Figure 2Top - Representative RAB7 expression in base of stomach units ( ZCs ) in vehicle or 24h HDT by immunohistochemistry . Arrowheads point to RAB7 vesicle aggregates . Scale bar 20µm . Bottom - Representative ATF3 expression in base of stomach units ( ZCs ) by immunohistochemistry . ATF3 appears in nuclei following 24h high - dose tamoxifen ( HDT ) . Scale bar 20µm . Stomach unit base outlined by dashed black line . Western blot of ATF3 expression over HDT and CER timecourses with β - Actin loading control . Top - Representative RAB7 staining in pancreatic acinar cells . Arrowheads indicate RAB7 vesicles . Scale bar 20µm . Bottom - Representative ATF3 expression in pancreatic acinar cells . Nuclei express ATF3 after 24h cerulein ( CER ) injury . Scale bar 20µm . ChIP - PCR on mouse stomach tissue at vehicle and 6 hour HDT . Chromatin probed with ATF3 , Histone H3 antibodies , or a Rabbit IgG control . Also included is the 2 % chromatin input and water to control for DNA contamination . Rab7b amplicon is from a conserved , putative ATF3 - binding site in the first intron . Amplicon from Atf3 is a positive control from a previously characterized ATF3 - binding site ; negative control is from a site in Rab9a lacking putative ATF3 binding motifs .

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Figure 2Top - Representative RAB7 expression in base of stomach units (ZCs) in vehicle or 24h HDT by immunohistochemistry. Arrowheads point to RAB7 vesicle aggregates. Scale bar 20µm. Bottom - Representative ATF3 expression in base of stomach units (ZCs) by immunohistochemistry. ATF3 appears in nuclei following 24h high-dose tamoxifen (HDT). Scale bar 20µm. Stomach unit base outlined by dashed black line.Western blot of ATF3 expression over HDT and CER timecourses with β-Actin loading control.Top - Representative RAB7 staining in pancreatic acinar cells. Arrowheads indicate RAB7 vesicles. Scale bar 20µm. Bottom - Representative ATF3 expression in pancreatic acinar cells. Nuclei express ATF3 after 24h cerulein (CER) injury. Scale bar 20µm.ChIP-PCR on mouse stomach tissue at vehicle and 6 hour HDT. Chromatin probed with ATF3, Histone H3 antibodies, or a Rabbit IgG control. Also included is the 2% chromatin input and water to control for DNA contamination. Rab7b amplicon is from a conserved, putative ATF3-binding site in the first intron. Amplicon from Atf3 is a positive control from a previously characterized ATF3-binding site; negative control is from a site in Rab9a lacking putative ATF3 binding motifs.


Figure 3mRNA expression of ATF3 by qRT - PCR in control , ATF3 knockdown ( KD ) , and ATF3 overexpression ( OE ) AGS cell lines generated using a PiggybacTM transposon system . Significance by one - way ANOVA , Dunnett post - hoc . Datapoint = mean of technical triplicates in a single mouse . n = 3 mice . Error bars denote standard deviation . mRNA expression of RAB7B by qRT - PCR in AGS cell lines from 3A . Statistics and plotting as for panel A . Top - Immunofluorescence for RAB7 ( red ) and DAPI ( blue , nuclei ) in control , ATF3 knockdown ( KD ) , and ATF3 overexpression ( OE ) AGS cells . Arrowhead indicates aggregated RAB7 vesicular formation similar to those observed in tissue . Bottom - Immunofluorescence for ATF3 ( purple ) and Cathepsin D ( CTSD , green , lysosomes ) in AGS cells . Scale bar , 20µm .

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Figure 3mRNA expression of ATF3 by qRT-PCR in control, ATF3 knockdown (KD), and ATF3 overexpression (OE) AGS cell lines generated using a PiggybacTM transposon system. Significance by one-way ANOVA, Dunnett post-hoc. Datapoint = mean of technical triplicates in a single mouse. n=3 mice. Error bars denote standard deviation. mRNA expression of RAB7B by qRT-PCR in AGS cell lines from 3A. Statistics and plotting as for panel A.Top - Immunofluorescence for RAB7 (red) and DAPI (blue, nuclei) in control, ATF3 knockdown (KD), and ATF3 overexpression (OE) AGS cells. Arrowhead indicates aggregated RAB7 vesicular formation similar to those observed in tissue. Bottom - Immunofluorescence for ATF3 (purple) and Cathepsin D (CTSD, green, lysosomes) in AGS cells. Scale bar, 20µm.


Figure 4Representative immunofluorescence images of gastric chief cells after vehicle or 24h HDT treatment stained for Pepsinogen C ( PGC , red , granules ) and MAP1LC3B ( green , autophagy marker ) . Top row is wild - type , bottom row is Atf3 − / − . Scale bar , 50μm . Stomach unit base outlined by dashed white line . Transmission electron micrograph images of zymogenic chief cells ( ZCs ) after 24h vehicle or HDT treatment in WT ( top ) and Atf3 − / − mice ( bottom ) . Scale bar , 1μm . Arrowheads indicate cell components being degraded . Quantification of degradation events per cell counted from EM images of ZCs after 24h HDT treatment in WT and Atf3 − / − mice as shown in panel 3D . Each data point = total granules in a cell in a single TEM section with cells from 2 separate mice plotted together ( data point colors indicate cells from a single mouse ) . Red line indicates mean across all the mice .

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Figure 4Representative immunofluorescence images of gastric chief cells after vehicle or 24h HDT treatment stained for Pepsinogen C (PGC, red, granules) and MAP1LC3B (green, autophagy marker). Top row is wild-type, bottom row is Atf3−/−. Scale bar, 50μm. Stomach unit base outlined by dashed white line.Transmission electron micrograph images of zymogenic chief cells (ZCs) after 24h vehicle or HDT treatment in WT (top) and Atf3−/− mice (bottom). Scale bar, 1μm. Arrowheads indicate cell components being degraded. Quantification of degradation events per cell counted from EM images of ZCs after 24h HDT treatment in WT and Atf3−/−mice as shown in panel 3D. Each data point=total granules in a cell in a single TEM section with cells from 2 separate mice plotted together (data point colors indicate cells from a single mouse). Red line indicates mean across all the mice.


Figure 5Representative immunofluorescence images of gastric units after vehicle or 72h high - dose tamoxifen ( HDT ) treatment stained for GIF ( red , granules ) , GSII ( green , neck cells and SPEM marker ) , BRDU ( white , proliferation ) and DAPI ( blue , nuclei ) . Top row is wild - type , bottom row is Atf3 − / − . Yellow arrowheads point to areas of base dropout where ZCs have been lost . Scale bar , 50μm . Stomach units outlined by dashed white line . Representative immunohistochemistry of Cleaved - Caspase 3 in pancreatic acinar cells marking apoptosis in vehicle or 24h cerulein ( CER ) treatment . Top row is WT , bottom row is Atf3 − / − . Scale bar , 50μm . Apoptosis quantified following 24h CER injury by the number of Cleaved - Caspase 3 + cells from a 40x field . Each data point is mean of positive cells in 4 - 5 40x fields in an individual mouse , n = 3 mice per treatment group . Significance by unpaired two - tailed t - test . Error bars denote standard deviation . Representative immunohistochemistry of BrdU in pancreatic acinar cells marking proliferation in vehicle or 7 days of repeated cerulein injury . Top row is WT , bottom row is Atf3 − / − . Scale bar , 50μm . Quantification of proliferation at peak ADM as counted by the number of BrdU + cells from a 20x field following 7D cerulein treatment . Plotting and statistical analysis as per panel F .

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Figure 5Representative immunofluorescence images of gastric units after vehicle or 72h high-dose tamoxifen (HDT) treatment stained for GIF (red, granules), GSII (green, neck cells and SPEM marker), BRDU (white, proliferation) and DAPI (blue, nuclei). Top row is wild-type, bottom row is Atf3−/−. Yellow arrowheads point to areas of base dropout where ZCs have been lost. Scale bar, 50μm. Stomach units outlined by dashed white line.Representative immunohistochemistry of Cleaved-Caspase 3 in pancreatic acinar cells marking apoptosis in vehicle or 24h cerulein (CER) treatment. Top row is WT, bottom row is Atf3−/−. Scale bar, 50μm. Apoptosis quantified following 24h CER injury by the number of Cleaved-Caspase 3+ cells from a 40x field. Each data point is mean of positive cells in 4-5 40x fields in an individual mouse, n=3 mice per treatment group. Significance by unpaired two-tailed t-test. Error bars denote standard deviation.Representative immunohistochemistry of BrdU in pancreatic acinar cells marking proliferation in vehicle or 7 days of repeated cerulein injury. Top row is WT, bottom row is Atf3−/−. Scale bar, 50μm. Quantification of proliferation at peak ADM as counted by the number of BrdU+ cells from a 20x field following 7D cerulein treatment. Plotting and statistical analysis as per panel F.


Figure 6Serial sections of human tissue . Representative immunohistochemistry of GSII ( top ; spasmolytic polypeptide - expressing metaplasia [ SPEM ] marker and neck cells ) counterstained with hematoxylin ( blue ) and eosin ( pink ) . Representative staining of ATF3 ( bottom ) counterstained with eosin ( pink ) . Tissue was taken from regions adjacent to gastric adenocarcinoma . Scale bars left - 50μm right - 20μm . Arrowheads indicate strongly expressing ATF3 cells and the corresponding region in the same gland labeled with GSII in a serial tissue section . GSII at the base of gastric glands in the body of the stomach indicate transition to SPEM .

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Figure 6Serial sections of human tissue. Representative immunohistochemistry of GSII (top; spasmolytic polypeptide-expressing metaplasia [SPEM] marker and neck cells) counterstained with hematoxylin (blue) and eosin (pink). Representative staining of ATF3 (bottom) counterstained with eosin (pink). Tissue was taken from regions adjacent to gastric adenocarcinoma. Scale bars left - 50μm right - 20μm. Arrowheads indicate strongly expressing ATF3 cells and the corresponding region in the same gland labeled with GSII in a serial tissue section. GSII at the base of gastric glands in the body of the stomach indicate transition to SPEM.


f1 ( a ) Structure of the GBA1 gene on chromosome 1 and schematic overview of gene correction using ZFNs . Black boxes represent exons . ( b ) Mutated and gene - corrected ( GC ) iPSCs after sequencing ( upper lanes : iPSCs with heterozygous mutations ; lower lanes : iPSCs with homozygous wt - GBA1 sequences as indicated by arrows ) .

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f1(a) Structure of the GBA1 gene on chromosome 1 and schematic overview of gene correction using ZFNs. Black boxes represent exons. (b) Mutated and gene-corrected (GC) iPSCs after sequencing (upper lanes: iPSCs with heterozygous mutations; lower lanes: iPSCs with homozygous wt-GBA1 sequences as indicated by arrows).


f2Immunostaining of indicated differentiated iPSC cultures at DIV65 . Cells were stained for TH ( red ) and β - TubIII , FOXA2 , NURR1 , GIRK2 and VMAT2 ( all green ) . Cell nuclei were counterstained with Hoechst ( blue ) . ( Bars , 20 μm ) .

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f2Immunostaining of indicated differentiated iPSC cultures at DIV65. Cells were stained for TH (red) and β-TubIII, FOXA2, NURR1, GIRK2 and VMAT2 (all green). Cell nuclei were counterstained with Hoechst (blue). (Bars, 20 μm).


f3a ) Gating strategy used for neuronal cell sorting . Live cells as determined by FSC / SSC gating were selected for CD184 - APC - negative / low surface expression and subsequently separated for CD24 - PE versus CD15 / CD29 / CD44 expression , detected in the FITC channel as indicated in the dot plots . Post - sort reanalysis shows representative samples of both fractions immediately after FACS enrichment , which were then plated for comparative in vitro analysis ( phase contrast images . Bars , 50 μm ) . ( b , c ) Immunocytochemistry at DIV2 post - sort for TH ( green ) and the neuronal marker MAP2 ( red ) performed on the neuronal ( b ) and mixed ( c ) populations of a representative control and GBA - PD line . Nuclei were counterstained with Hoechst ( blue ) . FACS enriches for both TH and neuronal marker positivity in the neuronal population compared with the non - neuronal population . ( Bars , 50 μm . )

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f3a) Gating strategy used for neuronal cell sorting. Live cells as determined by FSC/SSC gating were selected for CD184-APC-negative/low surface expression and subsequently separated for CD24-PE versus CD15/CD29/CD44 expression, detected in the FITC channel as indicated in the dot plots. Post-sort reanalysis shows representative samples of both fractions immediately after FACS enrichment, which were then plated for comparative in vitro analysis (phase contrast images. Bars, 50 μm).(b,c) Immunocytochemistry at DIV2 post-sort for TH (green) and the neuronal marker MAP2 (red) performed on the neuronal (b) and mixed (c) populations of a representative control and GBA-PD line. Nuclei were counterstained with Hoechst (blue). FACS enriches for both TH and neuronal marker positivity in the neuronal population compared with the non-neuronal population. (Bars, 50 μm.)


f4 ( a ) Ganglioside composition of human fibroblasts , iPSCs , differentiated iPSCs and enriched iPSC - derived neurons ( after FACS ) from control ( line C1 - 1 ) , GBA - PD ( line PD1 - 1 ) and GD ( line GD1 - 1 ) subjects . Cell lipids were metabolically labelled with [ 1 - 3H ] sphingosine and visualized by digital autoradiography . ( b ) Activities of glycohydrolases ( GCase , GBA2 , β - galactosidase , β - hexosaminidase ) in control iPSCs and iPSC - derived enriched neurons at DIV65 . Enzyme activities are expressed as pmoles mg − 1 h − 1 . Data are represented as mean + s . d . ; experiments were independently repeated three times in triplicate . * P0 . 01 ; Student ' s t - test . c ) GBA1 , GBA2 , β - galactosidase and HEXA and HEXB gene expression levels in control iPSCs and iPSC - derived enriched neurons at DIV65 . Data are represented as mean + s . d . ; experiments were independently repeated three times in triplicate . * P0 . 05 ; * * P0 . 01 , Student ' s t - test . ( d ) Representative western blots showing GCase , GBA2 , β - galactosidase and HEXA and HEXB protein levels in control iPSCs and corresponding iPSC - derived neurons ( DIV65 ) .

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f4(a) Ganglioside composition of human fibroblasts, iPSCs, differentiated iPSCs and enriched iPSC-derived neurons (after FACS) from control (line C1-1), GBA-PD (line PD1-1) and GD (line GD1-1) subjects. Cell lipids were metabolically labelled with [1-3H]sphingosine and visualized by digital autoradiography.(b) Activities of glycohydrolases (GCase, GBA2, β-galactosidase, β-hexosaminidase) in control iPSCs and iPSC-derived enriched neurons at DIV65. Enzyme activities are expressed as pmoles mg−1 h−1. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.01; Student's t-test.c) GBA1, GBA2, β-galactosidase and HEXA and HEXB gene expression levels in control iPSCs and iPSC-derived enriched neurons at DIV65. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.05; **P0.01, Student's t-test.(d) Representative western blots showing GCase, GBA2, β-galactosidase and HEXA and HEXB protein levels in control iPSCs and corresponding iPSC-derived neurons (DIV65).


f5 ( a ) LC - MS / MS analysis showing the level of Glc - Cer in control , GBA - PD , isogenic controls and GD iPSC - derived neurons . Data are represented as mean + s . d . ; n = 3 , * P0 . 01 , One - way ANOVA . ( b ) Control iPSCs ( line C1 - 1 ) were differentiated for 65 days and exposed to the GCase inhibitor CBE ( 25 μM ) for 48 h . Representative western blot showing increased levels of α - syn in CBE - treated iPSC - derived neurons compared to untreated neurons . ( c , d ) Representative western blots showing the protein levels of α - syn in control , GBA - PD , gene - corrected isogenic controls and GD iPSC - derived neurons . ( c , d ) Representative western blots showing the protein levels of α - syn in control , GBA - PD , gene - corrected isogenic controls and GD iPSC - derived neurons . ( e ) Optical densities of α - syn bands normalized by the averaged value of GAPDH and expressed as a percentage of the control ( CTRL ) line value . Data are represented as mean + s . d . ; experiments were independently repeated five times . * P0 . 01 , one - way ANOVA . ( f ) Average α - syn protein levels normalized by the averaged value of GAPDH and expressed as a percentage of the respective isogenic mutant lines . Data are represented as mean + s . d . ; experiments were independently repeated five times . * P0 . 01 , Student ' s t - test .

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f5(a) LC-MS/MS analysis showing the level of Glc-Cer in control, GBA-PD, isogenic controls and GD iPSC-derived neurons. Data are represented as mean+s.d.; n=3, *P0.01, One-way ANOVA.(b) Control iPSCs (line C1-1) were differentiated for 65 days and exposed to the GCase inhibitor CBE (25 μM) for 48 h. Representative western blot showing increased levels of α-syn in CBE-treated iPSC-derived neurons compared to untreated neurons.(c,d) Representative western blots showing the protein levels of α-syn in control, GBA-PD, gene-corrected isogenic controls and GD iPSC-derived neurons.(c,d) Representative western blots showing the protein levels of α-syn in control, GBA-PD, gene-corrected isogenic controls and GD iPSC-derived neurons.(e) Optical densities of α-syn bands normalized by the averaged value of GAPDH and expressed as a percentage of the control (CTRL) line value. Data are represented as mean+s.d.; experiments were independently repeated five times. *P0.01, one-way ANOVA. (f) Average α-syn protein levels normalized by the averaged value of GAPDH and expressed as a percentage of the respective isogenic mutant lines. Data are represented as mean+s.d.; experiments were independently repeated five times. *P0.01, Student's t-test.


f6 ( a ) Glycohydrolase enzyme activities ( GCase , GBA2 , β - galactosidase , β - hexosaminidase ) were measured in enriched neurons from control , GBA - PD , isogenic gene - corrected control and GD iPSC - derived neurons at DIV65 . Enzyme activities are expressed as pmoles mg − 1 h − 1 . Data are represented as mean + s . d . ; experiments were independently repeated three times in triplicate . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 001 compared with controls , one - way ANOVA . ( b , c ) Representative western blots of GCase protein levels in control , GBA - PD , corresponding gene - corrected and GD iPSC - derived neurons . Data are represented as mean + s . d . ; experiments were independently repeated three times . * P0 . 05 ; * * P0 . 01 , one - way ANOVA and Student ' s t - test .

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f6(a) Glycohydrolase enzyme activities (GCase, GBA2, β-galactosidase, β-hexosaminidase) were measured in enriched neurons from control, GBA-PD, isogenic gene-corrected control and GD iPSC-derived neurons at DIV65. Enzyme activities are expressed as pmoles mg−1 h−1. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.05; **P0.01; ***P0.001 compared with controls, one-way ANOVA.(b,c) Representative western blots of GCase protein levels in control, GBA-PD, corresponding gene-corrected and GD iPSC-derived neurons. Data are represented as mean+s.d.; experiments were independently repeated three times. *P0.05; **P0.01, one-way ANOVA and Student's t-test.


f7CSF glycohydrolase activities were measured in patients with idiopathic PD ( iPD ) , GBA - PD patients and age - matched unaffected individuals ( CTRL ) . Enzyme activities were normalized to total protein concentrations and expressed as pmoles mg − 1 h − 1 . Data are represented as mean + s . e . m . ; CTRL , n = 14 ; iPD , n = 15 ; GBA - PD , n = 15 . Experiments were performed in triplicate . * P0 . 05 ; * * P0 . 01 , Mann - Whitney U test .

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f7CSF glycohydrolase activities were measured in patients with idiopathic PD (iPD), GBA-PD patients and age-matched unaffected individuals (CTRL). Enzyme activities were normalized to total protein concentrations and expressed as pmoles mg−1 h−1. Data are represented as mean+s.e.m.; CTRL, n=14; iPD, n=15; GBA-PD, n=15. Experiments were performed in triplicate. *P0.05; **P0.01, Mann-Whitney U test.


f8 ( a ) Immunostaining of differentiated iPSC cultures at DIV65 under basal conditions . Cells were stained for TH ( green ) , β - TubIII ( magenta ) and LAMP1 ( red ) . Cell nuclei were counterstained with Hoechst ( blue ) . High magnification insets are shown in the right lane . ( Bars , 20 μm and 10 μm in the insets . ) ( b ) Average size and number of LAMP1 + particles in TH + neurons were quantified by ImageJ . Data are represented as mean + s . e . m . ; experiments were independently repeated three times in triplicate . * P0 . 01 , * * P0 . 001 , one - way ANOVA . ( c ) Western blot analysis for LC3 in iPSC - derived neuronal cultures at DIV65 , untreated ( − ) or treated with 200 μM leupeptin and 20 mM NH4Cl for 4 h ( + ) . ( d ) Quantification of the basal levels of LC3 - II relative to β - actin . Data are represented as mean + s . e . m . ; experiments were independently repeated three times . * P0 . 01 ; * * P0 . 001 , one - way ANOVA . ( e ) Quantification of LC3 flux normalized to actin . Data are represented as mean + s . e . m . ; experiments were independently repeated three times in triplicate . * P0 . 01 ; * * P0 . 001 , one - way ANOVA .

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f8(a) Immunostaining of differentiated iPSC cultures at DIV65 under basal conditions. Cells were stained for TH (green), β-TubIII (magenta) and LAMP1 (red). Cell nuclei were counterstained with Hoechst (blue). High magnification insets are shown in the right lane. (Bars, 20 μm and 10 μm in the insets.)(b) Average size and number of LAMP1+ particles in TH+ neurons were quantified by ImageJ. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01, **P0.001, one-way ANOVA.(c) Western blot analysis for LC3 in iPSC-derived neuronal cultures at DIV65, untreated (−) or treated with 200 μM leupeptin and 20 mM NH4Cl for 4 h (+).(d) Quantification of the basal levels of LC3-II relative to β-actin. Data are represented as mean+s.e.m.; experiments were independently repeated three times. *P0.01; **P0.001, one-way ANOVA.(e) Quantification of LC3 flux normalized to actin. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01; **P0.001, one-way ANOVA.


f9 ( a ) Cytosolic calcium levels were evaluated in iPSC - derived neurons at basal conditions by measuring the 340 / 380 nm ratio of Fura - 2 acetoxymethyl ester . Data are represented as mean + s . e . m . ; experiments were independently repeated three times in triplicate . * P0 . 01 ; Student ' s t - test . ( Number of cells analysed : CTRL , n = 58 ; RecNcil / wt , n = 36 ; RecNcil / wt GC , n = 107 ; L444P / wt , n = 82 ; L444P / wt GC , n = 33 ; L444P / L444P , n = 62 ) . ( b ) Caffeine ( 10 mM ) was applied to iPSC - derived neurons , and the peak cytosolic calcium concentration was measured as a change in Fura - 2 fluorescence . Data are represented as mean + s . e . m . ; experiments were independently repeated three times in triplicate . * P0 . 01 ; * * P0 . 001 , Student ' s t - test . ( Number of cells analysed : CTRL , n = 58 ; RecNcil / wt , n = 36 ; RecNcil / wt GC , n = 107 ; L444P / wt , n = 82 ; L444P / wt GC , n = 33 ; L444P / L444P , n = 62 ) . ( c ) iPSC - derived neurons were treated with A23187 ( 0 - 5 μM ) for 4 h and cytotoxicity was evaluated by released LDH activity . LDH release values were calculated as the percentage of untreated cells lysed by incubation with Triton X - 100 . Data are represented as mean + s . e . m . ; experiments were independently repeated four times in triplicate . * P0 . 05 , one - way ANOVA , control lines versus mutated lines ; δP0 . 05 , one - way ANOVA , mutated lines versus isogenic gene corrected controls . ( d ) Quantification of TH + cells treated with A23187 ( 1 μM ) for 4 h expressed as a percentage of DMSO - treated controls . Data are represented as mean + s . d . ; experiments were independently repeated three times in triplicate . * P0 . 01 , one - way ANOVA . ( e ) Knockdown efficiency of NECAB2 determined by qRT - PCR and normalized to scrambled non - targeting shRNA . Data are represented as mean + s . d . ; experiments were independently repeated three times in triplicate . * * * P0 . 0001 , Student ' s t - test . ( f ) Representative western blot for NECAB2 showing knockdown efficiency . 1 , scrambled non - targeting shRNA ; 2 , NECAB2 shRNA . ( g ) Quantification of TH + cells in GBA - PD and GD iPSC - derived neuronal cultures infected with scrambled non - targeting shRNA or NECAB2 shRNA lentivirus and treated with A23187 ( 1 μM for 4 h ) . Values were calculated as a percentage of scrambled shRNA - treated cells . Data are represented as mean + s . e . m . ; experiments were independently repeated three times in triplicate . * P0 . 01 , Student ' s t - test . ( h ) Representative images showing GBA - PD iPSC - derived DA neurons infected with scrambled non - targeting shRNA or NECAB2 shRNA lentivirus and treated with A23187 ( 1 μM for 4 h ) . Cells were stained for β - TubIII ( green ) and TH ( red ) . Nuclei were counterstained with Hoechst ( blue ) . ( Bar , 20 μm . )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f9(a) Cytosolic calcium levels were evaluated in iPSC-derived neurons at basal conditions by measuring the 340/380 nm ratio of Fura-2 acetoxymethyl ester. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01; Student's t-test. (Number of cells analysed: CTRL, n=58; RecNcil/wt, n=36; RecNcil/wt GC, n=107; L444P/wt, n=82; L444P/wt GC, n=33; L444P/L444P, n=62). (b) Caffeine (10 mM) was applied to iPSC-derived neurons, and the peak cytosolic calcium concentration was measured as a change in Fura-2 fluorescence. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01; **P0.001, Student's t-test. (Number of cells analysed: CTRL, n=58; RecNcil/wt, n=36; RecNcil/wt GC, n=107; L444P/wt, n=82; L444P/wt GC, n=33; L444P/L444P, n=62).(c) iPSC-derived neurons were treated with A23187 (0-5 μM) for 4 h and cytotoxicity was evaluated by released LDH activity. LDH release values were calculated as the percentage of untreated cells lysed by incubation with Triton X-100. Data are represented as mean+s.e.m.; experiments were independently repeated four times in triplicate. *P0.05, one-way ANOVA, control lines versus mutated lines; δP0.05, one-way ANOVA, mutated lines versus isogenic gene corrected controls.(d) Quantification of TH+ cells treated with A23187 (1 μM) for 4 h expressed as a percentage of DMSO-treated controls. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.01, one-way ANOVA.(e) Knockdown efficiency of NECAB2 determined by qRT-PCR and normalized to scrambled non-targeting shRNA. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. ***P0.0001, Student's t-test.(f) Representative western blot for NECAB2 showing knockdown efficiency. 1, scrambled non-targeting shRNA; 2, NECAB2 shRNA.(g) Quantification of TH+ cells in GBA-PD and GD iPSC-derived neuronal cultures infected with scrambled non-targeting shRNA or NECAB2 shRNA lentivirus and treated with A23187 (1 μM for 4 h). Values were calculated as a percentage of scrambled shRNA-treated cells. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01, Student's t-test.(h) Representative images showing GBA-PD iPSC-derived DA neurons infected with scrambled non-targeting shRNA or NECAB2 shRNA lentivirus and treated with A23187 (1 μM for 4 h). Cells were stained for β-TubIII (green) and TH (red). Nuclei were counterstained with Hoechst (blue). (Bar, 20 μm.)


Figure 2 ( B ) The number of metabolites that change significantly when wild - type yeast and mutants in the predicted target are treated with the indicated drugs at a concentration of 10 µM are shown . Drugs targeting GPR1 are written in black text , with drugs targeting SNF3 and RGT2 written in purple and green respectively . Orange filled bars are predicted antagonists for GPR1 . ( C ) The levels of trehalose compared to a DMSO treated control are shown for both wild - type yeast and gpr1 strains treated with predicted antagonists for GPR1 . The bar height indicates mean values , and error bars indicate the standard deviation of the mean . Asterisks indicate a p - value of less than 0 . 05 from a two - sided student ' s T - test ( n = 4 biological replicates ) when comparing The WT and gpr1 reponses to the drug . Results are representative of 3 independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) The number of metabolites that change significantly when wild-type yeast and mutants in the predicted target are treated with the indicated drugs at a concentration of 10 µM are shown. Drugs targeting GPR1 are written in black text, with drugs targeting SNF3 and RGT2 written in purple and green respectively. Orange filled bars are predicted antagonists for GPR1.(C) The levels of trehalose compared to a DMSO treated control are shown for both wild-type yeast and gpr1 strains treated with predicted antagonists for GPR1. The bar height indicates mean values, and error bars indicate the standard deviation of the mean. Asterisks indicate a p-value of less than 0.05 from a two-sided student's T-test (n = 4 biological replicates) when comparing The WT and gpr1 reponses to the drug. Results are representative of 3 independent experiments.


Figure 3 ( C ) The average quantified filamentous growth is shown for BY4741 and Σ1278b yeast spotted onto media supplemented with either a vehicle control ( DMSO 0 . 1 % ( v / v ) or the indicated compounds at a concentration of 20 µM across four biological replicates . Error bars represent the standard deviation of the mean between the averages of the replicates , and asterisks indicate p - value < 0 . 05 for a two - sided student ' s T - test comparing normalized values to the DMSO control ( n = 4 biological replicates ) . ( D ) The average quantified filamentous growth is shown for BY4741 and Σ1278b yeast spotted onto media supplemented with either a vehicle control ( DMSO 0 . 1 % ( v / v ) or the indicated concentrations of ibuprofen . Error bars represent the standard deviation of the mean between the averages of the replicates , and asterisks indicate p - value < 0 . 05 for a two - sided student ' s T - test comparing normalized values to the DMSO control ( n = 4 biological replicates ) . ( E ) The average relative amount of trehalose in ibuprofen - treated cells compared to a DMSO treatment is shown . The bar height indicates mean values , error bars indicate the standard deviation of the mean , and asterisks indicate p - value < 0 . 05 for a two - sided student ' s T - test comparing to the WT control ( n = 8 biological replicates ) . Results are representative of 3 independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(C) The average quantified filamentous growth is shown for BY4741 and Σ1278b yeast spotted onto media supplemented with either a vehicle control (DMSO 0.1% (v/v) or the indicated compounds at a concentration of 20 µM across four biological replicates. Error bars represent the standard deviation of the mean between the averages of the replicates, and asterisks indicate p-value < 0.05 for a two-sided student's T-test comparing normalized values to the DMSO control (n = 4 biological replicates).(D) The average quantified filamentous growth is shown for BY4741 and Σ1278b yeast spotted onto media supplemented with either a vehicle control (DMSO 0.1% (v/v) or the indicated concentrations of ibuprofen. Error bars represent the standard deviation of the mean between the averages of the replicates, and asterisks indicate p-value < 0.05 for a two-sided student's T-test comparing normalized values to the DMSO control (n = 4 biological replicates).(E) The average relative amount of trehalose in ibuprofen-treated cells compared to a DMSO treatment is shown. The bar height indicates mean values, error bars indicate the standard deviation of the mean, and asterisks indicate p-value < 0.05 for a two-sided student's T-test comparing to the WT control (n = 8 biological replicates). Results are representative of 3 independent experiments.


Figure 1 ( A ) Flow cytometry analysis of CD34 + CD1a - thymocytes cultured in FTOCs , in the presence of a control or anti - Notch1 blocking antibody . Numbers in dot plots indicate percentage of cells for the corresponding populations . Histograms show TCRγδ expression in CD3 + TCRαβ + ( blue ) and CD3 + TCRαβ - ( red ) cells . ( B , C ) ( B ) Mean frequency and ( C ) absolute cell numbers of human CD4 + CD8β + DP ( black shades ) , CD3 + TCRαβ + ( blue shades ) and CD3 + TCRγδ + ( red shades ) thymocytes developed in corresponding cultures from A ( n = 6 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 and ns ( not significant ) indicates p > 0 . 05 in a non - parametric paired Wilcoxon test ) . ( D ) Flow cytometry analysis of CD34 + CD1a - thymocytes submitted to FTOC with fetal thymic lobes deficient for Dll4 and / or Jag2 ( Cre + ) as indicated above the dot plots . Numbers in dot plots indicate percentage of cells for the corresponding populations . Histograms show TCRγδ expression in CD3 + TCRαβ + ( blue ) and CD3 + TCRαβ - ( red ) cells . ( E , F ) ( E ) Mean frequency and ( F ) absolute cell numbers of CD3 + TCRαβ + ( blue ) and CD3 + TCRγδ + ( red ) thymocytes developed in corresponding cultures from D ( n = 5 - 7 independent experiments , depending on the mouse genotype , error bars indicate SEM and * indicates p < 0 . 05 and ns ( not significant ) indicates p > 0 . 05 in a non - parametric Mann - Whitney U test ) .

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

Figure 1 (A) Flow cytometry analysis of CD34+CD1a- thymocytes cultured in FTOCs, in the presence of a control or anti-Notch1 blocking antibody. Numbers in dot plots indicate percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells. (B, C) (B) Mean frequency and (C) absolute cell numbers of human CD4+CD8β+ DP (black shades), CD3+TCRαβ+ (blue shades) and CD3+TCRγδ+ (red shades) thymocytes developed in corresponding cultures from A (n=6 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 and ns (not significant) indicates p>0.05 in a non-parametric paired Wilcoxon test).  (D) Flow cytometry analysis of CD34+CD1a- thymocytes submitted to FTOC with fetal thymic lobes deficient for Dll4 and/or Jag2 (Cre+) as indicated above the dot plots. Numbers in dot plots indicate percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells. (E, F) (E) Mean frequency and (F) absolute cell numbers of CD3+TCRαβ+ (blue) and CD3+TCRγδ+ (red) thymocytes developed in corresponding cultures from D (n=5-7 independent experiments, depending on the mouse genotype, error bars indicate SEM and * indicates p<0.05 and ns (not significant) indicates p>0.05 in a non-parametric Mann-Whitney U test).


Figure 2 ( A ) Line plots depicting fluctuations in gene expression levels from vsn normalized probe intensities across human T cell development for both αβ - and γδ - lineages ( n = 2 biological replicates and error bars indicate SEM ) . ( D - E ) Scatterplot displaying the up ( red ) or down ( blue ) regulation of probes ( log2 fold change > = 2 ) identified by sequential pairwise comparisons across either αβ ( D ) or γδ ( E ) development .

O O B-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Line plots depicting fluctuations in gene expression levels from vsn normalized probe intensities across human T cell development for both αβ- and γδ-lineages (n=2 biological replicates and error bars indicate SEM).(D-E) Scatterplot displaying the up (red) or down (blue) regulation of probes (log2 fold change >= 2) identified by sequential pairwise comparisons across either αβ (D) or γδ (E) development.


Figure 3 ( C - D ) Scatterplot displaying the up ( red ) or down ( blue ) regulation of miRNA ( log2 fold change > = 2 ) identified by sequential pairwise comparisons starting from CB towards either the αβ ( C ) or γδ ( D ) developmental lineages .

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

Figure 3 (C-D) Scatterplot displaying the up (red) or down (blue) regulation of miRNA (log2 fold change >= 2) identified by sequential pairwise comparisons starting from CB towards either the αβ (C) or γδ (D) developmental lineages.


Figure 4 ( A ) Line plots displaying averaged NOTCH1 , MYC and HES1 expression across T cell development ( n = 2 biological replicates and error bars indicate SEM ) . ( B ) Heatmap displaying transcript wise scaled expression of NOTCH1 , HES1 , MYC and miRNAs from ex vivo isolated subsets correlating ( spearman rho > = 0 . 5 ) with NOTCH1 gene expression Red arrows indicate all 6 members of the miR - 17 ~ 92 cluster ( n = 2 ) . ( C ) Heatmap visualizing Notch1 regulated gene expression of in vitro cultured thymocytes across multiple culturing conditions ( OP9 - control , OP9 - JAG1 , OP9 - JAG2 , OP9 - DLL1 and OP9 - DLL4 ) and time points ( day 0 , 5 and 10 ) . ( D ) NOTCH1 correlating miRNAs ( spearman rho > = 0 . 3 ) of in vitro cultured thymocytes across multiple culturing conditions and timepoints . The miRNA expression levels were scaled transcript wise and red arrows indicate members of miR - 17 ~ 92 cluster ( n = 2 ) . ( E ) CHIPseq data for Notch1 and RBPj binding at the miR - 17 ~ 92 locus in CUTTL1 cells . Red line underneath the ChIPseq peaks indicate significant enrichment compared to the input sample .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Line plots displaying averaged NOTCH1, MYC and HES1 expression across T cell development (n=2 biological replicates and error bars indicate SEM). (B) Heatmap displaying transcript wise scaled expression of NOTCH1, HES1, MYC and miRNAs from ex vivo isolated subsets correlating (spearman rho >= 0.5) with NOTCH1 gene expression Red arrows indicate all 6 members of the miR-17~92 cluster (n=2).  (C) Heatmap visualizing Notch1 regulated gene expression of in vitro cultured thymocytes across multiple culturing conditions (OP9-control, OP9-JAG1, OP9-JAG2, OP9-DLL1 and OP9-DLL4) and time points (day 0, 5 and 10). (D) NOTCH1 correlating miRNAs (spearman rho >= 0.3) of in vitro cultured thymocytes across multiple culturing conditions and timepoints. The miRNA expression levels were scaled transcript wise and red arrows indicate members of miR-17~92 cluster (n=2).(E) CHIPseq data for Notch1 and RBPj binding at the miR-17~92 locus in CUTTL1 cells. Red line underneath the ChIPseq peaks indicate significant enrichment compared to the input sample.


Figure 5 ( B ) Transfection of miR - 17 Pre - miR miRNA Precursor , but not of miR - 92a , specifically targets the wild - type ( black bars ) 3 ' UTR of BCL11B in a 3 ' UTR luciferase reporter assay following cotransfection of HEKT cells in comparison to the controls ( transfection of no miRNA or a control miRNA ) . Mutation of the predicted miR - 17 binding sites in the 3 ' UTR BCL11B reporter construct prevents miR - 17 dependent targeting ( grey bars ) . Graphs show mean firefly luciferase values ( from 3 ' UTR reporter constructs ) following normalization to the cotransfected control Renilla luciferase and relative to control sample without miRNA ( n = 5 independent experiments , error bars indicate SEM and * indicates p < 0 . 05 and ns ( not significant ) indicates p > 0 . 05 in a paired t - test ) . ( C ) Quantitative RT - PCR gene expression analysis of BCL11B in control ( black bar ) or miR - 17 ( grey bar ) transduced CD34 + thymocytes after 24h of culture on coated Fc - DLL1 plates , relative to the geometric mean of ACTIN and GAPDH and relative to the control - transduced sample ( n = 3 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 in a paired t - test ) . ( D ) Quantitative real - time RT - PCR gene expression analysis of BCL11B in control ( black bar ) or miR - 17 sponge ( white bar ) transduced CD34 + thymocytes , relative to the geometric mean of ACTIN and GAPDH ( n = 5 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 in a paired t - test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) Transfection of miR-17 Pre-miR miRNA Precursor, but not of miR-92a, specifically targets the wild-type (black bars) 3'UTR of BCL11B in a 3'UTR luciferase reporter assay following cotransfection of HEKT cells in comparison to the controls (transfection of no miRNA or a control miRNA). Mutation of the predicted miR-17 binding sites in the 3'UTR BCL11B reporter construct prevents miR-17 dependent targeting (grey bars). Graphs show mean firefly luciferase values (from 3'UTR reporter constructs) following normalization to the cotransfected control Renilla luciferase and relative to control sample without miRNA (n=5 independent experiments, error bars indicate SEM and * indicates p<0.05 and ns (not significant) indicates p>0.05 in a paired t-test). (C) Quantitative RT-PCR gene expression analysis of BCL11B in control (black bar) or miR-17 (grey bar) transduced CD34+ thymocytes after 24h of culture on coated Fc-DLL1 plates, relative to the geometric mean of ACTIN and GAPDH and relative to the control-transduced sample (n=3 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).  (D) Quantitative real-time RT-PCR gene expression analysis of BCL11B in control (black bar) or miR-17 sponge (white bar) transduced CD34+ thymocytes, relative to the geometric mean of ACTIN and GAPDH (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).


Figure 6 ( A ) Gene expression pattern of BCL11B ( red dots ) and HES1 ( blue squares ) during human T cell development , derived from microarray data as shown in Figure 2 . ( n = 2 biological replicates and error bars indicate SEM ) . Quantitative RT - PCR expression analysis of BCL11B in human after 5 days , respectively , of coculture on OP9 stromal cells expressing different Notch ligands . Graphs show mean expression , relative to the geometric mean of ACTIN and GAPDH and relative to the OP9 - DLL1 cultured cells ( n = 4 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 and ns ( not significant ) indicates p > 0 . 05 in a paired t - test ) . Quantitative RT - PCR expression analysis of BCL11B in human CD34 + thymocyte progenitors after 2 days , respectively , of coculture on OP9 stromal cells expressing different Notch ligands . Graphs show mean expression , relative to the geometric mean of ACTIN and GAPDH and relative to the OP9 - DLL1 cultured cells ( n = 4 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 and ns ( not significant ) indicates p > 0 . 05 in a paired t - test ) . ( D ) Quantitative RT - PCR expression analysis of BCL11B in human CD34 + thymocyte progenitors after 2 days of coculture on OP9 - DLL1 stromal cells in the presence of 5 µM DAPT ( GSI ) . Graphs show mean expression , relative to the geometric mean of ACTIN and GAPDH and relative to sample cultured in the presence of control DMSO ( n = 4 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 in a paired t - test ) . Quantitative RT - PCR expression analysis of miR - 17 in human CB CD34 + Lin - after 5 days , respectively , of coculture on OP9 stromal cells expressing different Notch ligands Graphs show mean expression , relative to the geometric mean of SNORD61 , SNORD95 and RNU6 - 2 and relative to the OP9 - DLL1 cultured cells ( n = 2 independent experiments with different donors ; error bars indicate SEM - the two data points are shown ) . Quantitative RT - PCR expression analysis of miR - 17 in human CD34 + thymocyte progenitors after 5 and 2 days , respectively , of coculture on OP9 - DLL1 stromal cells in the presence of 5 µM DAPT ( GSI ) . Graphs show mean expression , relative to the sample cultured in the presence of control DMSO ( F ) ( n = 2 independent experiments with different donors ; error bars indicate SEM - the two data points are shown ) . ( G ) Quantitative RT - PCR gene expression analysis of BCL11B in control ( black bar ) , GATA3 shRNA or TCF7 shRNA ( grey bars ) transduced CD34 + thymocytes after 48h of coculture on OP9 - DLL1 stromal cells , relative to the geometric mean of ACTIN and GAPDH and relative to the control - transduced sample ( n = 6 independent experiments with different donors for GATA3 shRNA , n = 4 independent experiments with different donors for TCF7 shRNA , error bars indicate SEM and * indicates p < 0 . 05 in a paired t - test ) .

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

Figure 6 (A) Gene expression pattern of BCL11B (red dots) and HES1 (blue squares) during human T cell development, derived from microarray data as shown in Figure 2. (n=2 biological replicates and error bars indicate SEM).  Quantitative RT-PCR expression analysis of BCL11B in human after 5 days, respectively, of coculture on OP9 stromal cells expressing different Notch ligands. Graphs show mean expression, relative to the geometric mean of ACTIN and GAPDH and relative to the OP9-DLL1 cultured cells (n=4 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 and ns (not significant) indicates p>0.05 in a paired t-test).  Quantitative RT-PCR expression analysis of BCL11B in human CD34+ thymocyte progenitors after 2 days, respectively, of coculture on OP9 stromal cells expressing different Notch ligands. Graphs show mean expression, relative to the geometric mean of ACTIN and GAPDH and relative to the OP9-DLL1 cultured cells (n=4 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 and ns (not significant) indicates p>0.05 in a paired t-test).  (D) Quantitative RT-PCR expression analysis of BCL11B in human CD34+ thymocyte progenitors after 2 days of coculture on OP9-DLL1 stromal cells in the presence of 5 µM DAPT (GSI). Graphs show mean expression, relative to the geometric mean of ACTIN and GAPDH and relative to sample cultured in the presence of control DMSO (n=4 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).  Quantitative RT-PCR expression analysis of miR-17 in human CB CD34+Lin- after 5 days, respectively, of coculture on OP9 stromal cells expressing different Notch ligands Graphs show mean expression, relative to the geometric mean of SNORD61, SNORD95 and RNU6-2 and relative to the OP9-DLL1 cultured cells (n=2 independent experiments with different donors; error bars indicate SEM - the two data points are shown).  Quantitative RT-PCR expression analysis of miR-17 in human CD34+ thymocyte progenitors after 5 and 2 days, respectively, of coculture on OP9-DLL1 stromal cells in the presence of 5 µM DAPT (GSI). Graphs show mean expression, relative to the sample cultured in the presence of control DMSO (F) (n=2 independent experiments with different donors; error bars indicate SEM - the two data points are shown).  (G) Quantitative RT-PCR gene expression analysis of BCL11B in control (black bar), GATA3 shRNA or TCF7 shRNA (grey bars) transduced CD34+ thymocytes after 48h of coculture on OP9-DLL1 stromal cells, relative to the geometric mean of ACTIN and GAPDH and relative to the control-transduced sample (n=6 independent experiments with different donors for GATA3 shRNA, n=4 independent experiments with different donors for TCF7 shRNA, error bars indicate SEM and * indicates p<0.05 in a paired t-test).


Figure 7 ( A ) Flow cytometry analysis of control and miR - 17 transduced CD34 + thymocytes , cultured in ATOs for 3 weeks . Numbers in contour plots indicate percentage of cells for the corresponding populations . Histograms show TCRγδ expression in CD3 + TCRαβ + ( blue ) and CD3 + TCRαβ - ( red ) cells . ( B ) Graphs show the mean frequency of CD4 + CD8β + DP , CD3 + TCRαβ + and CD3 + TCRγδ + thymocytes of control ( black bar ) or miR - 17 ( grey bar ) transduced CD34 + thymocytes in these cultures ( n = 6 and n = 8 independent experiments for control and miR - 17 , respectively . Error bars indicate SEM and ns ( not significant ) indicates p > 0 . 05 in a non - parametric Mann - Whitney U test ) . ( C ) Graphs show the mean absolute cell numbers of total , CD4 + CD8β + DP , CD3 + TCRαβ + and CD3 + TCRγδ + thymocytes that developed in these cultures ( n = 6 and n = 8 independent experiments for control and miR - 17 , respectively . Error bars indicate SEM and * indicates p < 0 . 05 in a non - parametric Mann - Whitney U test ) .

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

Figure 7 (A) Flow cytometry analysis of control and miR-17 transduced CD34+ thymocytes, cultured in ATOs for 3 weeks. Numbers in contour plots indicate percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells. (B) Graphs show the mean frequency of CD4+CD8β+ DP, CD3+TCRαβ+ and CD3+TCRγδ+ thymocytes of control (black bar) or miR-17 (grey bar) transduced CD34+ thymocytes in these cultures (n=6 and n=8 independent experiments for control and miR-17, respectively. Error bars indicate SEM and ns (not significant) indicates p>0.05 in a non-parametric Mann-Whitney U test). (C) Graphs show the mean absolute cell numbers of total, CD4+CD8β+ DP, CD3+TCRαβ+ and CD3+TCRγδ+ thymocytes that developed in these cultures (n=6 and n=8 independent experiments for control and miR-17, respectively. Error bars indicate SEM and * indicates p<0.05 in a non-parametric Mann-Whitney U test).


Figure 8 ( A ) Flow cytometry analysis showing mean of Median Fluorescence Intensity ( MFI ) of BCL11B protein levels in Jurkat cells , 72h post transduction with control shRNA ( black bar ) , BCL11B shRNA1 or BCL11B shRNA2 ( grey bars ) , each normalized to the FMO sample ( BCL11B stained - BCL11B unstained sample ) . ( n = 2 independent experiments ; error bars indicate SEM - the two data points are shown ) . ( B ) Flow cytometry analysis showing mean of MFI of BCL11B protein levels in CD4 + CD8β + DP ( black bar ) and CD4 - CD8β - DN CD3 + TCRγδ + ( grey bar ) ex vivo thymocytes , normalized to FMO sample ( BCL11B stained - BCL11B unstained sample ) ( n = 4 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 in a paired t - test ) . ( C ) Mean total cell numbers of control shRNA ( black bar ) , BCL11B shRNA1 or BCL11B shRNA2 ( grey bars ) transduced CD34 + thymocytes , cultured in ATOs for 3 weeks ( n = 5 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 in a non - parametric paired Wilcoxon test ) . ( D ) Flow cytometry analysis of CD4 + CD8β + DP , CD3 + TCRαβ + ( blue ) and CD3 + TCRαβ - ( red ) thymocytes that developed in corresponding cultures from C . Numbers in contour plots indicate the percentage of cells for the corresponding populations . Histograms show TCRγδ expression in CD3 + TCRαβ + ( blue ) and CD3 + TCRαβ - ( red ) cells . ( E ) Graphs show the mean frequency of CD3 + TCRαβ + and CD3 + TCRγδ + thymocytes that developed in these cultures ( n = 5 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 in a paired t - test ) . ( F - G ) Mean absolute cell numbers of ( F ) CD3 + TCRαβ + and ( G ) CD3 + TCRγδ + thymocytes that developed in these cultures ( n = 5 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 in a non - parametric paired Wilcoxon test ) . ( H ) CD3 + TCRγδ + / CD3 + TCRαβ + ratio from cultures depicted in D ( n = 5 independent experiments with different donors , error bars indicate SEM and * indicates p < 0 . 05 in a paired t - test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) Flow cytometry analysis showing mean of Median Fluorescence Intensity (MFI) of BCL11B protein levels in Jurkat cells, 72h post transduction with control shRNA (black bar), BCL11B shRNA1 or BCL11B shRNA2 (grey bars), each normalized to the FMO sample (BCL11B stained - BCL11B unstained sample). (n=2 independent experiments; error bars indicate SEM - the two data points are shown). (B) Flow cytometry analysis showing mean of MFI of BCL11B protein levels in CD4+CD8β+ DP (black bar) and CD4-CD8β- DN CD3+TCRγδ+ (grey bar) ex vivo thymocytes, normalized to FMO sample (BCL11B stained - BCL11B unstained sample) (n=4 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).  (C) Mean total cell numbers of control shRNA (black bar), BCL11B shRNA1 or BCL11B shRNA2 (grey bars) transduced CD34+ thymocytes, cultured in ATOs for 3 weeks (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a non-parametric paired Wilcoxon test).  (D) Flow cytometry analysis of CD4+CD8β+ DP, CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) thymocytes that developed in corresponding cultures from C. Numbers in contour plots indicate the percentage of cells for the corresponding populations. Histograms show TCRγδ expression in CD3+TCRαβ+ (blue) and CD3+TCRαβ- (red) cells. (E) Graphs show the mean frequency of CD3+TCRαβ+ and CD3+TCRγδ+ thymocytes that developed in these cultures (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test). (F-G) Mean absolute cell numbers of (F) CD3+TCRαβ+ and (G) CD3+TCRγδ+ thymocytes that developed in these cultures (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a non-parametric paired Wilcoxon test). (H) CD3+TCRγδ+/CD3+TCRαβ+ ratio from cultures depicted in D (n=5 independent experiments with different donors, error bars indicate SEM and * indicates p<0.05 in a paired t-test).


Figure 1Primary rat cortical neurons transfected with eGFP - GA50 were examined to determine at which time points preceding cell death aggregates are found in neurites . Two days post - transfection , aggregates formed by eGFP - GA50 ( green ) are detectable in neurites ( SMI - 32 staining in red ) . These aggregates remain localized to neurites at 96 hours ( 4 days ) and 288 hours ( 8 days ) . Colocalization is indicated by yellow overlay of colors ( right panels ) . Inset below each image shows enlargement of neurite regions containing aggregates , Representative fields from 60x magnification z - stack confocal images , scale bar indicates 20μm . Inset scale bars indicate 5μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Primary rat cortical neurons transfected with eGFP-GA50 were examined to determine at which time points preceding cell death aggregates are found in neurites. Two days post-transfection, aggregates formed by eGFP-GA50 (green) are detectable in neurites (SMI-32 staining in red). These aggregates remain localized to neurites at 96 hours (4 days) and 288 hours (8 days). Colocalization is indicated by yellow overlay of colors (right panels). Inset below each image shows enlargement of neurite regions containing aggregates, Representative fields from 60x magnification z-stack confocal images, scale bar indicates 20μm. Inset scale bars indicate 5μm.


Figure 2Primary rat cortical neurons underwent live - cell imaging after 48 hours of eGFP - GAn expression to determine aggregate mobility within neurites . A ) Example of a cortical neuron expressing eGFP - GA100 ( green ) with brightfield overlay , 60x magnification , scale bar indicates 10μm . Inset in upper right shows enlargement of boxed area , to better visualize aggregates within neuronal processes . Inset scale bar indicates 5μm . Primary rat motor neurons underwent live - cell imaging after 48 hours of eGFP - GAn expression to determine aggregate mobility within neurites . B ) Example of a motor neuron expressing eGFP - GA25 ( green ) with brightfield overlay , 60x magnification , scale bar indicates 10μm . Inset in upper right shows enlargement of boxed area , to better visualize aggregates within neuronal processes . Inset scale bar indicates 5μm . C ) Quantification of eGFP - GAn aggregate velocity within cortical neurons . D ) Quantification of eGFP - GAn aggregate velocity within motor neuron neurites .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O

Figure 2Primary rat cortical neurons underwent live-cell imaging after 48 hours of eGFP-GAn expression to determine aggregate mobility within neurites. A) Example of a cortical neuron expressing eGFP-GA100 (green) with brightfield overlay, 60x magnification, scale bar indicates 10μm. Inset in upper right shows enlargement of boxed area, to better visualize aggregates within neuronal processes. Inset scale bar indicates 5μm.Primary rat motor neurons underwent live-cell imaging after 48 hours of eGFP-GAn expression to determine aggregate mobility within neurites. B) Example of a motor neuron expressing eGFP-GA25 (green) with brightfield overlay, 60x magnification, scale bar indicates 10μm. Inset in upper right shows enlargement of boxed area, to better visualize aggregates within neuronal processes. Inset scale bar indicates 5μm.C) Quantification of eGFP-GAn aggregate velocity within cortical neurons.D) Quantification of eGFP-GAn aggregate velocity within motor neuron neurites.


Figure 3Primary rat neurons were co - transfected with Td - tomato and eGFP - GAn plasmids or eGFP - alone . The same neurons were imaged at 24 - hour intervals to assess viability . A ) Representative fields ( right ) of eGFP and eGFP - GA50 cortical neurons at days 1 , 7 , and 13 post - transfection follow the survival of individual cells over time ( white arrows ) . 20x magnification , scale bar indicates 25μm . B ) Kaplan - Meier survival analysis of cortical neurons followed for 13 days post - transfection . C ) Kaplan - Meier survival analysis ( left ) of motor neurons followed for 5 days post - transfection .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O

Figure 3Primary rat neurons were co-transfected with Td-tomato and eGFP-GAn plasmids or eGFP-alone. The same neurons were imaged at 24-hour intervals to assess viability. A) Representative fields (right) of eGFP and eGFP-GA50 cortical neurons at days 1, 7, and 13 post-transfection follow the survival of individual cells over time (white arrows). 20x magnification, scale bar indicates 25μm. B) Kaplan-Meier survival analysis of cortical neurons followed for 13 days post-transfection.C) Kaplan-Meier survival analysis (left) of motor neurons followed for 5 days post-transfection.


Figure 4Cortical neurons were loaded with FM4 - 64 dye 48 hours after transfection . Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl . A ) Representative dye loading showing defined puncta regions along neurites 40x magnification , scale bar indicates 10μm . B ) Representative intensities of FM4 - 64 puncta regions pre - and post - stimulation demonstrate loss of synaptic puncta fluorescence intensity following stimulation in GFP but not GA50 containing neurons . These images visually reflect the process performed in an automated manner through our imaging software to determine puncta fluorescence intensity . An unbiased constant threshold is placed on each image per experiment , then fluorescence intensity is measured over time . The threshold applied isolates puncta from background , giving distinct regions . 8 - bit images were thresholded and pseudo - color ( red ) was applied to enlarged puncta regions of interest , scale bar indicates 10μm . C ) Graphical representation is the change in fluorescence ( ΔF ) normalized to basal fluorescence ( F ) , ΔF / F . The final 10 seconds of baseline recording and 10 seconds of the depolarization phase are shown , arrow indicates start of depolarization . Non - linear regression was performed in GraphPad Prism7 using the plateau followed by one phase decay model . The decay constant ( k ) for GFP was 0 . 4971 s - 1 , while for GA50 it was 219 . 5 s - 1 . Similarly the tau ( τ ) values were 2 . 012 seconds for GFP and 0 . 00455 seconds for GA50 , respectively . Data presented as mean ± SEM for each imaged timepoint . Statistical significance was determined using the Sidak - Bonferroni method , * * * * p < 0 . 0001 , n = 20 puncta regions from 4 biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Cortical neurons were loaded with FM4-64 dye 48 hours after transfection. Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl. A) Representative dye loading showing defined puncta regions along neurites 40x magnification, scale bar indicates 10μm. B) Representative intensities of FM4-64 puncta regions pre- and post- stimulation demonstrate loss of synaptic puncta fluorescence intensity following stimulation in GFP but not GA50 containing neurons. These images visually reflect the process performed in an automated manner through our imaging software to determine puncta fluorescence intensity. An unbiased constant threshold is placed on each image per experiment, then fluorescence intensity is measured over time. The threshold applied isolates puncta from background, giving distinct regions. 8-bit images were thresholded and pseudo-color (red) was applied to enlarged puncta regions of interest, scale bar indicates 10μm.C) Graphical representation is the change in fluorescence (ΔF) normalized to basal fluorescence (F), ΔF/F. The final 10 seconds of baseline recording and 10 seconds of the depolarization phase are shown, arrow indicates start of depolarization. Non-linear regression was performed in GraphPad Prism7 using the plateau followed by one phase decay model. The decay constant (k) for GFP was 0.4971 s-1, while for GA50 it was 219.5 s-1. Similarly the tau (τ) values were 2.012 seconds for GFP and 0.00455 seconds for GA50, respectively. Data presented as mean ± SEM for each imaged timepoint. Statistical significance was determined using the Sidak-Bonferroni method, ****p < 0.0001, n=20 puncta regions from 4 biological replicates.


Figure 5Primary cortical or motor neurons were co - transfected with mCherry or mCherry - GA50 and GCaMP6f . After 48 hours , mCherry positive cells were determined . Green fluorescence intensity was then recorded from identified neurons . Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl . A ) Representative images of a co - transfected cortical neuron expressing mCherry - GA50 ( left panel ) and GCaMP6f ( right two panels ) . This image shows a representative demonstration of the increase in detectable green fluorescence from the GCaMP6f indicator pre - and post - stimulation ( center and right respectively ) . 40x magnification , scale bar indicates 25μm . B - C ) Graphical representation of the peak change in fluorescence ( ΔF ) following ACSF perfusion normalized to basal fluorescence ( F ) , ΔF / F . The contribution of Ca2 + influx was determined by experiments in which stimulation was performed in ACSF containing high KCl but no Ca2 + . Quantification of the peak change in fluorescence in cortical neurons ( B ) and motor neurons ( C ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Primary cortical or motor neurons were co-transfected with mCherry or mCherry-GA50 and GCaMP6f. After 48 hours, mCherry positive cells were determined. Green fluorescence intensity was then recorded from identified neurons. Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl. A) Representative images of a co-transfected cortical neuron expressing mCherry-GA50 (left panel) and GCaMP6f (right two panels). This image shows a representative demonstration of the increase in detectable green fluorescence from the GCaMP6f indicator pre- and post- stimulation (center and right respectively). 40x magnification, scale bar indicates 25μm.B-C) Graphical representation of the peak change in fluorescence (ΔF) following ACSF perfusion normalized to basal fluorescence (F), ΔF/F. The contribution of Ca2+ influx was determined by experiments in which stimulation was performed in ACSF containing high KCl but no Ca2+. Quantification of the peak change in fluorescence in cortical neurons (B) and motor neurons (C).


Figure 6eGFP - GAn dipeptides were expressed in primary cortical or motor neurons for 48 hours , then immunostained for neurofilament ( gray ) and SV2 ( red ) . A - B ) Representative z - stack confocal images of neurites from eGFP , eGFP - GA50 , and eGFP - GA100 expressing cortical ( A ) or motor ( B ) neurons . 60x magnification , scale bar indicates 5μm . C - D ) Quantification of SV2 puncta along neurites in cortical neurons ( C ) or motor neurons ( D ) by ImageJ manual counting , normalized to eGFP - only expressing cells . ( C ) Cortical neurons demonstrated a significant GA - length dependent reduction , as well as a reduction in all cells containing GA - repeats greater than 50 ( left panel ) . ( D ) Motor neurons demonstrated a significant GA - length dependent reduction , as well as a reduction in all cells containing GA - repeats greater than 100 ( left panel ) . Evaluation of neurite length by NeuronJ software revealed no differences in total neurite length per cell in cortical neurons ( C ) or motor neurons ( D ) expressing any GA - repeat length compared with eGFP alone ( right panels ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6eGFP-GAn dipeptides were expressed in primary cortical or motor neurons for 48 hours, then immunostained for neurofilament (gray) and SV2 (red). A-B) Representative z-stack confocal images of neurites from eGFP, eGFP-GA50, and eGFP-GA100 expressing cortical (A) or motor (B) neurons. 60x magnification, scale bar indicates 5μm. C-D) Quantification of SV2 puncta along neurites in cortical neurons (C) or motor neurons (D) by ImageJ manual counting, normalized to eGFP-only expressing cells. (C) Cortical neurons demonstrated a significant GA-length dependent reduction, as well as a reduction in all cells containing GA-repeats greater than 50 (left panel). (D) Motor neurons demonstrated a significant GA-length dependent reduction, as well as a reduction in all cells containing GA-repeats greater than 100 (left panel). Evaluation of neurite length by NeuronJ software revealed no differences in total neurite length per cell in cortical neurons (C) or motor neurons (D) expressing any GA-repeat length compared with eGFP alone (right panels).


Figure 7i3 cortical neurons from a C9orf72 - ALS patient and the paired isogenic control were differentiated for 21 days , then were immunostained for neurofilament ( red ) , and SV2 ( green ) ( A ) Shown are representative z - stack confocal images of neurites . B ) i3 cortical neurons from a C9orf72 - ALS patient and the paired isogenic control were differentiated for 21 days , then were immunostained for synaptophysin ( green ) ( B ) . Shown are representative z - stack confocal images of neurites . Quantification of SV2 ( C ) colocalized with neurofilament normalized to neurofilament area . This analysis revealed a significant reduction in SV2 levels in the C9orf72 line compared with the isogenic control , Data presented as mean ± SEM . Unpaired t - test , * p < 0 . 05 . 15 non - overlapping regions from 3 differentiated wells for each genotype were evaluated . Quantification of Synaptophysin ( D ) colocalized with neurofilament normalized to neurofilament area . This analysis revealed a significant reduction in SV2 levels in the C9orf72 line compared with the isogenic control , while synaptophysin remained unaltered . Data presented as mean ± SEM . Unpaired t - test , * p < 0 . 05 . 15 non - overlapping regions from 3 differentiated wells for each genotype were evaluated . E ) mRNA was Trizol extracted from 3 independent wells of C9 - ALS and isogenic i3 cortical neurons . RNA was converted to cDNA using the Superscript First - strand kit , and q - PCR was performed using PowerUp SYBR Green . Measurements were normalized to the housekeeping gene GAPDH and then to isogenic levels . Analysis was performed using the ΔΔCT method . 2ΔΔCT ± SE , is presented . Unpaired t - test , * * p < 0 . 01 . F ) Whole cell lysates were generated from 3 independent wells of C9 - ALS and isogenic i3 cortical neurons differentiated for 21 days . Lysates were immunoblotted for SV2 , and normalized to total protein loading . Quantification band intensities revealed a significant decrease in SV2 protein levels in the C9orf72 lines compared with the isogenic . Data presented as mean ± SEM . Unpaired t - test , * p < 0 . 05 . Whole cell lysates were generated from iMNs differentiated for 32 days . Lysates were immunoblotted for SV2 ( G ) Loading controls were β - actin Quantification band intensities normalized to loading controls revealed a trend towards reduction in SV2 protein levels in the C9orf72 cell lines compared with controls Data presented as mean ± SEM . Unpaired t - test . Samples from 5 independent control and 4 independent C9orf72 patient lines were used . Whole cell lysates were generated from iMNs differentiated for 32 days . Lysates were immunoblotted for synaptophysin ( H ) . Loading controls were total protein loading . synaptophysin levels remained unaltered . Data presented as mean ± SEM . Unpaired t - test . Samples from 5 independent control and 4 independent C9orf72 patient lines were used .

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Figure 7i3 cortical neurons from a C9orf72-ALS patient and the paired isogenic control were differentiated for 21 days, then were immunostained for neurofilament (red), and SV2 (green) (A) Shown are representative z-stack confocal images of neurites.B) i3 cortical neurons from a C9orf72-ALS patient and the paired isogenic control were differentiated for 21 days, then were immunostained for synaptophysin (green) (B). Shown are representative z-stack confocal images of neurites.Quantification of SV2 (C) colocalized with neurofilament normalized to neurofilament area. This analysis revealed a significant reduction in SV2 levels in the C9orf72 line compared with the isogenic control, Data presented as mean ± SEM. Unpaired t-test, *p < 0.05. 15 non-overlapping regions from 3 differentiated wells for each genotype were evaluated.Quantification of Synaptophysin (D) colocalized with neurofilament normalized to neurofilament area. This analysis revealed a significant reduction in SV2 levels in the C9orf72 line compared with the isogenic control, while synaptophysin remained unaltered. Data presented as mean ± SEM. Unpaired t-test, *p < 0.05. 15 non-overlapping regions from 3 differentiated wells for each genotype were evaluated.E) mRNA was Trizol extracted from 3 independent wells of C9-ALS and isogenic i3 cortical neurons. RNA was converted to cDNA using the Superscript First-strand kit, and q-PCR was performed using PowerUp SYBR Green. Measurements were normalized to the housekeeping gene GAPDH and then to isogenic levels. Analysis was performed using the ΔΔCT method. 2ΔΔCT ± SE, is presented. Unpaired t-test, **p < 0.01.F) Whole cell lysates were generated from 3 independent wells of C9-ALS and isogenic i3 cortical neurons differentiated for 21 days. Lysates were immunoblotted for SV2, and normalized to total protein loading. Quantification band intensities revealed a significant decrease in SV2 protein levels in the C9orf72 lines compared with the isogenic. Data presented as mean ± SEM. Unpaired t-test, *p < 0.05.Whole cell lysates were generated from iMNs differentiated for 32 days. Lysates were immunoblotted for SV2 (G) Loading controls were β-actin Quantification band intensities normalized to loading controls revealed a trend towards reduction in SV2 protein levels in the C9orf72 cell lines compared with controls Data presented as mean ± SEM. Unpaired t-test. Samples from 5 independent control and 4 independent C9orf72 patient lines were used.Whole cell lysates were generated from iMNs differentiated for 32 days. Lysates were immunoblotted for synaptophysin (H). Loading controls were total protein loading. synaptophysin levels remained unaltered. Data presented as mean ± SEM. Unpaired t-test. Samples from 5 independent control and 4 independent C9orf72 patient lines were used.


Figure 8A ) Cortical neurons were loaded with FM4 - 64 dye 48 hours after transfection with eGFP - GA50 and transduction with rSV2a - eGFP - pRRL lentivirus . Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl . Graphical representation is the change in fluorescence ( ΔF ) normalized to basal fluorescence ( F ) , ΔF / F . The final 60 seconds of baseline recording and 60 seconds of the depolarization phase are shown , arrow indicates start of depolarization . The lack of synaptic release in the case of GA50 expression ( red line ) was rescued back to levels comparable to eGFP controls ( pink line ) in the case of coexpression of rSV2a - eGFP - pRRL ( orange line ) . Statistical significance was determined using the Sidak - Bonferroni method , * * * p < 0 . 001 , n = 10 puncta regions from each of 4 biological replicates . B ) Cortical neurons were co - transfected with mCherry or mCherry - GA50 and GCaMP6f , then transduced with rSV2a - eGFP - pRRL . Green fluorescence intensity was recorded from identified mCherry positive neurons . Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl . Quantification of the peak change in fluorescence ( ΔF ) following ACSF perfusion normalized to basal fluorescence ( F ) , ΔF / F . The significant increase in Ca2 + influx observed following GA50 expression ( * p < 0 . 05 ) is resolved when rSV2a - eGFP - pRRL is coexpressed . Data presented as mean ± SEM . One - way ANOVA with Sidak ' s multiple comparisons test , at least 10 cells per condition , pooled from n = 3 independent biological experiments . No significant differences were observed between mCherry or mCherry + SV2 conditions , groups were merged for this analysis . Neurons were co - transfected with mCherry - GAn plasmids or mCherry - alone and transduced with rSV2a - eGFP - pRRL , then imaged at 24 - hour intervals . ( C ) Kaplan - Meier survival analysis of cortical neurons expressing GA50 or GA50 + SV2 followed for 13 days . The significant cellular toxicity induced by GA50 is rescued by SV2 replenishment . While the hazard ratio of GA50 was 1 . 473 , this was decreased to 0 . 7227 for GA50 + SV2 . At least 190 cells per condition were evaluated from a pooling of n = 3 independent experiments , log - rank Mantel - Cox test , * * * * p < 0 . 0001 . No significant differences were observed between eGFP or eGFP + SV2 conditions , and groups were merged for this analysis as Control . ( D ) A Kaplan - Meier survival analysis was performed for PR50 and PR50 + SV2 containing cells . Under these conditions significant toxicity is observed regardless of increased SV2 levels , with the hazard ratio of PR50 containing cells ( 1 . 772 ) remaining high for those expressing PR50 + SV2 ( 1 . 784 ) . At least 150 cells per condition were evaluated from a pooling of n = 3 independent experiments , log - rank Mantel - Cox test , * * * * p < 0 . 0001 . No significant differences were observed between eGFP or eGFP + SV2 conditions , and groups were merged for this analysis as Control . ( E ) Kaplan - Meier survival analysis of motor neurons expressing GA50 or GA50 + SV2 followed for 10 days . The significant cellular toxicity induced by GA50 is rescued by SV2 replenishment . While the hazard ratio of GA50 was 1 . 345 , this was decreased to 0 . 7769 for GA50 + SV2 . At least 200 cells per condition were evaluated from a pooling of n = 3 independent experiments , log - rank Mantel - Cox test , * * * * p < 0 . 0001 .

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Figure 8A) Cortical neurons were loaded with FM4-64 dye 48 hours after transfection with eGFP-GA50 and transduction with rSV2a-eGFP-pRRL lentivirus. Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl. Graphical representation is the change in fluorescence (ΔF) normalized to basal fluorescence (F), ΔF/F. The final 60 seconds of baseline recording and 60 seconds of the depolarization phase are shown, arrow indicates start of depolarization. The lack of synaptic release in the case of GA50 expression (red line) was rescued back to levels comparable to eGFP controls (pink line) in the case of coexpression of rSV2a-eGFP-pRRL (orange line). Statistical significance was determined using the Sidak-Bonferroni method, ***p < 0.001, n=10 puncta regions from each of 4 biological replicates.B) Cortical neurons were co-transfected with mCherry or mCherry-GA50 and GCaMP6f, then transduced with rSV2a-eGFP-pRRL. Green fluorescence intensity was recorded from identified mCherry positive neurons. Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl. Quantification of the peak change in fluorescence (ΔF) following ACSF perfusion normalized to basal fluorescence (F), ΔF/F. The significant increase in Ca2+ influx observed following GA50 expression (*p < 0.05) is resolved when rSV2a-eGFP-pRRL is coexpressed. Data presented as mean ± SEM. One-way ANOVA with Sidak's multiple comparisons test, at least 10 cells per condition, pooled from n= 3 independent biological experiments. No significant differences were observed between mCherry or mCherry+SV2 conditions, groups were merged for this analysis.Neurons were co-transfected with mCherry-GAn plasmids or mCherry-alone and transduced with rSV2a-eGFP-pRRL, then imaged at 24-hour intervals. (C) Kaplan-Meier survival analysis of cortical neurons expressing GA50 or GA50+SV2 followed for 13 days. The significant cellular toxicity induced by GA50 is rescued by SV2 replenishment. While the hazard ratio of GA50 was 1.473, this was decreased to 0.7227 for GA50+SV2. At least 190 cells per condition were evaluated from a pooling of n=3 independent experiments, log-rank Mantel-Cox test, ****p < 0.0001. No significant differences were observed between eGFP or eGFP+SV2 conditions, and groups were merged for this analysis as Control. (D) A Kaplan-Meier survival analysis was performed for PR50 and PR50+SV2 containing cells. Under these conditions significant toxicity is observed regardless of increased SV2 levels, with the hazard ratio of PR50 containing cells (1.772) remaining high for those expressing PR50+SV2 (1.784). At least 150 cells per condition were evaluated from a pooling of n=3 independent experiments, log-rank Mantel-Cox test, ****p < 0.0001. No significant differences were observed between eGFP or eGFP+SV2 conditions, and groups were merged for this analysis as Control.(E) Kaplan-Meier survival analysis of motor neurons expressing GA50 or GA50+SV2 followed for 10 days. The significant cellular toxicity induced by GA50 is rescued by SV2 replenishment. While the hazard ratio of GA50 was 1.345, this was decreased to 0.7769 for GA50+SV2. At least 200 cells per condition were evaluated from a pooling of n=3 independent experiments, log-rank Mantel-Cox test, ****p < 0.0001.