstb in (C). Nuclei (blue) are stained with DAPI.


Micrograph sections of d40 hCOs electroporated with GFP - CSTB expressing vector or GFP - R68X mutant and analyzed 5 dpe . Sections were immunostained for GFP and Ki67 . The dashed lines represent the apical surface of the cavities / ventricles ( V - like ) . The cortical plate like side is indicated ( CP - like ) . Quantification of the total number of proliferating KI67 + cells / area ( µm2 ) ( F ) and Ki67 + GFP + cells / area ( µm2 ) ( G ) of ventricles like structures transfected with GFP - empty vector control , GFP - CSTB or GFP - R68X mutant in ( E ) .

O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O B-TISSUE I-TISSUE I-TISSUE O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-TISSUE I-TISSUE O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O

Micrograph sections of d40 hCOs electroporated with GFP-CSTB expressing vector or GFP-R68X mutant and analyzed 5 dpe. Sections were immunostained for GFP and Ki67. The dashed lines represent the apical surface of the cavities/ventricles (V-like). The cortical plate like side is indicated (CP-like). Quantification of the total number of proliferating KI67+ cells/area(µm2)(F)and Ki67+GFP+ cells/area(µm2)(G) of ventricles like structures transfected with GFP-empty vector control, GFP-CSTB or GFP-R68X mutant in (E).


Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP - empty vector control , GFP - Cstb or GFP - R68X , analyzed 2 dpe and immunostained with GFP and Phospho - Histone H3 ( PH3 ) . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Distribution of electroporated GFP + cells in the mouse cortex . The cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side and Bin5 to pial side of the cortex and GFP + cells calculated in each Bin .

O O O O O O B-ORGANISM B-TISSUE I-TISSUE O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-TISSUE O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O O B-TISSUE O O O O B-GENEPROD O O O O B-ORGANISM B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-TISSUE O B-GENEPROD O O O O O O O

Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector control, GFP-Cstb or GFP-R68X, analyzed 2 dpe and immunostained with GFP and Phospho-Histone H3 (PH3). Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Distribution of electroporated GFP+ cells in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and GFP+ cells calculated in each Bin.


Distribution of mitotic progenitor PH3 + cells in the mouse cortex . The cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3 + cells calculated in each Bin .

O O O O O O O O O B-ORGANISM B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O

Distribution of mitotic progenitor PH3+ cells in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3+ cells calculated in each Bin.


K . Quantification of the total number of proliferating PH3 + cells / area ( µm2 ) of ventricular structures transfected with GFP - empty vector control , GFP - Cstb or GFP - R68X mutant in ( H ) .

O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-TISSUE O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O

K.Quantification of the total number of proliferating PH3+ cells/area(µm2)of ventricular structures transfected with GFP-empty vector control, GFP-Cstb or GFP-R68X mutant in (H).


Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP - empty vector control , GFP - Cstb or GFP - R68X mutant , analyzed 2 dpe and immunostained with GFP and Tbr2 . Ventricle ( V ) is indicated . The dashed lines represent the apical surface of the ventricles . Quantification of the total number of Tbr2 + cells / cortical area ( µm2 ) in ( L ) . Data shown as Z - scores relative to the mean of GFP control vector .

O O O O O O B-ORGANISM B-TISSUE I-TISSUE O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O O O O O O O O O O O O B-TISSUE O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O

Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector control, GFP-Cstb or GFP-R68X mutant, analyzed 2 dpe and immunostained with GFP and Tbr2. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of Tbr2+ cells/ cortical area (µm2) in (L). Data shown as Z-scores relative to the mean of GFP control vector.


Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co - electroporated with mCherry expressing vector and HA - Cstb or R68X , and analyzed 3 dpe . Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65 - GFP transgenic mouse line . Ventricle ( V ) is indicated . The dashed lines represent the apical surface of the ventricles . Quantification of the total number of GAD65 - GFP interneurons / cortical area ( µm2 ) in ( N ) . Data shown as Z - scores relative to the mean of GFP control vector . Nuclei ( blue ) are stained with DAPI .

O O O O O O B-ORGANISM B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O B-EXP_ASSAY B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O B-TISSUE O O O O O O O O O O O O O O O B-TISSUE O O O O O O O B-GENEPROD O B-GENEPROD B-CELL_TYPE O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O

Micrograph of coronal sections of E17 mouse cerebral cortices electroporated at E14 co-electroporated with mCherry expressing vector and HA-Cstb or R68X, and analyzed 3 dpe. Immunostaining with RFP to identify electroporated cells and GFP to identify migrating interneurons in the GAD65-GFP transgenic mouse line. Ventricle (V) is indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of GAD65-GFP interneurons/ cortical area (µm2) in (N). Data shown as Z-scores relative to the mean of GFP control vector. Nuclei (blue) are stained with DAPI.


Left panel shows representative images of hCOs from the different iPSC lines at the analyzed time points . Right panel shows the difference of the areas of f - CTRL , b - CTRL , UL1 and UL4 hCOs at different time in culture . Data shown as Z - scores relative to the mean of b - CTRL .

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Left panel shows representative images of hCOs from the different iPSC lines at the analyzed time points. Right panel shows the difference of the areas of f-CTRL, b-CTRL, UL1 and UL4 hCOs at different time in culture. Data shown as Z-scores relative to the mean of b-CTRL.


Micrograph sections of d30 b - CTRL , UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3 . The dashed lines represent the apical surface of the cavities / ventricles . Quantification of the total number of proliferating Ki67 + cells / area ( µm2 ) of ventricles like structures and of the number of PH3 + cells facing the ventricle / apical surface length ( µm ) . Data shown as Z - scores relative to the mean of b - CTRL . Nuclei ( blue ) are stained with DAPI .

O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O

Micrograph sections of d30 b-CTRL, UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the total number of proliferating Ki67+ cells/area (µm2) of ventricles like structures and of the number of PH3+ cells facing the ventricle/apical surface length (µm). Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.


Micrograph sections of d60 b - CTRL , UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3 . The dashed lines represent the apical surface of the cavities / ventricles . Quantification of the total number of proliferating KI67 + cells / area of ventricles like structures and of the number of PH3 + cells facing the ventricle / the apical surface length ( µm ) . Data shown as Z - scores relative to the mean of CTRL . Nuclei ( blue ) are stained with DAPI .

O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O

Micrograph sections of d60 b-CTRL, UL1 and UL4 hCOs immunostained for proliferation markers KI67 and PH3. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the total number of proliferating KI67+ cells/ area of ventricles like structures and of the number of PH3+ cells facing the ventricle/the apical surface length (µm). Data shown as Z-scores relative to the mean of CTRL. Nuclei (blue) are stained with DAPI.


Scheme of the experiment with conditioned medium exchange between b - CTRL and UL4 hCOs and quantification of the Ki67 + cells by FACS analysis on 36 days old dissociated hCOs . Four samples were analyzed as biological replicates .

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Scheme of the experiment with conditioned medium exchange between b-CTRL and UL4 hCOs and quantification of the Ki67+ cells by FACS analysis on 36 days old dissociated hCOs. Four samples were analyzed as biological replicates.


Micrograph sections of d30 b - CTRL , UL1 and UL4 hCOs immunostained for the neuronal marker DCX . The dashed lines represent the apical surface of the cavities / ventricles . Quantification of the DCX + area . The area of DCX + cells was calculated and related to the total area ( µm2 ) of the ventricular structure . Data shown as Z - scores relative to the mean of b - CTRL . Nuclei ( blue ) are stained with DAPI .

O O O O O O O O O O O O B-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O B-TISSUE O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O

Micrograph sections of d30 b-CTRL, UL1 and UL4 hCOs immunostained for the neuronal marker DCX. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the DCX+ area. The area of DCX+ cells was calculated and related to the total area (µm2) of the ventricular structure. Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.


Micrograph sections of d60 b - CTRL , UL1 and UL4 hCOs immunostained for neuronal marker DCX . The dashed lines represent the apical surface of the cavities / ventricles . Quantification of the DCX + area . The area ( µm2 ) of DCX + cells was calculated and related to the total area of the ventricular structure . Data shown as Z - scores relative to the mean of b - CTRL . Quantification of the number of neuronal DCX + cells by FACS sorting on 60 days old dissociated hCOs . Data shown as Z - scores relative to the mean of b - CTRL . Nuclei ( blue ) are stained with DAPI .

O O O O O O O O O O O O B-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O B-TISSUE O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O

Micrograph sections of d60 b-CTRL, UL1 and UL4 hCOs immunostained for neuronal marker DCX. The dashed lines represent the apical surface of the cavities/ventricles. Quantification of the DCX+ area. The area (µm2) of DCX+ cells was calculated and related to the total area of the ventricular structure. Data shown as Z-scores relative to the mean of b-CTRL. Quantification of the number of neuronal DCX+ cells by FACS sorting on 60 days old dissociated hCOs. Data shown as Z-scores relative to the mean of b-CTRL. Nuclei (blue) are stained with DAPI.


Micrograph sections of d30 fused - organoid ; ventral GFP - f - CTRL and dorsal UL1 .

O O O O O O O O O B-GENEPROD O O O O O O O O

Micrograph sections of d30 fused-organoid; ventral GFP-f-CTRL and dorsal UL1.


Micrographs of sections from d30 fused organoids , showing ventral GFP + cells in the dorsal area . The migrating GFP + cells were counted starting from 100 µm far from the ventral / dorsal border . Quantification of ventrally - derived GFP + cells in dorsal / thickness of the counted area ( µm3 ) . The counted area was always 100 µm far from the border between ventral and dorsal area as indicated in Fig . EV5D . Every dot represents a different hCOs and different areas were counted for each hCO . Nuclei ( blue ) are stained with DAPI .

O O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O

Micrographs of sections from d30 fused organoids, showing ventral GFP+ cells in the dorsal area. The migrating GFP+ cells were counted starting from 100 µm far from the ventral/dorsal border. Quantification of ventrally-derived GFP+ cells in dorsal/thickness of the counted area (µm3). The counted area was always 100 µm far from the border between ventral and dorsal area as indicated in Fig.EV5D. Every dot represents a different hCOs and different areas were counted for each hCO. Nuclei (blue) are stained with DAPI.


Volcano plot representing proteins identified by high - resolution nanoLC MS / MS analysis . Up - and down - regulated proteins ( - 0 . 3 ≥ log2FC ≥ 0 . 3 ) in human cerebral organoids ( UL1 vs b - CTRL ) are shown in red and green , respectively .

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Volcano plot representing proteins identified by high-resolution nanoLC MS/MS analysis. Up- and down-regulated proteins (-0.3≥log2FC≥0.3) in human cerebral organoids (UL1 vs b-CTRL) are shown in red and green, respectively.


A - C Calcium flux measured by FACScan for splenic B cells isolated from B1 - 8 transgenic mice after stimulation with ( A ) NIP15 - BSA ( 30 pM ) or 1NIP - pep ( see Figure EV1 , 80 nM ) ; ( B ) Ac146 antibody ( 12 . 5 nM ) or Ac146Fab ( 25 nM ) ; ( C ) Ac38 antibody ( 12 . 5 nM ) or Ac38Fab ( 25 nM ) . The addition of the stimuli to the cells is indicated by arrows .

O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-TISSUE B-CELL_TYPE I-CELL_TYPE O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

A-C Calcium flux measured by FACScan for splenic B cells isolated from B1-8 transgenic mice after stimulation with (A) NIP15-BSA (30 pM) or 1NIP-pep (see Figure EV1, 80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). The addition of the stimuli to the cells is indicated by arrows.


D Representative microscopy images showing Fab - PLA results measuring the BCR proximity for the IgM - BCR ( upper ) and the IgD - BCR ( lower ) on untreated or treated B1 - 8 splenic B cells . PLA signals are shown as red dots and the nuclei are visualized by DAPI staining . Scale bar : 5 μm . E The Fab - PLA results are quantified by Blobfinder software and presented as box plots . The median values are highlighted as thick lines and the whiskers represent the minimum and maximum value . PLA signals ( dots / cells ) were counted from at least 100 cells for each sample . Data from treated samples were compared to data from resting cells , p - values were calculated by Kruskal - Wallis one - way analysis of variance ( ANOVA ) .

O O O O O B-GENEPROD O B-EXP_ASSAY O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-TISSUE B-CELL_TYPE I-CELL_TYPE O B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Representative microscopy images showing Fab-PLA results measuring the BCR proximity for the IgM-BCR (upper) and the IgD-BCR (lower) on untreated or treated B1-8 splenic B cells. PLA signals are shown as red dots and the nuclei are visualized by DAPI staining. Scale bar: 5 μm.E The Fab-PLA results are quantified by Blobfinder software and presented as box plots. The median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. Data from treated samples were compared to data from resting cells, p-values were calculated by Kruskal-Wallis one-way analysis of variance (ANOVA).


A - C Calcium flux measured by FACScan for splenic B cells isolated from Lyn - deficient B1 - 8 transgenic mice after stimulation with ( A ) NIP15 - BSA ( 30 pM ) or 1NIP - pep ( 80 nM ) ; ( B ) Ac146 antibody ( 12 . 5 nM ) or Ac146Fab ( 25 nM ) ; ( C ) Ac38 antibody ( 12 . 5 nM ) or Ac38Fab ( 25 nM ) . Arrows indicate the addition of the stimuli to the cells .

O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-TISSUE B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O

A-C Calcium flux measured by FACScan for splenic B cells isolated from Lyn-deficient B1-8 transgenic mice after stimulation with (A) NIP15-BSA (30 pM) or 1NIP-pep (80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.


D Representative microscopy images showing Fab - PLA results monitoring the BCR proximity for the IgM - BCR ( upper ) and the IgD - BCR ( lower ) on untreated or treated B1 - 8 splenic B cells . Scale bar : 5 μm . E Quantified Fab - PLA results are presented as box plots , where the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value . PLA signals ( dots / cells ) were counted from at least 100 cells for each sample . p - values were calculated by Kruskal - Wallis one - way ANOVA .

O O O O O B-GENEPROD O B-EXP_ASSAY O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-TISSUE B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Representative microscopy images showing Fab-PLA results monitoring the BCR proximity for the IgM-BCR (upper) and the IgD-BCR (lower) on untreated or treated B1-8 splenic B cells. Scale bar: 5 μm.E Quantified Fab-PLA results are presented as box plots, where the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. p-values were calculated by Kruskal-Wallis one-way ANOVA.


A - D Proximity between IgM - BCR on the surface of 3046SM ( A , C ) or 3046M ( B , D ) cells before and after a 1 - min stimulation with the indicated reagents , assayed by Fab - PLA . Results are presented as representative microscopy images ( A , B ) and box plots after quantification ( C , D ) . Scale bar : 5 μm . In the box plots , the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value . PLA signals ( dots / cells ) were counted from at least 100 cells for each sample and p - values were calculated by Kruskal - Wallis one - way ANOVA .

O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O 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-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O 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-D Proximity between IgM-BCR on the surface of 3046SM (A, C) or 3046M (B, D) cells before and after a 1-min stimulation with the indicated reagents, assayed by Fab-PLA. Results are presented as representative microscopy images (A, B) and box plots after quantification (C, D). Scale bar: 5 μm. In the box plots, the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample and p-values were calculated by Kruskal-Wallis one-way ANOVA.


E - H Calcium flux measured by FACScan for 3046SM ( E , F ) and 3046M ( G , H ) cells after stimulation with NIP15 - BSA ( 30 pM ) , 1NIP - pep ( 80 nM ) , Ac146Fab ( 25 nM ) or Ac38Fab ( 25 nM ) . Arrows indicate the addition of the stimuli to the cells .

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E-H Calcium flux measured by FACScan for 3046SM (E, F) and 3046M (G, H) cells after stimulation with NIP15-BSA (30 pM), 1NIP-pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.


A - D Proximity between IgD - BCRs on the surface of 3046SD ( A , C ) or 3046D ( B , D ) cells before and after a 1 - min stimulation , assayed by Fab - PLA . Results are presented as representative microscopy images ( A , B ) and box plots after quantification ( C , D ) . PLA signals are shown as red dots and nuclei were visualized by DAPI staining . Scale bar : 5 μm . In the box plots , the median values were highlighted as thick lines and the whiskers represent the minimum and maximum value . PLA signals ( dots / cells ) were counted from at least 100 cells for each sample and p - values were calculated by Kruskal - Wallis one - way ANOVA .

O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O 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-GENEPROD O B-EXP_ASSAY 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-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O 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-EXP_ASSAY O O O O O O O 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-D Proximity between IgD-BCRs on the surface of 3046SD (A, C) or 3046D (B, D) cells before and after a 1-min stimulation, assayed by Fab-PLA. Results are presented as representative microscopy images (A, B) and box plots after quantification (C, D). PLA signals are shown as red dots and nuclei were visualized by DAPI staining. Scale bar: 5 μm. In the box plots, the median values were highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample and p-values were calculated by Kruskal-Wallis one-way ANOVA.


E - H Calcium flux measured by FACScan for 3046SD ( E , F ) and 3046D ( G , H ) cells after stimulation with NIP15 - BSA ( 30 pM ) , 1NIP - pep ( 80 nM ) , Ac146Fab ( 25 nM ) or Ac38Fab ( 25 nM ) . Arrows indicate the addition of the stimuli to the cells .

E-H Calcium flux measured by FACScan for 3046SD (E, F) and 3046D (G, H) cells after stimulation with NIP15-BSA (30 pM), 1NIP-pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.


Calcium flux measured by FACScan for 3046M ( A , D ) , 3046SD ( B , E ) and 3046D ( C , F ) cells stimulated with anti - HC ( 2 μl / ml ) ( A , B , C ) , or anti - λ ( 2 μl / ml ) ( D , E , F ) antibodies with or without 1 min prestimulation with 1NIP - pep ( 80 nM ) . The addition of antibodies is indicated by black arrows . The 1NIP - pep was added immediately before recording . Data are representative of three independent experiments .

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Calcium flux measured by FACScan for 3046M (A, D), 3046SD (B, E) and 3046D (C, F) cells stimulated with anti-HC (2 μl/ml) (A, B, C), or anti-λ (2 μl/ml) (D, E, F) antibodies with or without 1 min prestimulation with 1NIP-pep (80 nM). The addition of antibodies is indicated by black arrows. The 1NIP-pep was added immediately before recording. Data are representative of three independent experiments.


A - E B1 - 8 B cells , 3046SM cells , 3046M cells , 3046SD cells , or 3046D cells were stimulated with 1NIP - pep ( 80 nM ) , or anti - CD19 antibody ( 62 nM ) , or costimulated with 1NIP - pep and anti - CD19 antibody . The calcium fluxes were measured by FACScan .

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A-E B1-8 B cells, 3046SM cells, 3046M cells, 3046SD cells, or 3046D cells were stimulated with 1NIP-pep (80 nM), or anti-CD19 antibody (62 nM), or costimulated with 1NIP-pep and anti-CD19 antibody. The calcium fluxes were measured by FACScan.


F - J B1 - 8 B cells , 3046SM cells , 3046M cells , 3046SD cells , or 3046D cells were stimulated with 1NIP - pep ( 80 nM ) , or anti - CD20 antibody ( 62 nM ) , or costimulated with 1NIP - pep and anti - CD20 antibody . The calcium fluxes were measured by FACScan .

O O O B-ORGANISM I-ORGANISM I-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O

F-J B1-8 B cells, 3046SM cells, 3046M cells, 3046SD cells, or 3046D cells were stimulated with 1NIP-pep (80 nM), or anti-CD20 antibody (62 nM), or costimulated with 1NIP-pep and anti-CD20 antibody. The calcium fluxes were measured by FACScan.


( D ) Validation by western blot of changes in specific phosphorylation events on kinases . Left , Immunoblots of cortical lysates of CTL and micTNFα - KO at ZT6 for : ( top ) phosphorylated GSK3α / β ( pGSK3α at Ser21 and pGSK3β at Ser9 ) and total GSK3α ; and ( bottom ) phosphorylated MAPK ( pMAPK , MAPK3 at Thr203 / Tyr205 and MAPK1 at Thr183 / Tyr185 ) and total MAPK . Right , Ratio between phosphorylated and total kinase normalized to CTL at ZT18 and ZT6 . n = 5 mice per group . * * * p = 0 . 000009 ( GSK3α ) , multiple t - test with Benjamini - Hochberg correction .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O B-TISSUE O O O O B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O

(D) Validation by western blot of changes in specific phosphorylation events on kinases. Left, Immunoblots of cortical lysates of CTL and micTNFα-KO at ZT6 for: (top) phosphorylated GSK3α/β (pGSK3α at Ser21 and pGSK3β at Ser9) and total GSK3α; and (bottom) phosphorylated MAPK (pMAPK, MAPK3 at Thr203/Tyr205 and MAPK1 at Thr183/Tyr185) and total MAPK. Right, Ratio between phosphorylated and total kinase normalized to CTL at ZT18 and ZT6. n=5 mice per group. ***p=0.000009 (GSK3α), multiple t-test with Benjamini-Hochberg correction.


( E ) Kinases with altered activity between CTL and micTNF - KOs predicted by robust kinase activity inference ( RoKAI ) at ZT18 and ZT6 . Graph shows the z - score value of each kinase assigned by RoKAI . Kinases above and below the dotted line represent prediction of up - and downregulated activity in micTNFα - KO , respectively . Kinases are color - coded according to kinase group as described in ( C ) .

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(E) Kinases with altered activity between CTL and micTNF-KOs predicted by robust kinase activity inference (RoKAI) at ZT18 and ZT6. Graph shows the z-score value of each kinase assigned by RoKAI. Kinases above and below the dotted line represent prediction of up- and downregulated activity in micTNFα-KO, respectively. Kinases are color-coded according to kinase group as described in (C).


( F Validation by western blot of altered activity of predicted kinases between CTL and micTNF - KOs . ( F ) Left , Immunoblots of cortical lysates of CTL and micTNFα - KO at ZT6 using antibodies specific to target phosphorylation motifs of MAPK / CDK ( PXS * P , S * PX ( K / R ) ) and PKC ( ( K / R ) XS * X ( K / R ) ) . Dashed boxes indicate quantified signal . Right , Ratio between phosphorylated substrates and loading control normalized to CTL at ZT18 and ZT6 . ( F , n = 5 mice per group . * p = 0 . 022 ( MAPK / CDK phospho sub ) , * p = 0 . 014 ( PKC phospho sub ) multiple t - test with Benjamini - Hochberg correction .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-CELL_TYPE O O O O O O O O B-EXP_ASSAY O B-TISSUE O O O O B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O

(F Validation by western blot of altered activity of predicted kinases between CTL and micTNF-KOs. (F) Left, Immunoblots of cortical lysates of CTL and micTNFα-KO at ZT6 using antibodies specific to target phosphorylation motifs of MAPK/CDK (PXS*P, S*PX(K/R)) and PKC ((K/R)XS*X(K/R)). Dashed boxes indicate quantified signal. Right, Ratio between phosphorylated substrates and loading control normalized to CTL at ZT18 and ZT6. (F, n=5 mice per group. *p=0.022 (MAPK/CDK phospho sub), *p=0.014 (PKC phospho sub) multiple t-test with Benjamini-Hochberg correction.


G ) Validation by western blot of altered activity of predicted kinases between CTL and micTNF - KOs . ( G ) Left , Immunoblots show signal of phosphorylated threonine in MARKs activation loop . Right , Ratio between phosphorylated MARK activation loop and loading control normalized to CTL at ZT18 and ZT6 . G ) n = 5 mice per group . * * p = 0 . 0091 ( MARK ) , multiple t - test with Benjamini - Hochberg correction .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-CELL_TYPE O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O

G) Validation by western blot of altered activity of predicted kinases between CTL and micTNF-KOs. (G) Left, Immunoblots show signal of phosphorylated threonine in MARKs activation loop. Right, Ratio between phosphorylated MARK activation loop and loading control normalized to CTL at ZT18 and ZT6. G) n=5 mice per group. **p=0.0091 (MARK), multiple t-test with Benjamini-Hochberg correction.


( A ) 3D time ‐ lapse microscopy of mitochondria undergoing fission in a dendritic arbor of a neuron . Neurons were transfected with Mito ‐ DsRed2 , pretreated with the pan ‐ caspase inhibitor zVAD ‐ fmk methyl ester ( 100 μM ) , and exposed to SNOC ( 200 μM ) . Images were 3D iso ‐ surface rendered . Frames depict representative time points of the movie demonstrating mitochondrial fragmentation within 3 h of NO exposure ( upper panels ; scale bar , 15 μm ) and closeup views ( lower panels ; scale bar , 3 μm ) . See Supplementary Video 1 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O 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) 3D time‐lapse microscopy of mitochondria undergoing fission in a dendritic arbor of a neuron. Neurons were transfected with Mito‐DsRed2, pretreated with the pan‐caspase inhibitor zVAD‐fmk methyl ester (100 μM), and exposed to SNOC (200 μM). Images were 3D iso‐surface rendered. Frames depict representative time points of the movie demonstrating mitochondrial fragmentation within 3 h of NO exposure (upper panels; scale bar, 15 μm) and closeup views (lower panels; scale bar, 3 μm). See Supplementary Video 1.


( B ) Mitochondrial fragmentation within the field of view shown in ( A ) results in increased mitochondrial numbers .

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(B) Mitochondrial fragmentation within the field of view shown in (A) results in increased mitochondrial numbers.


( C ) NO induces dose ‐ dependent mitochondrial fission . Neurons expressing Mito ‐ DsRed2 were exposed to SNOC and fixed at 60 min . The fraction of neurons displaying fragmented mitochondria is shown as the mean ± s . e . m .

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(C) NO induces dose‐dependent mitochondrial fission. Neurons expressing Mito‐DsRed2 were exposed to SNOC and fixed at 60 min. The fraction of neurons displaying fragmented mitochondria is shown as the mean±s.e.m.


( D ) Mito ‐ DsRed2 ‐ expressing corticalneurons were pretreated with 1 mM nitro ‐ L ‐ arginine ( 20 min ) and exposed to 25 μM NMDA . The fraction of neurons exhibiting fragmented mitochondria at 60 min after NMDA treatment is shown as the mean ± s . e . m . of quintuplicate samples from a representative experiment ( † † † significance at P0 . 001 or n . s . † , not significant compared to control ; * * * significance at P0 . 001 as compared to NMDA treatment ) .

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(D) Mito‐DsRed2‐expressing corticalneurons were pretreated with 1 mM nitro‐L‐arginine (20 min) and exposed to 25 μM NMDA. The fraction of neurons exhibiting fragmented mitochondria at 60 min after NMDA treatment is shown as the mean±s.e.m. of quintuplicate samples from a representative experiment (†††significance at P0.001 or n.s.†, not significant compared to control; ***significance at P0.001 as compared to NMDA treatment).


Mitochondrial fission occurs before dendritic injury and without AIF or cytochrome c release from mitochondria . Neurons were co ‐ transfected with vectors encoding ( A ) Mito ‐ DsRed2 and ( B ) MyrPalm ‐ mCFP plus ( C ) AIF ‐ GFP and the effects of SNOC were recorded using 3D time ‐ lapse imaging . Projection images zoomed in onto the dendritic arbor of one neuron ( representative of n = 42 ) . Arrows indicate dendritic spines . The inset shows the soma at appropriate grayscale levels . The apparent brightening of AIF ‐ GFP fluorescence in the nucleus at 5 h was due to nuclear and cell body shrinkage rather than nuclear translocation of AIF . Scale bars , 10 μm . See also Supplementary Video 2 .

B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-CELL_TYPE O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O O O O O O O

Mitochondrial fission occurs before dendritic injury and without AIF or cytochrome c release from mitochondria. Neurons were co‐transfected with vectors encoding (A) Mito‐DsRed2 and (B) MyrPalm‐mCFP plus (C) AIF‐GFP and the effects of SNOC were recorded using 3D time‐lapse imaging. Projection images zoomed in onto the dendritic arbor of one neuron (representative of n=42). Arrows indicate dendritic spines. The inset shows the soma at appropriate grayscale levels. The apparent brightening of AIF‐GFP fluorescence in the nucleus at 5 h was due to nuclear and cell body shrinkage rather than nuclear translocation of AIF. Scale bars, 10 μm. See also Supplementary Video 2.


( D ) 3D time ‐ lapse image reconstructions of mitochondria and cytochrome c in a dendritic arbor at indicated time points following SNOC addition . Scale bar , 10 μm . Five mitochondria were selected and motion tracked throughout the imaging series .

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(D) 3D time‐lapse image reconstructions of mitochondria and cytochrome c in a dendritic arbor at indicated time points following SNOC addition. Scale bar, 10 μm. Five mitochondria were selected and motion tracked throughout the imaging series.


( E ) Traces show the ratio of mean GFP to DsRed2 emission intensity from each mitochondrion . Each trace color corresponds to the same colored mitochondrion . ( representative of n = 9 )

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(E) Traces show the ratio of mean GFP to DsRed2 emission intensity from each mitochondrion. Each trace color corresponds to the same colored mitochondrion. (representative of n=9)


( F - G ) Immunocytochemistry for staining anti ‐ AIF antibodies ( red ) anti ‐ NeuN antibodies ( green ) and nuclei labeled with Hoechst 33342 dye ( blue ) after ( F ) aged SNOC or ( G ) SNOC ( 150 μM ; 4 h ) treatment . Volume rendered 3D reconstruction of confocal image stacks are shown above , and a single confocal plane in grayscale below . Grid , 3 . 3 μm for ( F ) and 1 . 9 μm for ( G ) , scale bars , 5 μm .

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(F-G) Immunocytochemistry for staining anti‐AIF antibodies (red) anti‐NeuN antibodies (green) and nuclei labeled with Hoechst 33342 dye (blue) after (F) aged SNOC or (G) SNOC (150 μM; 4 h) treatment. Volume rendered 3D reconstruction of confocal image stacks are shown above, and a single confocal plane in grayscale below. Grid, 3.3 μm for (F) and 1.9 μm for (G), scale bars, 5 μm.


( H ) Ratio of nuclear to somal AIF signal was measured as mean fluorescence intensity in the center of nuclei divided by the mean fluorescence intensity in the cell body , outlined by the NeuN staining ( excluding the nucleus ) . The bar diagram summarizes the mean ± s . e . m . of 282 and 262 neurons measured in n = 19 and 20 image stacks in three independent experiments for aged SNOC and SNOC , respectively ( P ∼ 0 . 99 not significant by Student ' s t ‐ test ) .

O O O O O B-SUBCELLULAR O B-SUBCELLULAR B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(H) Ratio of nuclear to somal AIF signal was measured as mean fluorescence intensity in the center of nuclei divided by the mean fluorescence intensity in the cell body, outlined by the NeuN staining (excluding the nucleus). The bar diagram summarizes the mean±s.e.m. of 282 and 262 neurons measured in n=19 and 20 image stacks in three independent experiments for aged SNOC and SNOC, respectively (P∼0.99 not significant by Student's t‐test).


( I ) DEVDase activity . Purified cortical neurons were exposed to either 200 μM SNOC or 1 μM staurosporine , as positive control . The rate of zDEVD ‐ AMC caspase substrate cleavage per minute was monitored and expressed in arbitrary fluorescence units . Data show mean ± s . e . m . for four independent experiments ( * significance at P0 . 01 by ANOVA ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-SUBCELLULAR O O O O O O O O O O O O O O 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) DEVDase activity. Purified cortical neurons were exposed to either 200 μM SNOC or 1 μM staurosporine, as positive control. The rate of zDEVD‐AMC caspase substrate cleavage per minute was monitored and expressed in arbitrary fluorescence units. Data show mean±s.e.m. for four independent experiments (*significance at P0.01 by ANOVA).


( A ) Elongated mitochondrion ( arrow ) in neuronal process of the unaffected hemisphere ( top ) . Small mitochondrial segments ( arrows ) are in close proximity to each other in a neuronal process of the ischemic brain region ( bottom ) . Images are representative of three analyzed mice . Scale bars , 500 nm .

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(A) Elongated mitochondrion (arrow) in neuronal process of the unaffected hemisphere (top). Small mitochondrial segments (arrows) are in close proximity to each other in a neuronal process of the ischemic brain region (bottom). Images are representative of three analyzed mice. Scale bars, 500 nm.


( B ) Histogram comparing mitochondrial cross ‐ section length of contralateral , control ( red ) and ischemic ( black ) hemispheres . The lower bins of the histogram ( 2000 nm ) were compared with χ2 ‐ test and were not different . The table shows the presence of long mitochondrial cross ‐ sections ( > 2000 nm ) in the control hemisphere and their absence in the ischemic hemisphere ( P0 . 01 ; Fisher ' s exact test ) .

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(B) Histogram comparing mitochondrial cross‐section length of contralateral, control (red) and ischemic (black) hemispheres. The lower bins of the histogram (2000 nm) were compared with χ2‐test and were not different. The table shows the presence of long mitochondrial cross‐sections (>2000 nm) in the control hemisphere and their absence in the ischemic hemisphere (P0.01; Fisher's exact test).


( C ) Proximities of mitochondria comparing the contralateral ( red ) and ischemic ( black ) hemispheres . Bar diagram shows percents of mitochondrial proximities found to be smaller or larger than 400 nm . The same data set is summarized as a contingency table on the right , ( P0 . 001 ; two ‐ way Fisher ' s exact test ) .

O O O O O B-SUBCELLULAR O O O O O O O O O O O B-TISSUE O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O 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) Proximities of mitochondria comparing the contralateral (red) and ischemic (black) hemispheres. Bar diagram shows percents of mitochondrial proximities found to be smaller or larger than 400 nm. The same data set is summarized as a contingency table on the right, (P0.001; two‐way Fisher's exact test).


Fission is associated with ultrastructural changes in mitochondria . EM tomography of mitochondria in cultured corticalneurons demonstrates injury associated with mitochondrial fission . Cortical cultures were exposed to aged SNOC control ( A - D ) or 200 μM SNOC ( E - L ) for 6 h in the presence of 80 μM zVAD ‐ fmk methyl ester . A slice from each tilt series is shown ( A , E and I ) , followed by orthogonal views of 3D reconstructions after segmentation of the OMM ( blue ) and IMM ( gray ) , with cristae ( various arbitrary colors ) ( B - D , F - H and J - L ) . For clarity , 10 representative cristae are shown in ( B , D , F , G , J and K ) , while all cristae are displayed in ( C , H and L ) . The control mitochondrion ( A - D ) is elongated with a dark matrix , indicating no swelling , and 34 cristae . The NO ‐ exposed mitochondrion in ( E - H ) is dividing , and the lower mitochondrion of the couplet shows severe damage , recognized by an outer membrane rupture , a regionally confined inner membrane blowout , slight matrix swelling and cristae fragmentation . Each of the daughter mitochondria is shorter and rounder than controls . Unlike the upper daughter , few of the cristae extend throughout the lower mitochondrion . Additionally , cristae are considerably smaller and regionally confined . The lower mitochondrion of the couplet contains 82 cristae , while the upper contains only 39 , indicating cristae fragmentation in the former . The NO ‐ exposed mitochondrial triplet in ( I - L ) displays further damage , including membrane degradation , accumulation of an electron dense mass , greater cristae fragmentation , and apparent mitochondrial fragmentation . All cristae are smaller than controls , with 73 in the top mitochondrion , 112 in the middle , and 47 in the bottom . Scale bars , 400 nm . See Supplementary Video 3 , 4 and 5 of tomographic reconstruction .

O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O 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-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fission is associated with ultrastructural changes in mitochondria. EM tomography of mitochondria in cultured corticalneurons demonstrates injury associated with mitochondrial fission. Cortical cultures were exposed to aged SNOC control (A-D) or 200 μM SNOC (E-L) for 6 h in the presence of 80 μM zVAD‐fmk methyl ester. A slice from each tilt series is shown (A, E and I), followed by orthogonal views of 3D reconstructions after segmentation of the OMM (blue) and IMM (gray), with cristae (various arbitrary colors) (B-D, F-H and J-L). For clarity, 10 representative cristae are shown in (B, D, F, G, J and K), while all cristae are displayed in (C, H and L). The control mitochondrion (A-D) is elongated with a dark matrix, indicating no swelling, and 34 cristae. The NO‐exposed mitochondrion in (E-H) is dividing, and the lower mitochondrion of the couplet shows severe damage, recognized by an outer membrane rupture, a regionally confined inner membrane blowout, slight matrix swelling and cristae fragmentation. Each of the daughter mitochondria is shorter and rounder than controls. Unlike the upper daughter, few of the cristae extend throughout the lower mitochondrion. Additionally, cristae are considerably smaller and regionally confined. The lower mitochondrion of the couplet contains 82 cristae, while the upper contains only 39, indicating cristae fragmentation in the former. The NO‐exposed mitochondrial triplet in (I-L) displays further damage, including membrane degradation, accumulation of an electron dense mass, greater cristae fragmentation, and apparent mitochondrial fragmentation. All cristae are smaller than controls, with 73 in the top mitochondrion, 112 in the middle, and 47 in the bottom. Scale bars, 400 nm. See Supplementary Video 3, 4 and 5 of tomographic reconstruction.


( A ) Projection image of two neurons expressing Mito ‐ DsRed2 ( red ) and MyrPalm ‐ CFP ( blue ) . Images were high ‐ pass filtered and z ‐ stacks were projected over maximum intensity .

O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

(A) Projection image of two neurons expressing Mito‐DsRed2 (red) and MyrPalm‐CFP (blue). Images were high‐pass filtered and z‐stacks were projected over maximum intensity.


( B ) Changes in mitochondrial dynamics were evaluated as mean skeletal length of mitochondria or ( C ) as mitochondrial numbers .

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(B) Changes in mitochondrial dynamics were evaluated as mean skeletal length of mitochondria or (C) as mitochondrial numbers.


( D ) Magnified representative frames of the time ‐ lapse recording of the area within the white rectangle of ( A ) indicate mitochondrial morphology at time 0 , 20 min and 1 h . These time points are depicted in ( B ) and ( C ) by red arrows . Data are representative of 26 neurons , which exhibited reversible mitochondrial fission from a total of 55 analyzed neurons ( six experiments ) . Scale bar , 10 μm . See Supplementary Video 6 .

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-SUBCELLULAR O O O O O O O O O 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-CELL_TYPE O O O O B-SUBCELLULAR O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O

(D) Magnified representative frames of the time‐lapse recording of the area within the white rectangle of (A) indicate mitochondrial morphology at time 0, 20 min and 1 h. These time points are depicted in (B) and (C) by red arrows. Data are representative of 26 neurons, which exhibited reversible mitochondrial fission from a total of 55 analyzed neurons (six experiments). Scale bar, 10 μm. See Supplementary Video 6.


( E ) Autophagy in neurites at 6 h after SNOC exposure . Arrows indicate autophagosomes engulfing injured mitochondria next to an intact mitochondrion in the left image . The arrowhead in the right panel indicates membrane wrappers characteristic of autophagosomes . White arrowheads in the insets show the double membranes of the autophagosome and those of mitochondria within the autophagosome . Scale bars , 500 nm .

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(E) Autophagy in neurites at 6 h after SNOC exposure. Arrows indicate autophagosomes engulfing injured mitochondria next to an intact mitochondrion in the left image. The arrowhead in the right panel indicates membrane wrappers characteristic of autophagosomes. White arrowheads in the insets show the double membranes of the autophagosome and those of mitochondria within the autophagosome. Scale bars, 500 nm.


( A ) Mitochondrial fission is linked to a drop in ATP . Purified corticalneurons were exposed to increasing SNOC concentrations or mitochondrial inhibitors ( 2 μM rotenone plus 2 μg / ml oligomycin ; Rot / Olig ) . ATP concentrations are shown as the mean ± s . e . m . normalized to the plating density of neurons ( n = 6 )

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(A) Mitochondrial fission is linked to a drop in ATP. Purified corticalneurons were exposed to increasing SNOC concentrations or mitochondrial inhibitors (2 μM rotenone plus 2 μg/ml oligomycin; Rot/Olig). ATP concentrations are shown as the mean±s.e.m. normalized to the plating density of neurons (n=6)


( B ) [ ATP ] / [ ADP ] ratios in purified corticalneurons exposed to increasing SNOC concentrations ( n = 4 ) .

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(B) [ATP]/[ADP] ratios in purified corticalneurons exposed to increasing SNOC concentrations (n=4).


( C ) The bar graph depicts ethidine fluorescence density in arbitrary units of aged and fresh SNOC treated neurons at 2 . 5 h .

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(C) The bar graph depicts ethidine fluorescence density in arbitrary units of aged and fresh SNOC treated neurons at 2.5 h.


( D ) Cortical neurons expressing Mito ‐ GFP ( green ) were exposed to aged or fresh 200 μM SNOC solution . After 2 . 5 h cultures were loaded with hydroethidine . Images are representative of more that 20 neurons analyzed ( in D ) for each condition from at least three independent experiments . Scale bar , 10 μm .

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(D) Cortical neurons expressing Mito‐GFP (green) were exposed to aged or fresh 200 μM SNOC solution. After 2.5 h cultures were loaded with hydroethidine. Images are representative of more that 20 neurons analyzed (in D) for each condition from at least three independent experiments. Scale bar, 10 μm.


( E ) Reduced glutathione ( GSH ) partially blocks mitochondrial fission . Neurons were transfected with Mito pretreated for 2 h with 2 mM GSH monoethyl ester , washed once and then exposed to 35 μM SNOC . Mitochondrial fission was scored after 1 h . Data are means ± s . e . m . of quintuplicate samples from one representative experiment of a total of three ( † † † significance at P0 . 001 or n . s . , † not significant compared to control ; * significance at P0 . 05 ) .

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(E) Reduced glutathione (GSH) partially blocks mitochondrial fission. Neurons were transfected with Mito pretreated for 2 h with 2 mM GSH monoethyl ester, washed once and then exposed to 35 μM SNOC. Mitochondrial fission was scored after 1 h. Data are means±s.e.m. of quintuplicate samples from one representative experiment of a total of three (†††significance at P0.001 or n.s., †not significant compared to control; *significance at P0.05).


( A ) Representative fluorescence micrographs of mitochondrial morphology before and after SNOC ( 175 μM ; 7 h ) or rotenone ( 30 nM ; 2 h ) treatments and a comparison of the effects of Aβ25 - 35 versus Aβ35 - 25 ( 10 μM , 6 h ) , as indicated . Scale bar , 20 μm .

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(A) Representative fluorescence micrographs of mitochondrial morphology before and after SNOC (175 μM; 7 h) or rotenone (30 nM; 2 h) treatments and a comparison of the effects of Aβ25-35 versus Aβ35-25(10 μM, 6 h), as indicated. Scale bar, 20 μm.


( B ) Percentage of mitochondrial fission and cell death at 18 h ( † and † † † significance at P0 . 05 and 0 . 001 as compared to control pcDNA3 transfection ; * , * * and * * * significance at P0 . 05 , 0 . 01 and 0 . 001 , respectively , compared to SNOC treated , pcDNA3 transfected neurons ; n = 3 independent experiments ) . Dying neurons were recognized by their shrunken and condensed nuclei after Hoechst 33342 staining .

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(B) Percentage of mitochondrial fission and cell death at 18 h († and ††† significance at P0.05 and 0.001 as compared to control pcDNA3 transfection; *,** and *** significance at P0.05 , 0.01 and 0.001, respectively, compared to SNOC treated, pcDNA3 transfected neurons; n=3 independent experiments). Dying neurons were recognized by their shrunken and condensed nuclei after Hoechst 33342 staining.


( C ) Enforced Drp1 or Fis1 expression ( 60 h ) evokes fission and neuronal cell death ( * , * * and * * * significance at P0 . 05 , 0 . 01 and 0 . 001 , respectively , compared to pcDNA3 transfected neurons ; n = 3 independent experiments ) .

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(C) Enforced Drp1 or Fis1 expression (60 h) evokes fission and neuronal cell death (*,** and *** significance at P0.05, 0.01 and 0.001, respectively, compared to pcDNA3 transfected neurons; n=3 independent experiments).


( D ) Rotenone induces dose ‐ dependent mitochondrial fission . The fraction of neurons displaying fissioned mitochondria is shown as the mean ± s . e . m . ( * significance at P0 . 05 , n = 4 ) .

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(D) Rotenone induces dose‐dependent mitochondrial fission. The fraction of neurons displaying fissioned mitochondria is shown as the mean ±s.e.m. (*significance at P0.05, n=4).


( E ) Mfn1 or Drp1K38A inhibits 100 nM rotenone ‐ induced mitochondrial fission ( 4 h ) and cell death ( 48 h ) . Data indicate means ± s . e . m . of triplicate measurements ( † † and † † † significance at P0 . 01 and 0 . 001 as compared to untreated control ; * * * significance at P0 . 001 as compared to rotenone treatment with pcDNA3 transfection ; n = 3 ) .

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(E) Mfn1 or Drp1K38A inhibits 100 nM rotenone‐induced mitochondrial fission (4 h) and cell death (48 h). Data indicate means±s.e.m. of triplicate measurements (†† and ††† significance at P0.01 and 0.001 as compared to untreated control; ***significance at P0.001 as compared to rotenone treatment with pcDNA3 transfection; n=3).


( F ) Mitochondrial fission by Aβ35 - 25 or Aβ25 - 35 exposure . The fraction of neurons exhibiting fragmented mitochondria is shown as the mean ± s . e . m . ( † † significance at P0 . 01 compared to pcDNA3 transfected , Aβ35 − 25 treated control ; * and * * significance at P0 . 05 and 0 . 001 , respectively , compared to pcDNA3 transfected , Aβ25 - 35 treated neurons ; n = 3 ) .

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(F) Mitochondrial fission by Aβ35-25 or Aβ25-35 exposure. The fraction of neurons exhibiting fragmented mitochondria is shown as the mean±s.e.m. (††significance at P0.01 compared to pcDNA3 transfected, Aβ35−25 treated control; * and ** significance at P0.05 and 0.001, respectively, compared to pcDNA3 transfected, Aβ25-35 treated neurons; n=3).


Therapeutic leads generated using PDN were directly tested for survival benefit using a murine model of endotoxemia . Select compounds were injected 24 hours before and on the day of LPS administration , using routes and doses specified in the methods . C57bl / 6 female mice were injected with a high - lethality dose of E . coli LPS ( 38 - 40 μg / g ) followed by a subcutaneous injection of sterile saline . Significant differences in concentration between drug and vehicle - treated pre - and post - pubertal mice are labeled with * * * * ( p < 0 . 0001 ) , * * * ( p < 0 . 001 , * * ( p < 0 . 01 ) , or * ( p < 0 . 05 ) . Percent survival was compared using a log rank Mantel Cox test

O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O O B-DISEASE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-SMALL_MOLECULE O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O

Therapeutic leads generated using PDN were directly tested for survival benefit using a murine model of endotoxemia. Select compounds were injected 24 hours before and on the day of LPS administration, using routes and doses specified in the methods. C57bl/6 female mice were injected with a high-lethality dose of E. coli LPS (38-40 μg/g) followed by a subcutaneous injection of sterile saline. Significant differences in concentration between drug and vehicle-treated pre- and post-pubertal mice are labeled with **** (p<0.0001), *** (p<0.001, ** (p<0.01), or * (p<0.05). Percent survival was compared using a log rank Mantel Cox test


A Representative images of alkaline phosphatase ( ALP ) , Alizarin Red S ( ARS ) and Van Gieson ' s staining of human

A Representative images of alkaline phosphatase (ALP), Alizarin Red S (ARS) and Van Gieson's staining of human MSCs


B , C Quantitative analyses of the ALP activity and calcium mineralization . Results are shown as mean ± SEM ; n = 6 ; * * : p < 0 . 01 and * * * : p < 0 . 001 by t test

O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O 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 Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=6; **: p<0.01 and ***: p<0.001 by t test