H . Representative image of a Drosophila Dronc KO intestine 7 days after transfer to 29 oC . Note , transcription of Su ( H ) in large GFP ( - ) , suggestive of an aberrant upregulation of Notch - signalling within fully differentiated ECs . Genotype : w1118 , Su ( H ) GBE - LacZ ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA -

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

H. Representative image of a Drosophila Dronc KO intestine 7 days after transfer to 29 oC. Note, the transcription of Su(H) in large cells GFP (-), suggestive of an aberrant upregulation of Notch-signalling within fully differentiated ECs. Genotype: w1118, Su(H)GBE-LacZ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry


I . Representative image of a 7d Drosophila Dronc heterozygous intestines following a 16hr treatment with paraquat . Note absence of large Su ( H ) positive , GFP ( - ) compared to ( H ) . arrows indicate enlarged depicted in insets . Genotypes : + / - : w1118 , Su ( H ) GBE - LacZ ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / + - / - : w1118 , Su ( H ) GBE - LacZ ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA -

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

I. Representative image of a 7d Drosophila Dronc heterozygous intestines following a 16hr treatment with paraquat. Note the absence of large Su(H) positive, GFP (-) cells compared to (H). The white arrows indicate the enlarged area depicted in the insets. Genotypes: +/-: w1118, Su(H)GBE-LacZ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / + -/-: w1118, Su(H)GBE-LacZ ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry


a . Lipid droplet time course . Representative photomicrographs showing lipid droplet abundance in ARPE19 treated with DFP starting at 7 h maximal at 24 h . Neutral lipids were revealed by BODIPY 493 / 503 . Nuclei are counterstained with Hoescht33342 . Inset shows detail of LDs in a treated with DFP for 24 h . Arrows highlight LD accumulation . Scale bars = 5 μm .

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

a. Lipid droplet time course. Representative photomicrographs showing lipid droplet abundance in ARPE19 cells treated with DFP starting at 7 h and maximal at 24 h. Neutral lipids were revealed by BODIPY 493/503. Nuclei are counterstained with Hoescht33342. Inset shows detail of LDs in a cell treated with DFP for 24 h. Arrows highlight LD accumulation. Scale bars =5 μm.


b . Mitophagy time course . Representative photomicrographs showing time course of DFP treatment in mitophagy reporter ( human ARPE - 19 with stable expression of mito - QC ) . Mitochondrial networks are visible in yellow mitolysosomes as red - only signifying mitophagy . Nuclei are counterstained with Hoescht33342 . Mitophagy appears maximal after 24 h of DFP treatment , with little difference between control 7 h conditions . Inset shows details of mitolysosomes within mitochondrial network of a highly mitophagic following DFP treatment . Arrows highlight mitolysosomes . Scale bar = 5 μm .

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

b. Mitophagy time course. Representative photomicrographs showing time course of DFP treatment in mitophagy reporter cells (human ARPE-19 cells with stable expression of mito-QC). Mitochondrial networks are visible in yellow and mitolysosomes as red-only puncta signifying mitophagy. Nuclei are counterstained with Hoescht33342. Mitophagy appears maximal after 24 h of DFP treatment, with little difference between control and 7 h conditions. Inset shows details of mitolysosomes within the mitochondrial network of a highly mitophagic cell following DFP treatment. Arrows highlight mitolysosomes. Scale bar = 5 μm.


c . Lipid droplets accumulate at mitochondria when are treated with DFP . Representative photomicrographs showing lipid droplet mitochondria in ARPE19 treated with DFP at 7 h at 24 h . Neutral lipids were revealed by BODIPY 493 / 503 mitochondria by Mitotracker . Nuclei are counterstained with Hoescht33342 . Inset shows detail of LDs in close proximity to mitochondria in treated with DFP for 24 h . Scale bar = 5 μm .

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

c. Lipid droplets accumulate at mitochondria when cells are treated with DFP. Representative photomicrographs showing lipid droplet and mitochondria in ARPE19 cells treated with DFP at 7 h and at 24 h. Neutral lipids were revealed by BODIPY 493/503 and mitochondria by Mitotracker. Nuclei are counterstained with Hoescht33342. Inset shows detail of LDs in close proximity to mitochondria in cells treated with DFP for 24 h. Scale bar = 5 μm.


d . of lipid droplet per over time . Lipid droplet was quantified per using a pipeline generated in CellProfiler . n = 3 with at least 50 quantified per biological replicate for each condition . Data represent means ± .

O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-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

d. Quantification of lipid droplet area per cell over time. Lipid droplet area was quantified per cell area using a pipeline generated in CellProfiler. n=3 with at least 50 cells quantified per biological replicate for each condition. Data represent means ±SEM.


e . of mitophagy over time . Mitophagy index per is measured as a of number of mitolysosomes per mitochondrial content . n = 3 with at least 50 quantified per biological replicate for each condition . Data represent means ± .

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

e. Quantification of mitophagy over time. Mitophagy index per cell is measured as a ratio of the number of mitolysosomes per mitochondrial content. n=3 with at least 50 cells quantified per biological replicate for each condition. Data represent means ±SEM.


f . of lipid droplet mitochondria proximity over time . n = 2 with at least 50 quantified per biological replicate for each condition . Ratios between Control 24h DFP ; P < 0 . 05 .

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 B-SMALL_MOLECULE O O O O O O O

f. Quantification of lipid droplet and mitochondria proximity over time. n=2 with at least 50 cells quantified per biological replicate for each condition. Ratios between Control and 24h DFP; P<0.05.


d . DFP - induced Lipid droplet accumulation is dependent on DGAT1 . Representative photomicrographs of ARPE19 treated with DFP ( 1mM ) for 7 h followed by treatment with LD inhibitors ( 5 μM ) as indicated . were fixed after 24 h of DFP treatment . Neutral lipid droplets were stained by BODIPY493 / 503 imaged by confocal microscopy after 24 h of DFP treatment ( ' i ' denotes presence of inhibitor e . g . DGAT1i = DGAT1 - inhibition ) . Scale bar = 5 μm .

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

d. DFP-induced Lipid droplet accumulation is dependent on DGAT1. Representative photomicrographs of ARPE19 cells treated with DFP (1mM) for 7 h followed by treatment with LD inhibitors (5 μM) as indicated. Cells were fixed after 24 h of DFP treatment. Neutral lipid droplets were stained by BODIPY493/503 and imaged by confocal microscopy after 24 h of DFP treatment ('i' denotes the presence of inhibitor e.g. DGAT1i = DGAT1-inhibition). Scale bar = 5 μm.


e . Quantitation of lipid droplets per from experiments in 2d . Data are represented as mean ± ( n = 3 with at least 80 analysed per condition in each biological replicate ) .

O O O O B-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

e. Quantitation of lipid droplets area per cell from experiments in 2d. Data are represented as mean ± SEM (n=3 with at least 80 cells analysed per condition in each biological replicate).


a . ARPE - 19 ULK1 CRISPR KO with / without re - introduction of FLAG - ULK1 were treated with DFP for 24h as indicated . were fixed stained with BODIPY493 / 503 to visualise lipid droplets ( green ) or DAPI to visualise nuclei ( blue ) . Scale bar , 10 μm .

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

a. ARPE-19 ULK1 CRISPR KO cells with/without re-introduction of FLAG-ULK1 were treated with DFP for 24h as indicated. Cells were fixed and stained with BODIPY493/503 to visualise lipid droplets (green) or DAPI to visualise nuclei (blue). Scale bar, 10 μm.


b . from 5a , n = 3 independent experiments , statical by one - way ANOVA . * = P < 0 . 05 ; * * P = < 0 . 01 . Median represented by dashed , quartiles by dotted lines .

O O O O O O O O O O O O O O O O O O O O O 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. Quantification from 5a, n=3 independent experiments, statical analysis by one-way ANOVA. *=P<0.05; **P=<0.01. Median represented by dashed line, quartiles by dotted lines.


c . Authentication of lines in 5a by immunoblot of lysates .

c. Authentication of cell lines in 5a by immunoblot of protein lysates.


f . Representative confocal photomicrographs showing pattern of endogenous mitochondrial NIX in human ARPE - 19 treated with DFP ± DGAT1i / 2i . Samples were immunolabelled with antibodies to NIX ( green ) ATPB ( red ) . Scale bar = 2 μm . g . Representative confocal photomicrograpghs of Cathepsin - reactive endolysosomes in human ARPE - 19 treated with with DFP ± DGAT1i / 2i . Inset shows examples of lysosomal . Scale bar = 5 μm . h . Colocalization quantitation for experiments shown in 6g . One - way ANOVA with Bonferroni ' s post hoc . * * * * P < 0 . 0001 . Median represented by dashed , quartiles by dotted lines n = 2 biological replicates . i . Colocalization quantitation for ULK1 / WIPI2 immunolabelling in human ARPE - 19 treated with DFP ± DGAT1i / 2i . Median represented by dashed , quartiles by dotted lines n = 2 biological replicates . j . Quantitation of lysosome positioning in DFP ± DGAT1i / 2i . One - way ANOVA with Bonferroni ' s post hoc . * * * * = P < 0 . 0001 . Median represented by dashed , quartiles by dotted lines n = 2 biological replicates .

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

f. Representative confocal photomicrographs showing the pattern of endogenous mitochondrial NIX in human ARPE-19 cells treated with DFP ±DGAT1i/2i. Samples were immunolabelled with primary antibodies to NIX (green) and ATPB (red). Scale bar = 2 μm. g. Representative confocal photomicrograpghs of Cathepsin-reactive endolysosomes in human ARPE-19 cells treated with with DFP ±DGAT1i/2i. Inset shows examples of lysosomal phenotypes. Scale bar = 5 μm. h. Colocalization quantitation for experiments shown in 6g. One-way ANOVA with Bonferroni's post hoc test. ****P<0.0001. Median represented by dashed line, quartiles by dotted lines n=2 biological replicates. i. Colocalization quantitation for ULK1/WIPI2 immunolabelling in human ARPE-19 cells treated with DFP ±DGAT1i/2i. Median represented by dashed line, quartiles by dotted lines n=2 biological replicates. j. Quantitation of lysosome positioning in DFP ±DGAT1i/2i. One-way ANOVA with Bonferroni's post hoc test. ****=P<0.0001. Median represented by dashed line, quartiles by dotted lines n=2 biological replicates.


c . Representative photomicrographs of larval neuronal bodies expressing matrix - QC reporter RNAi for mdy or control driven with nSyb - GAL4 . GFP is shown in green , mCherry is shown in magenta . Mitolysosomes ( mCherry - only ) are shown in greyscale . Scale bar = 5 μm .

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

c. Representative photomicrographs of larval neuronal cell bodies expressing the matrix-QC reporter and RNAi for mdy or control driven with nSyb-GAL4. GFP is shown in green, mCherry is shown in magenta. Mitolysosomes (mCherry-only puncta) are shown in greyscale. Scale bar = 5 μm.


d . Quantitative of mitophagy ( mitolysosome per neuronal ) . Data are shown as violin plot with median ( dashed ) quartile range ( dotted lines ) ; n = 5 - 6 , with 23 - 40 per . One - way ANOVA with Šidák ' s post - correction for multiple samples ; * * = P < 0 .

O O O O B-EXP_ASSAY O B-SUBCELLULAR 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 O O O O O O O O O O O O O O

d. Quantitative analysis of mitophagy (mitolysosome per neuronal cell body). Data are shown as violin plot with median (dashed line) and quartile range (dotted lines); n = 5-6 animals, with 23-40 cells per animal. One-way ANOVA with Šidák's post-test correction for multiple samples; **=P<0.01


B Average seizure score of KA - treated WT Fxr2 KO mice over time from t = 0 ( injection time ) to 2 h . * p < 0 . 05 compared to WT mice ( two - way Repeated Measures ANOVA ; interaction effect of time genotype ; F1 , 666 = 10 . 05 , p = 0 . 0016 ) . KA - treated WT mice ( n = 16 ) , KA - treated Fxr2 KO mice ( n = 12 ) . Data are presented as mean ± Standard Error of Mean ( ) . D Average seizure score of pilocarpine - treated WT Fxr2 KO mice over time from t = 0 ( injection time ) to 2 h ( two - way Repeated Measures ANOVA ) . Pilocarpine - treated WT mice ( n = 6 ) , pilocarpine - treated Fxr2 KO mice ( n = 6 ) . Data are presented as mean ± .

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

B Average seizure score of KA-treated WT and Fxr2 KO mice over time from t=0 (injection time) to 2 h. * p < 0.05 compared to WT mice (two-way Repeated Measures ANOVA; interaction effect of time and genotype; F1,666 = 10.05, p = 0.0016). KA-treated WT mice (n=16), KA-treated Fxr2 KO mice (n=12). Data are presented as mean ± Standard Error of the Mean (SEM). D Average seizure score of pilocarpine-treated WT and Fxr2 KO mice over time from t=0 (injection time) to 2 h (two-way Repeated Measures ANOVA). Pilocarpine-treated WT mice (n=6), pilocarpine-treated Fxr2 KO mice (n=6). Data are presented as mean ± SEM.


B PSD - 95 mRNA had lower read in Fxr2 KO samples ( n = 3 ) compared to WT samples ( n = 3 ) ( + adjusted p < 0 . 1 , two - Student ' s t - ) . Data are presented as mean ± .

O B-GENEPROD I-GENEPROD I-GENEPROD 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

B PSD-95 mRNA had lower read counts in Fxr2 KO samples (n=3) compared to WT samples (n=3) (+ adjusted p < 0.1, two-tailed Student's t-test). Data are presented as mean ± SEM.


A Representative Western blot of GluK5 , PSD - 95 , GluN2B mGluR5 . Endogenous GluK5 , PSD - 95 , GluN2B mGluR5 in Fxr2 KO mice ( n = 9 ) compared to WT mice ( n = 11 ) . * p < 0 . 05 , * * p < 0 . 01 , significant difference between WT Fxr2 KO ( two - Student ' s t - : GluK5 : t18 = 0 . 87 , p = 0 . 394 ; PSD - 95 : t18 = 2 . 29 , p < 0 . 05 ; GluN2B : t18 = 3 . 09 , p < 0 . 01 ; mGluR5 : t18 = 3 . 74 , p < 0 . 01 ) . Quantified bands are highlighted with an asterisk or squared bracket . Data are presented as mean ± .

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

A Representative Western blot and quantification of GluK5, PSD-95, GluN2B and mGluR5 proteins. Endogenous GluK5, PSD-95, GluN2B and mGluR5 in Fxr2 KO mice (n=9) compared to WT mice (n=11). * p < 0.05, ** p < 0.01, significant difference between WT and Fxr2 KO (two-tailed Student's t-test: GluK5: t18 = 0.87, p = 0.394; PSD-95: t18 = 2.29, p < 0.05; GluN2B: t18 = 3.09, p < 0.01; mGluR5: t18 = 3.74, p < 0.01). Quantified bands are highlighted with an asterisk or squared bracket. Data are presented as mean ± SEM.


A Bar plot showing phospho - ERK1 / 2 over total ERK1 / 2 in vehicle - KA - treated WT ( closed open black circles , vehicle - treated WT n = 6 ; KA - treated WT n = 6 ) Fxr2 KO mice ( closed open magenta circles , vehicle - treated Fxr2 KO n = 5 ; KA - treated Fxr2 KO n = 6 ) . * * * p < 0 . 001 comparison between vehicle - KA - treated WT ( two - way ANOVA : interaction effect between genotype treatment : F1 , 19 = 24 . 66 , p < 0 . 001 ) . Data are presented as mean ± . B Phospho - ERK1 / 2 in vehicle - pilocarpine - treated WT ( n = 4 - 6 ) Fxr2 KO mice ( n = 4 - 6 ) . Both genotypes responded with long - lasting seizures upon pilocarpine administration * p < 0 . 05 ( two - way ANOVA : main effect for treatment : F1 , 16 = 10 . 98 , p < 0 . 01 ) . Data are presented as mean ± .

O O O O B-EXP_ASSAY O B-GENEPROD O O O 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 O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O 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 O O O O 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 B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-ORGANISM O 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 O O O O O O 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 Bar plot showing phospho-ERK1/2 over total ERK1/2 levels in vehicle- and KA-treated WT (closed and open black circles, vehicle-treated WT n=6; KA-treated WT n=6) and Fxr2 KO mice (closed and open magenta circles, vehicle-treated Fxr2 KO n=5; KA-treated Fxr2 KO n=6). *** p < 0.001 comparison between vehicle- and KA-treated WT (two-way ANOVA: interaction effect between genotype and treatment: F1,19 = 24.66, p < 0.001). Data are presented as mean ± SEM. B Phospho-ERK1/2 levels in vehicle- and pilocarpine-treated WT (n=4-6) and Fxr2 KO mice (n=4-6). Both genotypes responded with long-lasting seizures upon pilocarpine administration * p < 0.05 (two-way ANOVA: main effect for treatment: F1,16 = 10.98, p < 0.01). Data are presented as mean ± SEM.


( b ) Total sequencing of cenRNAs in yeasts . Left ; read per million tracks for CEN16 in wildtype ( black , top ) DAD1E50D humanized ( red , bottom ) yeasts . Right ; of cenRNAs transcripts per million across chromosomes in wildtype DAD1E50D humanized strains . Centromere VI is marked with an asterisk to indicate that plasmid encoding histone plasmid also encodes a CEN6 , which reads likely

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

(b) Total RNA sequencing of cenRNAs in yeasts. Left; read counts per million tracks for CEN16 in wildtype (black, top) and DAD1E50D humanized (red, bottom) yeasts. Right; heatmap of cenRNAs transcripts per million counts across all chromosomes in wildtype and DAD1E50D humanized strains. Centromere VI is marked with an asterisk to indicate that the plasmid encoding the histone plasmid also encodes a CEN6 sequence, which reads likely emanant


( c ) RT - PCRs of indicated RNAs . Lane 1 is genomic , lane 2 is wildtype shuffle ( yDT67 ) , lane 3 is BY4742 , lane 4 is DAD1E50D humanized , lane 5 is scc4D65Y humanized , lanes 6 - 9 are same as 2 - 5 , but without reverse transcriptase added , lane 10 is no .

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

(c) RT-PCRs of the indicated RNAs. Lane 1 is genomic DNA, lane 2 is wildtype shuffle strain (yDT67), lane 3 is BY4742, lane 4 is DAD1E50D humanized, lane 5 is scc4D65Y humanized, lanes 6-9 are the same as 2-5, but without reverse transcriptase added, lane 10 is no DNA.


( d ) Example from strains with tags on microtubule organizing center ( Spc110 - GFP ) kinetochore ( Nuf2 - RFP ) . were taken from cultures in mid - log phase of growth .

O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O

(d) Example images from strains with fluorescent tags on the microtubule organizing center (Spc110-GFP) and the kinetochore (Nuf2-RFP). Images were taken from cultures in mid-log phase of growth.


( e ) DASH mutants increase histone - humanization in scc4D65Y background . Humanization rates of scc4D65Y mutant with without DAD1E50D or DAM1N80Y . Significance of mean difference in 5 - FOAR frequency was determined with Mann - Whitney . Each dot represents one biological replicate ( n = 8 ) , central band represents median , box extends from 25th to 75th percentiles , whiskers represent minimum maximum .

O O O B-SUBCELLULAR O O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-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 O O O O O O O O O O

(e) DASH mutants increase histone-humanization in the scc4D65Y background. Humanization rates of the scc4D65Y mutant with and without DAD1E50D or DAM1N80Y. Significance of the mean difference in 5-FOAR frequency was determined with the Mann-Whitney test. Each dot represents one biological replicate (n = 8), central band represents the median, box extends from the 25th to 75th percentiles, and whiskers represent the minimum and maximum.


( f ) Ploidy by flow cytometry in scc4D65Y humanized mutants from panel d . Flow cytometry data from humanized lineages yHs5 yHs7 are shown for aneuploid euploid controls , respectively .

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

(f) Ploidy analysis by flow cytometry in the scc4D65Y humanized mutants from panel d. Flow cytometry data from humanized lineages yHs5 and yHs7 are shown for aneuploid and euploid controls, respectively.


( a ) Growth assays of wild type , ipl1 - 2 mutants , ipl1 - 2 kinetochore double mutants at permissive ( 24ºC ) non - permissive ( 32ºC ) . Ten - fold serial dilutions of ~ 1 . 0 OD600 yeast cultures were spotted onto YPD solid media grown at indicated temperatures for four three days , respectively .

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

(a) Growth assays of wild type, ipl1-2 mutants, and ipl1-2 kinetochore double mutants at permissive (24ºC) and non-permissive temperature (32ºC). Ten-fold serial dilutions of ~1.0 OD600 yeast cultures were spotted onto YPD solid media and grown at the indicated temperatures for four and three days, respectively.


( b ) Flow cytometry of wild type , ipl1 - 2 mutants , ipl1 - 2 kinetochore double mutants at permissive ( 24 ˚ C ) non - permissive ( 30ºC ) . Yeast cultures were grown to logarithmic phase at 24 ˚ C divided grown at 24 ˚ C 30 ˚ C for 6 hours before were collected processed for content using Sytox Green stain .

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

(b) Flow cytometry analysis of wild type, ipl1-2 mutants, and ipl1-2 kinetochore double mutants at the permissive (24˚C) and non-permissive temperature (30ºC). Yeast cultures were grown to logarithmic phase at 24˚C and divided and grown at 24˚C and 30˚C for 6 hours before cells were collected and processed for DNA content analysis using Sytox Green stain.


( c ) Growth assays of wild type , mad3 ∆ mutants , mad3 ∆ DAD1E50D double mutants on YPD with without 30 µg / ml benomyl . Ten - fold serial dilutions of ~ 1 . 0 OD600 yeast cultures were spotted onto indicated solid media grown at 30 ˚ C for three days .

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

(c) Growth assays of wild type, mad3∆ mutants, and mad3∆ DAD1E50D double mutants on YPD with and without 30 µg/ml benomyl. Ten-fold serial dilutions of ~1.0 OD600 yeast cultures were spotted onto the indicated solid media and grown at 30˚C for three days.


( b ) Histone - humanization for various dam1N80X mutants . 5 - FOA is used to counter - select yeast histone plasmid , forcing growth with human histone plasmid . Yeast were serially diluted from a starting culture of 1 . 0 OD600 .

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

(b) Histone-humanization assay for various dam1N80X mutants. 5-FOA is used to counter-select the yeast histone plasmid, forcing growth with the human histone plasmid. Yeast were serially diluted from a starting culture of 1.0 OD600.


( f ) Rupture force from experiments at three concentrations of DASH incubated with beads . Briefly , 3 . 5 pM of beads were incubated with indicated concentrations of DASH ( representing a of DASH to beads of 714 : 1 , 1428 : 1 , 2860 : 1 ) . These DASH - decorated beads were then washed before suspending in a reaction mix containing free tubulin 5 nM free DASH . Two way ANOVA of mean difference in rupture force are shown with multiple corrections ( false discovery rate ) . Each dot represents an rupture force measurement ( n ≥ 21 ; exact number shown below each box between parentheses ) , central band represents median , box extends from 25th to 75th percentiles , whiskers represent minimum maximum .

O O O B-EXP_ASSAY I-EXP_ASSAY 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 B-SUBCELLULAR 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 B-GENEPROD 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 O O O O O O O O O O O O O O O O O O O O O

(f) Rupture force assay from experiments at three concentrations of DASH complex incubated with the beads. Briefly, 3.5 pM of beads were incubated with the indicated concentrations of DASH complex (representing a ratio of DASH complex to beads of 714:1, 1428:1, and 2860:1). These DASH-decorated beads were then washed before suspending in a reaction mix containing free tubulin and 5 nM free DASH complex. Two way ANOVA tests of the mean difference in rupture force are shown with multiple test corrections (false discovery rate). Each dot represents an individual rupture force measurement (n ≥ 21; exact number shown below each box between parentheses), central band represents the median, box extends from the 25th to 75th percentiles, and whiskers represent the minimum and maximum.


( g ) Survival curves for each DASH are shown at indicated concentrations . Log - rank were performed to whether survival curves were significantly different from one another . Error bars represent standard error are displayed as shaded .

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

(g) Survival curves for each DASH complex are shown at the indicated concentrations. Log-rank tests were performed to test whether the survival curves were significantly different from one another. Error bars represent the standard error and are displayed as the shaded area.


( a ) Sporulation efficiency after five days in sporulation medium at 25 ˚ C . Example are shown to left for both wildtype DAD1E50D SK1 strains . Right , of average sporulation efficiency from . Two unpaired t of mean difference in sporulation efficiency . Each dot represents fraction of sporulated in a single field of view ( technical replicates ; n ≥ 15 ) , central band represents median , box extends from 25th to 75th percentiles , whiskers represent minimum maximum .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 O O O O O O 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) Sporulation efficiency after five days in sporulation medium at 25 ˚C. Example micrographs are shown to the left for both wildtype and DAD1E50D SK1 strains. Right, quantification of average sporulation efficiency from all micrographs. Two tailed unpaired t test of the mean difference in sporulation efficiency. Each dot represents the fraction of cells sporulated in a single field of view (technical replicates; n ≥15), central band represents the median, box extends from the 25th to 75th percentiles, and whiskers represent the minimum and maximum.


( b ) Spore viability . tetrads were micromanipulated on a agar surface spores were dissected arrayed . Genotype was determined by PCR genotyping of DAD1 locus . from tetrad dissections performed , total are shown below . one - probability of observing 51 % spore viability in DAD1E50D SK1 over a total of 139 tetrads is p < 0 . 000001 , assuming a spore viability of ~ 90 % from wild type SK1 .

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

(b) Spore viability assay. Individual tetrads were micromanipulated on a agar surface and spores were dissected and arrayed. Genotype was determined by PCR genotyping of the DAD1 locus. Quantification from all tetrad dissections performed, total are shown below. The one-tailed probability of observing 51% spore viability in DAD1E50D SK1 over a total of 139 tetrads is p < 0.000001, assuming a spore viability of ~90% from wild type SK1.


Double whole mount in situ hybridization ( WM - ISH ) to detect her6 ( green ) her9 ( magenta ) ; coronal view ( left panel ) transversal section ( right panel ) , scale bar 20μm ; 30 - 32hpf ; annotations denote anterior ( A ) , posterior ( P ) , otic vesicle ( ov ) , dorsal ( D ) ventral ( V ) .

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

Double fluorescent whole mount in situ hybridization (WM-ISH) to detect her6 (green) and her9 (magenta); coronal view (left panel) and transversal section (right panel), scale bar 20μm; 30-32hpf; annotations denote anterior (A), posterior (P), otic vesicle (ov), dorsal (D) and ventral (V).


Representative time series example of Her6 : : Venus expression during development , in midbrain hindbrain . Confocal represented as 2D Maximum projection ; longitudinal view ; scale bar 50μm ; otic vesicle ( ov ) ; also included in Movie EV1 . r1 : rhombomere 1 , r2 : rhombomere 2 , r3 : rhombomere 3 , r4 : rhombomere 4 , r5 : rhombomere 5 , r6 : rhombomere 6 .

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

Representative time series example of Her6::Venus expression during development, in the midbrain and hindbrain. Confocal images represented as 2D Maximum projection; longitudinal view; scale bar 50μm; otic vesicle (ov); also included in Movie EV1. r1: rhombomere 1, r2: rhombomere 2, r3: rhombomere 3, r4: rhombomere 4, r5: rhombomere 5, r6: rhombomere 6.


mean of Her6 : : Venus per rhombomere over development grouped by expression level , related to r1 - r6 regions in panel ( c ) : ( d ) r1 r2 ; ( e ) r3 r4 ; ( f ) r5 r6 .

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

Intensity mean of Her6::Venus per rhombomere area over development grouped by expression level, related to the r1-r6 regions in panel (c) : (d) r1 and r2; (e) r3 and r4; (f) r5 and r6.


Transversal view of r6 in Her6 : : Venus over time ; Her6 : : Venus expression domains : a ventral domain ( arrows ) a more dorsal lateral domain ( arrowheads ) ; caax - mRFP was used as marker ( magenta ) ; scale bars 20μm ; at 30 - 40hpf are maximum projection of 4 z - stacks from Movie EV2 .

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

Transversal view of r6 in Her6::Venus embryos over time; Her6::Venus protein expression domains: a ventral domain (arrows) and a more dorsal lateral domain (arrowheads); the caax-mRFP was used as membrane marker (magenta); scale bars 20μm; images at 30-40hpf are maximum projection of 4 z-stacks from Movie EV2.


of Her6 : : Venus ( + ) number ( green ) compared to total number ( black ) over development .

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

Quantification of Her6::Venus(+) cell number (green) compared to total cell number (black) over development.


Proportional changes in Her6 : : Venus ( + ) numbers during development ; bars indicate median interquartile range of collected from 3 different z - stacks per one per condition ; one - way ANOVA with Bonferroni multiple comparison with significance p < 0 .

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

Proportional changes in Her6::Venus(+) cell numbers during development; bars indicate median and interquartile range of counts collected from 3 different z-stacks per embryo and one embryo per condition; one-way ANOVA with Bonferroni multiple comparison test with significance p<0.01


abundance of Her6 : : Venus in homozygous at different stages during development measured by correlation spectroscopy ( FCS ) ; bars indicate median interquartile range of ( 19hpf : 6 , 50 ) , ( 29hpf : 6 , 86 ) , ( 33hpf : 5 , 76 ) , ( 35hpf : 6 , 59 ) ( 48hpf : 6 , 89 ) ; Kruskal - Wallis with Dunn ' s multiple comparison , significance p < 0 . 05 * , p < 0 . 001 * * * , p < 0 . 0001 * * * * .

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

Nuclear abundance and nuclear concentration of Her6::Venus protein in homozygous embryos at different stages during development measured by fluorescence correlation spectroscopy (FCS); bars indicate median and interquartile range of (19hpf: 6 embryos, 50 cells), (29hpf: 6 embryos, 86 cells), (33hpf: 5 embryos, 76 cells), (35hpf: 6 embryos, 59 cells) and (48hpf: 6 embryos, 89 cells); Kruskal-Wallis with Dunn's multiple comparison test, significance p<0.05*, p<0.001***, p<0.0001****.


3D confocal representative of a single neural progenitor ( red arrow ) tracked over time , data starting from 34hpf ; scale bar 10μm , from Movie EV3 .

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

3D confocal representative images of a single neural progenitor cell (red arrow) tracked over time, data starting from 34hpf; scale bar 10μm, images from Movie EV3.


Single time series of Her6 : : Venus , mKeima - H2B Her6 : : Venus signal normalized by mKeima - H2B corresponding to in ( c ) .

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

Single cell time series of Her6::Venus, mKeima-H2B and Her6::Venus signal normalized by mKeima-H2B corresponding to the cell in (c).


Chromogenic WM - ISH of miR - 9 using miR - 9 LNA 5 ' - Dig observed at different stages during development ; longitudinal view , anterior to left .

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

Chromogenic WM-ISH of miR-9 using miR-9 LNA 5'-Dig observed at different stages during development; longitudinal view, anterior to the left.


Transverse section of double WM - ISH for her6 ( green ) mir - 9 - 4 ( magenta ) imaged in fixed at 31hpf ; scale bar 30μm .

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

Transverse section of double fluorescent WM-ISH for her6 (green) and mir-9-4 (magenta) imaged in fixed embryo at 31hpf; scale bar 30μm.


Confocal imaging of CTRL MBSm showing Her6 : : Venus expression in hindbrain , rhombomeres 3 to 6 ( r3 - r6 ) over course of development ; longitudinal view , scale 30μm , otic vesicle ( ov ) . are representing 2D maximum projection .

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

Confocal imaging of CTRL and MBSm embryo showing Her6::Venus expression in the hindbrain, rhombomeres 3 to 6 (r3-r6) over the course of development; longitudinal view, scale 30μm, otic vesicle (ov). Images are representing 2D maximum projection.


Her6 : : Venus expression ( red arrows ) in hindbrain , rhombomere 6 of live CTRL MBSm at 52hpf ; edge of ventricular zone shown in yellow ; transversal view ; scale 30μm .

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

Her6::Venus expression (red arrows) in hindbrain, rhombomere 6 of live CTRL and MBSm embryos at 52hpf; edge of ventricular zone shown in yellow; transversal view; scale 30μm.


Her6 : : Venus abundance in CTRL versus MBSm homozygous observed in hindbrain , r6 between 34hpf 69hpf ; bars indicate median interquartile range of ( 34hpf CTRL : 36 , 1 ) , ( 34hpf MBSm : 35 , 1 ) , ( 48 ± 2hpf CTRL : 172 , 7 ) , ( 48 ± 2hpf MBSm : 124 , 5 ) , ( 56 ± 2hpf CTRL : 69 , 4 ) , ( 56 ± 2hpf MBSm : 31 , 2 ) , ( 69hpf CTRL : 12 , 1 ) , ( 69hpf MBSm : 6 , 2 ) ; Kruskal - Wallis with Dunn ' s multiple comparison , significance : p < 0 . 05 * , p < 0 . 001 * * * , p < 0 . 0001 * * * * .

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

Her6::Venus protein abundance in CTRL versus MBSm homozygous embryos observed in hindbrain, r6 between 34hpf and 69hpf; bars indicate median and interquartile range of (34hpf CTRL: 36 cells, 1 embryo), (34hpf MBSm: 35 cells, 1 embryo), (48±2hpf CTRL: 172 cells, 7 embryos), (48±2hpf MBSm: 124 cells, 5 embryos), (56±2hpf CTRL: 69 cells, 4 embryos), (56±2hpf MBSm: 31 cells, 2 embryos), (69hpf CTRL: 12 cells, 1 embryo), (69hpf MBSm: 6 cells, 2 embryos); Kruskal-Wallis with Dunn's multiple comparison test, significance: p<0.05*, p<0.001***, p<0.0001****.


Comparative profiles of Her6 abundance over development in CTRL MBSm quantified as median of single abundance data in ( f ) .

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

Comparative profiles of Her6 protein abundance over development in CTRL and MBSm embryos quantified as median of single cell abundance data in (f).


Representative examples of single oscillators observed in CTRL ( a ) MBSm ( b ) imaged from 34hpf onwards ; time series represent Her6 : : Venus expression relative to mKeima - H2B ( top panel ) detrended relative signal ( bottom panel ) ; parameters reported for log - likelihood ( LLR ) period correspond to KOUosc .

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

Representative examples of single cell oscillators observed in CTRL (a) and MBSm (b) embryos imaged from 34hpf onwards; time series represent Her6::Venus expression relative to mKeima-H2B (top panel) and detrended relative signal (bottom panel); parameters reported for log-likelihood ratio (LLR) and period correspond to KOUosc .


Pairwise comparing proportion of oscillators in CTRL versus MBSm indicated as dots ; paired t with two tail significance p < 0 . 01 * * .

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

Pairwise analysis comparing proportion of oscillators in CTRL versus MBSm embryos indicated as dots; paired t test with two tail significance p<0.01**.


Pairwise comparing periods observed in CTRL versus MBSm oscillatory ; dots indicate median per experiment per condition ; paired t with two tail not significant .

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

Pairwise analysis comparing periods observed in CTRL versus MBSm oscillatory cells; dots indicate median per experiment per condition; paired t test with two tail not significant.


Representative examples of detrended Her6 : : Venus relative to mKeima - H2B in non - oscillatory single observed in CTRL ( f ) MBSm ( g ) imaged from 34hpf onwards .

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

Representative examples of detrended Her6::Venus relative to mKeima-H2B in non-oscillatory single cells observed in CTRL (f) and MBSm (g) embryos imaged from 34hpf onwards.


Representative examples of triple whole mount WM - ISH labelling of gfap ( green ) , elavl3 ( magenta ) her6 ( grey ) domains of expression ( top panels ) in hindbrain rhombomere6 ( r6 ) in wild - type observed at 34hpf ; merged indicate how her6 expression domain overlaps with progenitor zone ( NP = gfap ( + ) / elavl3 ( - ) ) , transition zone ( T = gfap ( + ) / elavl3 ( + ) ) neurogenic zone ( N = gfap ( - ) / elavl3 ( + ) ) ; ( bottom - right panel ) schematic representation showing her6 domain spanning NP , T N zones with of distances from dorsal ; transversal view ; annotations denote dorsal ( D ) ventral ( V ) ; scale bar 30μm .

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

Representative examples of triple fluorescent whole mount WM-ISH labelling of gfap (green), elavl3 (magenta) and her6 (grey) domains of expression (top panels) in hindbrain rhombomere6 (r6) in wild-type embryo observed at 34hpf; merged images indicate how the her6 expression domain overlaps with the progenitor zone (NP=gfap(+)/elavl3(-)), transition zone (T=gfap(+)/elavl3(+)) and neurogenic zone (N=gfap(-)/elavl3(+)); (bottom-right panel) schematic representation showing the her6 domain spanning the NP, T and N zones with quantification of distances from dorsal; transversal view; annotations denote dorsal (D) and ventral (V); scale bar 30μm.


Single molecule in situ hybridization ( smFISH ) showing her6 ( green ) , elavl3 ( magenta ) dapi ( blue ) obtained from hindbrain ( r6 ) sections of wild type at 34hpf ; head arrows indicates examples of co - existence of transcriptional active sites for her6 elavl3 ; scale bar 3μm .

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

Single molecule fluorescent in situ hybridization (smFISH) showing her6 (green), elavl3 (magenta) and dapi nuclear staining (blue) obtained from hindbrain (r6) sections of wild type embryo at 34hpf; head arrows indicates examples of co-existence of transcriptional active sites for her6 and elavl3; scale bar 3μm.


Triple WM - ISH for gfap ( green ) elavl3 ( magenta ) her6 ( grey ) in hindbrain ( r6 ) of CTRL ( top ) MBSm ( bottom ) at 52hpf ; dorsal ( D ) ; ventral ( V ) ; scale bar 30μm . Magnification of inset from merged gfap / elavl3 in ( c ) showing CTRL ( top ) MBSm ( bottom ) with corresponding NP / T / N NP / T zones respectively ; dorsal ( D ) ; ventral ( V ) ; scale bar 30μm . Normalized mean of elavl3 gfap along DV axis spanning NP / T / N zones in CTRL NP / T zones in MBSm respectively ; region of interest ( ROI ) delineates high elavl3 versus gfap in CTRL ( T N zones ) while in MBSm only T zone is observed , overlap of elavl3 gfap mean peaks .

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

Triple fluorescent WM-ISH for gfap (green) and elavl3 (magenta) and her6 (grey) in the hindbrain (r6) of CTRL (top) and MBSm (bottom) embryos at 52hpf; dorsal (D); ventral (V); scale bar 30μm. Magnification of inset from merged gfap/elavl3 in (c) showing CTRL (top) and MBSm (bottom) embryos with corresponding NP/T/ N and NP/T zones respectively; dorsal (D); ventral (V); scale bar 30μm. Normalized intensity mean of elavl3 and gfap along the DV axis spanning the NP/T/N zones in CTRL and NP/T zones in MBSm respectively; region of interest (ROI) delineates high elavl3 versus gfap in CTRL (T and N zones) while in MBSm only the T zone is observed, overlap of elavl3 and gfap intensity mean peaks.


Normalized mean of elavl3 gfap intensities in ROI observed in : ( f ) uninjected ( UI ) ( 5 , 62 slices ) , ( g ) control ( CTRL ) ( 5 , 35 slices ) ( h ) MBSm ( 8 , 62 slices ) conditions ; bars represent mean ; multiple t - with Benjamini , Krieger Yekuteli discovery , significance : p < 0 . 05 * , p < 0 . 001 * * * , p < 0 . 0001 * * * * .

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

Normalized mean of elavl3 and gfap intensities in ROI observed in: (f) uninjected (UI) (5 embryos, 62 slices), (g) control (CTRL) (5 embryos, 35 slices) and (h) MBSm (8 embryos, 62 slices) conditions; bars represent mean and SEM; multiple t-test with Benjamini, Krieger and Yekuteli discovery, significance: p<0.05*, p<0.001***, p<0.0001****.


Transverse sections of double WM - ISH for gfap ( green ) neuroD4 ( blue ) in hindbrain ( r6 ) of CTRL ( top ) MBSm ( bottom ) at 52hpf ; dorsal ( D ) ; ventral ( V ) ; scale bar 30μm .

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

Transverse sections of double fluorescent WM-ISH for gfap (green) and neuroD4 (blue) in the hindbrain (r6) of CTRL (top) and MBSm (bottom) embryos at 52hpf; dorsal (D); ventral (V); scale bar 30μm.


Chromogenic WM - ISH showing neuroD4 expression in : uninjected ( UI ) 51 / 56 ( 91 % ) , CTRL 50 / 54 ( 93 % ) mutant ( MBSm ) ; 36 / 65 ( 55 % ) ; longitudinal view , scale bar 100μm .

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

Chromogenic WM-ISH showing neuroD4 expression intensity in: uninjected (UI) 51/56 embryos (91%), CTRL 50/54 embryos (93%) and mutant (MBSm); 36/65 embryos (55 %); longitudinal view, scale bar 100μm.


( A ) Murine Ntras enrichment in endothelial versus other heart - derived types ( data acquired from GSE95755 ( n = 4 mice ) . Data information : In ( A , data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( A ) One - way

O O O B-ORGANISM B-GENEPROD O O B-CELL_TYPE O O B-TISSUE 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 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) Murine Ntras enrichment in endothelial cells versus other heart-derived cell types (data acquired from GSE95755 (n = 4 mice). Data information: In (A, data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (A) One-way ANOVA


( B ) Changes in lncRNA expression from HUVECs exposed to hypoxia ( 0 . 2 % O2 for 12 h or 24 h ) , determined by ribo - minus deep sequencing ( data acquired from GSE107033 ; NTRAS is highlighted ( n = 2 independent biological replicates ) .

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

(B) Changes in lncRNA expression from HUVECs exposed to hypoxia (0.2 % O2 for 12 h or 24 h), determined by ribo-minus RNA deep sequencing (data acquired from GSE107033 ; NTRAS is highlighted (n = 2 independent biological replicates).


( C ) Validation of hypoxia - induced NTRAS expression at indicated time points by RT - qPCR ( n = 4 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . two - unpaired t -

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

(C) Validation of hypoxia-induced NTRAS expression at the indicated time points by RT-qPCR (n = 4 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. two-tailed unpaired t-test


( D ) Subcellular localization of NTRAS control transcripts , assayed by - fractionation of HUVECs RT - qPCR ( n = 3 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . two - unpaired t -

O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O B-CELL_LINE B-EXP_ASSAY I-EXP_ASSAY I-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 O O O O O O O O O O O O O O O O O O O

(D) Subcellular localization of NTRAS and control transcripts, assayed by nuclear-cytoplasmic fractionation of HUVECs and RT-qPCR (n = 3 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. two-tailed unpaired t-test


( E ) FITC - dextran - in vitro permeability comparing control NTRAS - silenced HUVECs ( n = 5 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . two - unpaired t - ,

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) FITC-dextran-based in vitro permeability comparing control and NTRAS-silenced HUVECs (n = 5 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. two-tailed unpaired t-test,


( F ) Cumulative in vitro sprout lengths of control NTRAS - silenced HUVECs under basal conditions VEGFA stimulation ( n = 3 - 10 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(F) Cumulative in vitro sprout lengths of control and NTRAS-silenced HUVECs under basal conditions and VEGFA stimulation (n = 3-10 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( G ) cycle in Ntras - silenced H5V ( n = 3 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ( G ) two - way ANOVA ,

O O O B-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Cell cycle analysis in Ntras-silenced H5V cells (n = 3 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (G) two-way ANOVA,


( H ) TMR - dextran - assessment of vascular permeability in vivo , comparing heart homogenates from control Ntras - silenced mice . Data normalized to organ ( n = 11 - 12 mice per group ) . Experimental outline on left . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . H ) two - unpaired t -

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

(H) TMR-dextran-based assessment of vascular permeability in vivo, comparing heart homogenates from control and Ntras-silenced mice. Data normalized to organ and body weight (n = 11-12 mice per group). Experimental outline on the left. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. H) two-tailed unpaired t-test


( I ) Survival of control Ntras - silenced mice under basal conditions ( black , n = 21 - 23 mice per group ) in hind limb ischemia ( HLI ; red , n = 8 mice per group ) . Number of survivors is indicated ; experimental outline on left . Data information : ( I ) Mantel - Cox - .

O O O B-EXP_ASSAY O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O B-DISEASE I-DISEASE I-DISEASE O B-DISEASE 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

(I) Survival of control and Ntras-silenced mice under basal conditions (black, n = 21-23 mice per group) and in hind limb ischemia (HLI; red, n = 8 mice per group). Number of survivors is indicated; experimental outline on the left. Data information: (I) Mantel-Cox-test.


( A ) Separation of mock proteinase K - treated HeLa extracts by sucrose density gradient ultracentrifugation followed by NTRAS detection using RT - qPCR . Fractions 1 14 represent top bottom of gradient , respectively ( n = 1 ) .

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

(A) Separation of mock and proteinase K-treated HeLa nuclear extracts by sucrose density gradient ultracentrifugation followed by NTRAS detection using RT-qPCR. Fractions 1 and 14 represent top and bottom of the gradient, respectively (n = 1).


( B ) Enrichment of NTRAS by antisense affinity selection from HeLa lysate followed by RT - qPCR , comparing a control an NTRAS - specific probe ( n = 5 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * * p < 0 . 001 . two - unpaired t - .

O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 O O O O O O

(B) Enrichment of NTRAS by antisense affinity selection from HeLa cell lysate followed by RT-qPCR, comparing a control and an NTRAS-specific probe (n = 5 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, ***p < 0.001. two-tailed unpaired t-test.


( D ) RT - qPCR - validation of NTRAS - hnRNPL - interaction by anti - hnRNPL RIPs from HUVEC fractions ( n = 6 independent biological replicates ) . Representative western blot on right . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * * p < 0 . 001 . two - unpaired t - .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE 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 O O O O O O O O O O O O O O O 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) RT-qPCR-based validation of NTRAS-hnRNPL-interaction by anti-hnRNPL RIPs from HUVEC nuclear fractions (n = 6 independent biological replicates). Representative western blot on the right. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, ***p < 0.001. two-tailed unpaired t-test.


( E ) RT - qPCR of NTRAS expression in HUVECs upon silencing of hnRNPL ( n = 4 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * * p < 0 . 001 . two - unpaired t - .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE 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 O O O

(E) RT-qPCR analysis of NTRAS expression in HUVECs upon silencing of hnRNPL (n = 4 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, ***p < 0.001. two-tailed unpaired t-test.


( F ) RPLP0 - normalized NTRAS - CA16 motif expression after hnRNPL silencing determined by RT - PCR ( n = 4 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * * p < 0 . 001 . two - unpaired t - .

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

(F) RPLP0-normalized NTRAS-CA16 motif expression after hnRNPL silencing determined by RT-PCR (n = 4 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, ***p < 0.001. two-tailed unpaired t-test.


( H ) NTRAS hnRNPL co - regulated alternative splicing ( AS ) events . Data points with an FDR < 0 . 05 are circled ( n = 2 independent biological replicates ) .

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

(H) NTRAS and hnRNPL co-regulated alternative splicing (AS) events. Data points with an FDR < 0.05 are circled (n = 2 independent biological replicates).


( I ) RT - PCR - of TJP1 exon 20 inclusion upon silencing of NTRAS in HUVECs ( n = 7 independent biological replicates ) . Representative agarose gels on right . Data information : , I , data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * * p < 0 . 001 . I , two - unpaired t - .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O B-GENEPROD O B-CELL_LINE 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 O O O O O O O O O O O O O O O O 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) RT-PCR-based analysis of TJP1 exon 20 inclusion upon silencing of NTRAS in HUVECs (n = 7 independent biological replicates). Representative agarose gels on the right. Data information: , I, data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, ***p < 0.001. I, two-tailed unpaired t-test.


( J ) RT - PCR - of TJP1 exon 20 inclusion upon silencing of hnRNPL in HUVECs ( n = 4 independent biological replicates ) . Representative agarose gels on right . Data information : , J ) data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * * p < 0 . 001 . J ) two - unpaired t - .

(J) RT-PCR-based analysis of TJP1 exon 20 inclusion upon silencing of hnRNPL in HUVECs (n = 4 independent biological replicates). Representative agarose gels on the right. Data information: , J) data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, ***p < 0.001. J) two-tailed unpaired t-test.


( B ) In vitro splicing efficiency of TJP1 splice substrate , comparing mock , NTRAS - depleted , NTRAS - CA16 motif add - back conditions ( n = 7 - 12 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . ( B two - unpaired t - ,

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

(B) In vitro splicing efficiency of the TJP1 splice substrate, comparing mock, NTRAS-depleted, and NTRAS-CA16 motif add-back conditions (n = 7-12 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. (B two-tailed unpaired t-test,


( C ) Co - precipitation of TJP1 pre - mRNA in anti - hnRNPL RIPs , using lysates from control NTRAS - silenced HUVECs ( n = 6 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - paired t -

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-precipitation of TJP1 pre-mRNA in anti-hnRNPL RIPs, using nuclear lysates from control and NTRAS-silenced HUVECs (n = 6 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed paired t-test


( D ) Co - precipitation of TJP1 pre - mRNA in anti - hnRNPL RIPs , using lysates from control NTRAS - overexpressing ( n = 5 independent biological replicates ) . Representative western blot on right . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - paired t - ,

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD 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 O O O O O O O O O O O O O O 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) Co-precipitation of TJP1 pre-mRNA in anti-hnRNPL RIPs, using nuclear lysates from control and NTRAS-overexpressing cells (n = 5 independent biological replicates). Representative western blot on the right. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed paired t-test,


( E ) Co - precipitation of TJP1 pre - mRNA in anti - hnRNPL RIPs , using lysates from control NTRAS - CA16 motif overexpressing ( n = 5 independent biological replicates ) . Representative western blot on right . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - paired t - ,

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

(E) Co-precipitation of TJP1 pre-mRNA in anti-hnRNPL RIPs, using nuclear lysates from control and NTRAS-CA16 motif overexpressing cells (n = 5 independent biological replicates). Representative western blot on the right. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed paired t-test,


( F ) RT - PCR - of TJP1 exon 20 inclusion upon NTRAS overexpression overexpression of NTRAS - CA16 motif ( n = 8 independent biological replicates ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . one - way ANOVA .

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

(F) RT-PCR-based analysis of TJP1 exon 20 inclusion upon NTRAS overexpression and overexpression of the NTRAS-CA16 motif (n = 8 independent biological replicates). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. one-way ANOVA.


( G ) Endothelial resistance of NTRAS - silenced HUVECs ( n = 3 - 5 independent biological replicates ) , analyzed by electrical - substrate impedance sensing ( ECIS ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - unpaired t -

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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

(G) Endothelial resistance of NTRAS-silenced HUVECs (n = 3-5 independent biological replicates), analyzed by electrical cell-substrate impedance sensing (ECIS). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed unpaired t-test


( H ) Endothelial resistance of hnRNPL - silenced HUVECs ( n = 3 independent biological replicates ) , analyzed by ECIS . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - unpaired t - ,

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE 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

(H) Endothelial resistance of hnRNPL-silenced HUVECs (n = 3 independent biological replicates), analyzed by ECIS. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed unpaired t-test,


( I ) of TJP1 exon 20 inclusion by RT - PCR upon transfection of HUVECs with a control SSO or an SSO masking exon 20 - intron 20 boundary ( E20 SSO ) ( n = 9 independent biological replicates ) . Representative agarose gel on right . Schematic outline at top right . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - unpaired t - ,

O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O O O 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-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 O O O O O O O O O O O O O O O O O O O O

(I) Analysis of TJP1 exon 20 inclusion by RT-PCR upon transfection of HUVECs with a control SSO or an SSO masking the exon 20 - intron 20 boundary (E20 SSO) (n = 9 independent biological replicates). Representative agarose gel on the right. Schematic outline at the top right. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed unpaired t-test,


( J ) Endothelial resistance of control SSO - or E20 SSO - transfected HUVECs ( n = 4 independent biological replicates ) , analyzed by ECIS . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 . two - unpaired t - ,

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-CELL_LINE 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

(J) Endothelial resistance of control SSO- or E20 SSO-transfected HUVECs (n = 4 independent biological replicates), analyzed by ECIS. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01. two-tailed unpaired t-test,


( A ) Validation of Ntras - hnRNPL - interaction by anti - hnRNPL RIPs from H5V lysates , followed by RT - qPCR ( n = 4 independent biological replicates ) . Representative western blot on right . Data information : In ( A , data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( A ) two - paired t -

O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Validation of Ntras-hnRNPL-interaction by anti-hnRNPL RIPs from H5V lysates, followed by RT-qPCR (n = 4 independent biological replicates). Representative western blot on the right. Data information: In (A, data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. (A) two-tailed paired t-test


( B ) RT - PCR - of murine Tjp1 exon 20 inclusion in cardiac tissue from control Ntras - silenced mice ( n = 8 mice per group ) . Experimental outline on left . Data information : In data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( B , two - unpaired t - ,

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

(B) RT-PCR-based analysis of murine Tjp1 exon 20 inclusion in cardiac tissue from control and Ntras-silenced mice (n = 8 mice per group). Experimental outline on the left. Data information: In data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. (B, two-tailed unpaired t-test,


( D ) Co - precipitation of Ntras in anti - hnRNPL RIPs using whole heart lysates from NtrasCA / CA Ntras ∆ CA / ∆ CA mice ( n = 3 - 4 mice per group ) . Representative western blot on right . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . two - unpaired t - ,

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

(D) Co-precipitation of Ntras in anti-hnRNPL RIPs using whole heart lysates from NtrasCA/CA and Ntras∆CA/∆CA mice (n = 3-4 mice per group). Representative western blot on the right. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. two-tailed unpaired t-test,


( E ) RT - PCR - of TJP1 exon 20 inclusion in hearts from NtrasCA / CA Ntras ∆ CA / ∆ CA mice ( n = 9 - 14 mice per group ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . two - unpaired t - ,

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

(E) RT-PCR-based analysis of TJP1 exon 20 inclusion in hearts from NtrasCA/CA and Ntras∆CA/∆CA mice (n = 9-14 mice per group). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. two-tailed unpaired t-test,


( F ) Retinal angiogenesis assessed in P7 NtrasCA / CA Ntras ∆ CA / ∆ CA pups by immunostaining of isolectin B4 . Vascularized areas were normalized to total retinal ( n = 4 - 8 mice per group ) . Representative are shown . Scale bars are 200 µm . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . two - unpaired t - ,

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

(F) Retinal angiogenesis assessed in P7 NtrasCA/CA and Ntras∆CA/∆CA pups by immunostaining of isolectin B4. Vascularized areas were normalized to total retinal area (n = 4-8 mice per group). Representative micrographs are shown. Scale bars are 200 µm. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. two-tailed unpaired t-test,


( G ) FTSC TMR - dextran in vivo permeability assays , comparing homogenates of hearts from NtrasCA / CA Ntras ∆ CA / ∆ CA mice . Data normalized to organ ( n = 11 - 18 mice per group ) . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . two - unpaired t - ,

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

(G) FTSC and TMR-dextran in vivo permeability assays, comparing homogenates of hearts from NtrasCA/CA and Ntras∆CA/∆CA mice. Data normalized to organ and body weight (n = 11-18 mice per group). Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. two-tailed unpaired t-test,


( H ) of CD45 + ( red ) infiltration into cardiac tissue from NtrasCA / CA Ntras ∆ CA / ∆ CA mice normalized to DAPI . Isolectin B4 ( green ) was used to label endothelial ( n = 4 - 5 mice per group ) . Representative are shown . Scale bars are 50 µm . Insets arrows indicate sites of CD45 + infiltration . Data information : data are represented as mean ± S . E . M . n . s . : non - significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . two - unpaired t - ,

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

(H) Quantification of CD45+ cell (red) infiltration into cardiac tissue from NtrasCA/CA and Ntras∆CA/∆CA mice normalized to DAPI. Isolectin B4 (green) was used to label endothelial cells (n = 4-5 mice per group). Representative micrographs are shown. Scale bars are 50 µm. Insets and arrows indicate sites of CD45+ cell infiltration. Data information: data are represented as mean ± S.E.M. n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001. two-tailed unpaired t-test,


( I ) of control Ntras - silenced mice showing cardiac pathologies ( n = 15 - 17 mice per group ) . Prevalence is indicated above bars . H & E of murine heart sections from control Ntras - silenced mice are shown . Insets arrows indicate sites of lymphocytic infiltration . Scale bars are 100 µm . Data information : ( I ) Chi - square .

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

(I) Percentage of control and Ntras-silenced mice showing cardiac pathologies (n = 15-17 mice per group). Prevalence is indicated above the bars. H&E micrographs of murine heart sections from control and Ntras-silenced mice are shown. Insets and arrows indicate sites of lymphocytic infiltration. Scale bars are 100 µm. Data information: (I) Chi-square test.


A EN qRT - PCR of murine liver sinusoidal endothelial ( LSEC ) , Kupffer ( KC ) , hepatocytes ( Hep ) , HSC ( n = 3 per type ) .

A EN qRT-PCR of murine liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC), hepatocytes (Hep), and HSC (n = 3 per cell type).


B EN qRT - PCR of human hepatocytes ( n = 1 ) HSC LX - 2 ( n = 3 ) .

B EN qRT-PCR of human hepatocytes (n = 1) and HSC cell line LX-2 (n = 3).


C EN immunofluorescence of LX - 2 . Scale bar : 25 μm .

O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O

C EN immunofluorescence staining of LX-2 cells. Scale bar: 25 μm.


D EN qRT - PCR of normal liver ( NL ) fibrotic liver ( FL , n = 8 each ) , active or inactive cirrhotic liver ( CL + / − , n = 10 / n = 2 ) .

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

D EN qRT-PCR of normal liver (NL) and fibrotic liver (FL,n = 8 each), and active or inactive cirrhotic liver (CL+/−, n = 10/ n = 2).


E - H EN immunohistochemistry of human normal liver ( E ) , fibrotic liver ( F ) , cirrhotic liver ( G , H ) . Weak EN expression in portal tracts of normal liver ( E ) . Abundant EN expression detected in activated HSC in fibrous septa of fibrotic ( F ) , active cirrhotic ( G ) , inactive cirrhotic liver ( H ) . Scale bars : 500 μm . I - N Higher magnification of EN - stained normal liver ( I ) , fibrotic liver ( K ) , ( active ) cirrhotic liver ( M ) compared to alpha - smooth muscle actin ( αSMA ) immunohistochemistry on serial slides ( J , L N ) . Scale bars : 100 μm .

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

E-H EN immunohistochemistry staining of human normal liver (E), fibrotic liver (F), and cirrhotic liver (G, H). Weak EN expression in portal tracts of normal liver (E). Abundant EN expression detected in activated HSC in fibrous septa of fibrotic (F), active cirrhotic (G), and inactive cirrhotic liver (H). Scale bars: 500 μm.I-N Higher magnification of EN-stained normal liver (I), fibrotic liver (K), and (active) cirrhotic liver (M) compared to alpha-smooth muscle actin (αSMA) immunohistochemistry on serial slides (J, L and N). Scale bars: 100 μm.


O EN / αSMA mRNA in normal liver , fibrotic liver , active cirrhotic liver .

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

O EN/αSMA mRNA ratio in normal liver, fibrotic liver, and active cirrhotic liver.


P Collagen 1a ( Col1a ) expression in normal , fibrotic , active cirrhotic liver determined by qRT - PCR .

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

P Collagen 1a (Col1a) expression in normal, fibrotic, and active cirrhotic liver determined by qRT-PCR.


A , B qRT - PCR for EN ( A ) αSMA ( B ) of liver lysates from single high - dose CCl4 - or oil - treated wild - type ( WT ) mice ( n = 2 per time point ) .

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

A, B qRT-PCR for EN (A) and αSMA (B) of liver lysates from single high-dose CCl4- or oil-treated wild-type (WT) mice (n = 2 per time point).


C Early activated long - term - activated HSC isolated by differential density gradient centrifugation ( 8 . 26 % versus 11 . 5 % density layer ) . qRT - PCR for EN from early activated ( 8 . 26 % ) long - term - activated HSC ( 11 . 5 % ) from 4 - week CCL4 - treated mice vehicle control mice ( n = 3 - 4 per data point ) .

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

C Early activated and long-term-activated HSC isolated by differential density gradient centrifugation (8.26% versus 11.5% density layer). qRT-PCR for EN from early activated (8.26%) and long-term-activated HSC (11.5%) from 4-week CCL4-treated mice and vehicle control mice (n = 3-4 per data point).


D EN expression in liver lysates from 2 - , 4 - , 6 - week CCl4 - treated mice ( n = 5 - 8 ) vehicle control mice ( n = 3 ) determined by qRT - PCR .

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

D EN expression in liver lysates from 2-, 4-, and 6-week CCl4-treated mice (n = 5-8) and vehicle control mice (n = 3) determined by qRT-PCR.


E - G METAVIR score of 2 - , 4 - , 6 - week CCl4 - treated mice ( F - G ) Col1a F4 / 80 immunofluorescence of 6 - week CCl4 - treated vehicle control WT ( F ) ENKO ( G ) mice . H Mean Col1a + per high power field ( HPF ) shown as of total ( n = 5 - 8 ) .

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

E-G METAVIR score of 2-, 4-, and 6-week CCl4-treated mice (F-G) Col1a and F4/80 immunofluorescence of 6-week CCl4-treated and vehicle control WT (F) and ENKO (G) mice.H Mean Col1a+ area per high power field (HPF) shown as percentage of the total area (n = 5-8).


I Western blot for collagen 1a ( Col1a ) , platelet - derived growth factor receptor beta ( PDGFRβ ) β - actin of total liver lysates from 6 - week CCl4 - treated or control vehicle WT ENKO mice ( n = 3 - 5 ) .

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

I Western blot analysis for collagen 1a (Col1a), platelet-derived growth factor receptor beta (PDGFRβ) and β-actin of total liver protein lysates from 6-week CCl4-treated or control vehicle WT and ENKO mice (n = 3-5).


J - L Double immunofluorescence of 6 - week CCl4 - treated vehicle control WT ( J ) ENKO ( K ) mice for desmin αSMA ( L ) shown as of total ( n = 5 - 8 ) .

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

J-L Double immunofluorescence of 6-week CCl4-treated and vehicle control WT (J) and ENKO (K) mice for desmin and αSMA (L) shown as percentage of the total area (n = 5-8).


M Serum liver enzymes ( GPT / ALT GOT / AST ) from 2 - , 4 - , 6 - week CCl4 - treated WT ENKO mice ( n = 5 - 8 ) .

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

M Serum liver enzymes (GPT/ALT and GOT/AST) from 2-, 4-, and 6-week CCl4-treated WT and ENKO mice (n = 5-8).


N qRT - PCR of endosialin ( EN ) , αSMA , collagen 1a1 / 6a1 from lentiviral - mediated endosialin knockdown ( shEN ) control - transfected ( CTL ) human hepatic stellate ( LX - 2 ) .

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

N qRT-PCR of endosialin (EN), αSMA, and collagen 1a1/6a1 from lentiviral-mediated endosialin knockdown (shEN) and control-transfected (CTL) human hepatic stellate cells (LX-2).


O Lentiviral - mediated endosialin knockdown ( shEN ) control ( CTL ) in LX - 2 ( n = 3 ) . Magnification 10 × .

O B-ORGANISM O O B-GENEPROD O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O

O Lentiviral-mediated endosialin knockdown (shEN) and control (CTL) in LX-2 cells (n = 3). Magnification 10×.


A Representative of Ki - 67 immunohistochemistry after 2 , 4 , 6 of CCl4 treatment of WT ENKO mice ( n = 5 - 8 mice per group time point ) . Scale bars : 75 μm . B Quantitation of Ki67 + hepatocytes / HPF after 2 , 4 , 6 of CCl4 treatment .

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

A Representative images of Ki-67 immunohistochemistry after 2, 4, and 6 weeks of CCl4 treatment of WT and ENKO mice (n = 5-8 mice per group and time point). Scale bars: 75 μm.B Quantitation of Ki67+ hepatocytes/HPF after 2, 4, and 6 weeks of CCl4 treatment.


C Time course of EN expression after partial hepatectomy ( 2 / 3 PHx ) determined by qRT - PCR of total liver lysates ( n = 5 / time point , sham : n = 3 ) .

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

C Time course of EN expression after partial hepatectomy (2/3 PHx) determined by qRT-PCR of total liver lysates (n = 5/time point, sham: n = 3).


D Representative of Ki - 67immunohistochemistry 2 days after 2 / 3 PHx of WT ENKOmice ( n = 5 - 9 mice per group time point ) . E Quantitation of Ki - 67 + hepatocytes / HPF determined 1 , 2 , 4 , 8 days after 2 / 3 PHx . F Number of mitotic figures 2 days after 2 / 3 PHx of WT ENKOmice ( n = 5 - 9 mice per group time point ) .

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

D Representative images of Ki-67immunohistochemistry 2 days after 2/3 PHx of WT and ENKOmice (n = 5-9 mice per group and time point).E Quantitation of Ki-67+hepatocytes/HPF determined 1, 2, 4, and 8 days after 2/3 PHx.F Number of mitotic figures 2 days after 2/3 PHx of WT and ENKOmice (n = 5-9 mice per group and time point).


G qRT - PCR of total liver lysates 1 day after PHx for IGF2 .

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

G qRT-PCR analysis of total liver lysates 1 day after PHx for IGF2.


H Western blot of phosphorylated insulin - like growth factor receptor 1 ( pIGFR1 ) , phosphorylated insulin receptor substrate 1 ( pIRS1 ) β - actin 2 days after PHx .

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

H Western blot analysis of phosphorylated insulin-like growth factor receptor 1 (pIGFR1), phosphorylated insulin receptor substrate 1 (pIRS1) and β-actin 2 days after PHx.


I Western blot of phosphorylated total extracellular signal - regulated kinase ( ERK ) AKT 2 days after PHx .

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

I Western blot analysis of phosphorylated and total extracellular signal-regulated kinase (ERK) and AKT 2 days after PHx.


J , K qRT - PCR of IGF2 in isolated liver sinusoidal endothelial ( LSEC ) ( J ) or hepatocytes ( HEP ) ( K ) 1 day after PHx .

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

J, K qRT-PCR of IGF2 in isolated liver sinusoidal endothelial cells (LSEC)(J) or hepatocytes (HEP)(K) 1 day after PHx.


L EdU - positive hepatocytes ( in % ) after 24 - h stimulation with conditioned medium ( CM ) from either WT or ENKO HSC ( 3 , 5 , or 7 days of activation ) . HGF ( 40 ng / ml ) TGF - β ( 10 ng / ml ) serve as positive or negative control , respectively .

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

L EdU-positive hepatocytes (in %) after 24-h stimulation with conditioned medium (CM) from either WT or ENKO HSC (3, 5, or 7 days of activation). HGF (40 ng/ml) and TGF-β (10 ng/ml) serve as positive or negative control, respectively.


M EdU - positive WT ENKO hepatocytes ( in % ) after stimulation with IGF2 ( 100 ng / ml ) . HGF ( 40 or 10 ng / ml ) TGF - β ( 10 ng / ml ) serve as positive or negative control , respectively .

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

M EdU-positive WT and ENKO hepatocytes (in %) after stimulation with IGF2 (100 ng/ml). HGF (40 or 10 ng/ml) and TGF-β (10 ng/ml) serve as positive or negative control, respectively.


A Up , Representative western blot from whole extracts showing H2AX in wt junD - / - fibroblasts ( Left ) or in wt Nfe2l2 - / - fibroblasts ( Right ) . Two representative clones , with their corresponding controls , are shown per genotype . H2B was used as negative control . Down , Bar plot showing H2AX as assessed by densitometry of western blots ( as shown above ) . n ≥ 3 independent experiments .

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

A Up, Representative western blot from whole cell extracts showing H2AX protein levels in wt and junD-/- fibroblasts (Left) or in wt and Nfe2l2-/- fibroblasts (Right). Two representative clones, with their corresponding controls, are shown per genotype. H2B was used as negative control. Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above). n ≥ 3 independent experiments.


B Up , Representative western blot from kidney whole extracts showing H2AX in - matched wt junD - / - mice . Mice are 2 months old ( young ) 18 months old ( old ) mice , respectively . Down , Bar plot showing H2AX as assessed by densitometry of western blots ( as shown above ) . N ≥ 5 mice per per genotype .

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

B Up, Representative western blot from kidney whole cell extracts showing H2AX protein levels in age-matched wt and junD-/- mice. Mice are 2 months old (young) and 18 months old (old) mice, respectively. Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above). N ≥ 5 mice per age and per genotype.


C Up , Representative western blot from kidney whole extracts , showing H2AX in 24 months old wt junD - / - mice , either untreated ( - ) or treated with anti - oxidant agent , N - acetyl - cysteine ( NAC ) . Down , Bar plot showing H2AX as assessed by densitometry of western blots ( as shown above ) . N ≥ 4 mice per treatment per genotype .

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

C Up, Representative western blot from kidney whole cell extracts, showing H2AX protein levels in 24 months old wt and junD-/- mice, either untreated (-) or treated with the anti-oxidant agent, N-acetyl-cysteine (NAC). Down, Bar plot showing H2AX protein levels as assessed by densitometry analysis of western blots (as shown above). N ≥ 4 mice per treatment and per genotype.


D , E Left , Representative western blots from whole extract showing γ - H2AX , H2AX Kap1 phosphorylation ( P - Kap1 ) in wt junD - / - fibroblasts ( D ) or in wt Nfe2l2 - / - fibroblasts ( E ) , after H2O2 exposure for indicated times ( hours , h ) . Right , Bar plots showing γ - H2AX H2AX , as well as γ - H2AX / H2AX as assessed by densitometry of western blots ( as shown on left ) . n = 3 independent experiments .

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

D,E Left, Representative western blots from whole cell extract showing γ-H2AX, H2AX protein levels and Kap1 phosphorylation (P-Kap1) in wt and junD-/- fibroblasts (D) or in wt and Nfe2l2-/- fibroblasts (E), after H2O2 exposure for the indicated times (hours, h). Right, Bar plots showing γ-H2AX and H2AX protein levels, as well as γ-H2AX/H2AX ratio as assessed by densitometry analysis of western blots (as shown on the left). n = 3 independent experiments.