Representative image of a Drosophila posterior midgut showing the expression of Armadillo ( red membranes ) and Actin - Dronc - GFP - Myc ( green , anti - GFP ) ; note the preferential enrichment of Dronc expression in the small groups of cells formed by intestinal progenitor cells ( arrow ) . High magnification of the picture shown in B . The white arrow indicates a presumptive ISC showing low levels of Dronc compared to the flanking GFP + EBs . The arrow indicates a presumptive small ISC expressing high levels of Arm and low levels of Dronc - GFP - Myc . Transversal Z - section of the cells shown in C . The arrow indicates a presumptive small ISC expressing high levels of Arm and low levels of Dronc - GFP - Myc .

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

Representative image of a Drosophila posterior midgut showing the expression of Armadillo (red membranes) and Actin-Dronc-GFP-Myc (green, anti-GFP); note the preferential enrichment of Dronc expression in the small groups of cells formed by intestinal progenitor cells (arrow). High magnification of the picture shown in B. The white arrow indicates a presumptive ISC showing low levels of Dronc compared to the flanking GFP+ EBs. The arrow indicates a presumptive small ISC expressing high levels of Arm and low levels of Dronc-GFP-Myc. Transversal Z-section of the cells shown in C. The arrow indicates a presumptive small ISC expressing high levels of Arm and low levels of Dronc-GFP-Myc.


E . The enteroblast marker Su ( H ) ( red , immunostaining against Beta - galactosidase ) strongly co - localises with high levels of Dronc expression ( green , immunostaining against GFP ) ; white arrow indicates the enlarged area depicted in the insets .

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

E. The enteroblast marker Su(H) (red, immunostaining against Beta-galactosidase) strongly co-localises with high levels of Dronc expression (green, immunostaining against GFP); white arrow indicates the enlarged area depicted in the insets.


F . There is extensive overlap between the expression of the EB marker Su ( H ) ( green , immunostaining against Beta - galactosidase ; white arrow ) and the apical caspase reporter DBS - S - QF ( red , immunostaining against HA ) , but it is also present in progenitor cells without Su ( H ) expression ( yellow arrowheads ) . DAPI ( Blue ) labels cell nuclei in all the panels .

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

F. There is extensive overlap between the expression of the EB marker Su(H) (green, immunostaining against Beta-galactosidase; white arrow) and the apical caspase reporter DBS-S-QF (red, immunostaining against HA), but it is also present in progenitor cells without Su(H) expression (yellow arrowheads). DAPI (Blue) labels cell nuclei in all the panels.


A . Representative ReDDM labeling of a Drosophila Dronc heterozygous ( + / - ) intestine overexpressing an RNAi against Notch for 3 days ; note the lack of fully differentiated Histone - RFP cells as EC ( red ) without esg expression ( green , GFP ) . DAPI staining labels cell nuclei in panels All of the experiments described in the figure were performed in Oxford medium following an experimental regime that protects the epithelial integrity . Genotype : w1118 UAS - Notch - RNAi ( Joaquin Navascués ) ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / + B . Representative ReDDM labelling of a Drosophila Dronc mutant homozygous ( - / - ) intestine overexpressing an RNAi against Notch for 3 post temperature shift at 29 oC ; note the lack of differentiated Histone - RFP cells as EC ( red ) without esg expression ( green , GFP ) , as well as the increase of GFP - positive cells ( compare A to B ) . Genotype : w1118 UAS - Notch - RNAi ( Joaquin Navascués ) ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry C . Quantification of the number of Histone - RFP cells normalised to DAPI ( proxy of progenitor cell proliferation obtained from the experiments shown in A and B panels ) ; note the statically significant increase in Histone - RFP positive cells in Dronc homozygous mutant intestines compared to controls ( * * ; P = 0 . 0080 ) ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - tailed t test , + / - n = 8 , - / - n = 15 ) . Error bars represent Standard Deviation of the mean . Genotypes : + / - : w1118 UAS - Notch - RNAi ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / + - / - : w1118 UAS - Notch - RNAi ( Joaquin Navascués ) ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry

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

A. Representative ReDDM labeling of a Drosophila Dronc heterozygous (+/-) intestine overexpressing an RNAi against Notch for 3 days; note the lack of fully differentiated Histone-RFP cells as EC (red) without esg expression (green, GFP). DAPI staining labels cell nuclei in panels All of the experiments described in the figure were performed in Oxford medium following an experimental regime that protects the epithelial integrity. Genotype: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / + B. Representative ReDDM labelling of a Drosophila Dronc mutant homozygous (-/-) intestine overexpressing an RNAi against Notch for 3 post temperature shift at 29 oC; note the lack of differentiated Histone-RFP cells as EC (red) without esg expression (green, GFP), as well as the increase of GFP-positive cells (compare A to B). Genotype: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry C. Quantification of the number of Histone-RFP cells normalised to DAPI (proxy of progenitor cell proliferation obtained from the experiments shown in A and B panels); note the statically significant increase in Histone-RFP positive cells in Dronc homozygous mutant intestines compared to controls (**; P =0.0080) (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, +/- n = 8, -/- n = 15). Error bars represent Standard Deviation of the mean. Genotypes: +/-: w1118 UAS-Notch-RNAi; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / + -/-: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry


D . Drosophila Dronc heterozygous intestine overexpressing the Notch intracellular domain for 7 days post temperature shift at 29 oC ; intestinal esg - positive progenitor cells ( green ( GFP ) and red ( Histone - RFP ) . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - Notchintra ( Joaquin Navascués ) ; TubG80ts UAS - Histone - RFP DroncKO / + E . Drosophila Dronc homozygous intestine overexpressing the Notch intracellular domain for 7d post temperature shift at 29 oC ; notice that the Dronc deficiency accelerates the elimination of intestinal progenitor cells induced by Notch overactivation ( compare D and E ) . The white arrows indicate the position of insets 500μm from the posterior region . Note the complete loss of esg labelled cells in this region . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - Notchintra ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry F . Relative number of esg - positive cells to DAPI in either heterozygous or homozygous Dronc mutant esg cells overexpressing Notch - Intra ; note the significant reduction of esg - expressing cells ( * * * * ; P < 0 . 0001 ) ( Quantifications were made using N ≥ 2 biological replicates ; Mann - Whitney test , + / - n = 17 , - / - n = 17 ) . Error bars represent Standard Deviation of the mean . Genotypes : + / - : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - Notchintra ; TubG80ts UAS - Histone - RFP DroncKO / + - / - : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - Notchintra ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA -

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

D. Drosophila Dronc heterozygous intestine overexpressing the Notch intracellular domain for 7 days post temperature shift at 29 oC; intestinal esg-positive progenitor cells (green (GFP) and red (Histone-RFP). Genotype: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra (Joaquin Navascués); TubG80ts UAS-Histone-RFP DroncKO / + E. Drosophila Dronc homozygous intestine overexpressing the Notch intracellular domain for 7d post temperature shift at 29 oC; notice that the Dronc deficiency accelerates the elimination of intestinal progenitor cells induced by Notch overactivation (compare D and E). The white arrows indicate the position of insets 500μm from the posterior region. Note the complete loss of esg labelled cells in this region. Genotype: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry F. Relative number of esg-positive cells to DAPI in either heterozygous or homozygous Dronc mutant esg cells overexpressing Notch-Intra; note the significant reduction of esg-expressing cells (****; P < 0.0001) (Quantifications were made using N ≥ 2 biological replicates; Mann-Whitney test, +/- n = 17, -/- n = 17). Error bars represent Standard Deviation of the mean. Genotypes: +/-: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra ; TubG80ts UAS-Histone-RFP DroncKO / + -/-: w1118; esg-Gal4 UAS-CD8 -GFP / UAS-Notchintra; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry


D . Drosophila Dronc heterozygous intestine overexpressing the Notch intracellular domain for 7 days post temperature shift at 29 oC ; intestinal esg - positive progenitor cells ( green ( GFP ) and red ( Histone - RFP ) . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - Notchintra ( Joaquin Navascués ) ; TubG80ts UAS - Histone - RFP DroncKO / +

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

D. Drosophila Dronc heterozygous intestine overexpressing the Notch intracellular domain for 7 days post temperature shift at 29 oC; intestinal esg-positive progenitor cells (green (GFP) and red (Histone-RFP). Genotype: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra (Joaquin Navascués); TubG80ts UAS-Histone-RFP DroncKO / +


G . Representative image of a Drosophila Dronc heterozygous intestines 7 days after transfer to 29 oC . esg expression in green ( GFP ) labels all intestinal progenitors cells , whilst Su ( H ) - lacZ ( red ) distinguishes a subpopulation of EBs within the esg - expressing cells . Genotype : w1118 , Su ( H ) GBE - LacZ ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / +

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

G. Representative image of a Drosophila Dronc heterozygous intestines 7 days after transfer to 29 oC. esg expression in green (GFP) labels all intestinal progenitors cells, whilst Su(H)-lacZ (red) distinguishes a subpopulation of EBs within the esg-expressing cells. Genotype: w1118, Su(H)GBE-LacZ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / +


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 -

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

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 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 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 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 .

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 O B-SMALL_MOLECULE O 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 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 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 .

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 O B-ORGANISM B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR O 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 O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O 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 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 .

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

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

O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O 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 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 . 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 .

O O O O B-SUBCELLULAR I-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 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 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 .

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 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 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 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 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 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 ) .

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 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 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 .

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD I-GENEPROD O 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 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 . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O 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 cell lines in 5a by immunoblot of protein lysates .

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


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 .

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

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

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

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

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 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 .

O B-GENEPROD I-GENEPROD I-GENEPROD 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

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 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 .

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

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

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

(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 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 .

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 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 O B-GENEPROD I-GENEPROD O 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 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 .

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

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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O 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 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 , 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 .

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

(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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O 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 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 B-EXP_ASSAY I-EXP_ASSAY I-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 , 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 .

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

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

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

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

(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 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 .

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 O O O B-GENEPROD 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 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

(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 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 .

O O O B-CELL_TYPE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY 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 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 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 ) .

O 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 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 B-TISSUE I-TISSUE O 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 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 .

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


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 .

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

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

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.


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

O O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-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 ( + ) 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 .

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


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 * * * * .

B-SUBCELLULAR O O B-SUBCELLULAR O O B-GENEPROD O O B-GENEPROD O O O B-TISSUE O O O O O O O B-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 B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O 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 images of a single neural progenitor cell ( red arrow ) tracked over time , data starting from 34hpf ; scale bar 10μm , images 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 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 cell time series of Her6 : : Venus , mKeima - H2B and Her6 : : Venus signal normalized by mKeima - H2B corresponding to the cell in ( c ) .

O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O 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 the 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 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 fluorescent WM - ISH for her6 ( green ) and mir - 9 - 4 ( magenta ) imaged in fixed embryo at 31hpf ; scale bar 30μm .

O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-TISSUE 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 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 .

B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O B-GENEPROD O O B-GENEPROD O 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 O B-TISSUE I-TISSUE O 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 and MBSm embryos 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 B-TISSUE 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 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 * * * * .

B-GENEPROD O O B-GENEPROD O O O O O O O B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-TISSUE O O O O O O O O O O B-TISSUE O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O 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 protein abundance over development in CTRL and MBSm embryos quantified as median of single cell abundance data in ( f ) .

O 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

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 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 .

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

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

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 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 .

O O O O O 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 cells observed in CTRL ( f ) and MBSm ( g ) embryos 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 B-TISSUE 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 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 .

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

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

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

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

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

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 B-TISSUE O O O O O 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

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 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

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

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

O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-CELL_LINE 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 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 - 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 ,

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY 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 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 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 .

O O O O O O O O O O O B-GENEPROD O O B-CELL_LINE 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 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 ) 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 ,

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

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

O O O B-EXP_ASSAY 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 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 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 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 ) .

O O O O O O O B-GENEPROD I-GENEPROD O O B-CELL_LINE B-SUBCELLULAR 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 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 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 .

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

(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 - 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 .

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

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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 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 - tailed unpaired t - test .

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 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 and 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 O B-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 - 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O 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 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 - 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 .

(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 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 ,

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

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 B-SUBCELLULAR 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 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 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 ,

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 B-SUBCELLULAR O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ,

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 B-SUBCELLULAR O O O O B-GENEPROD O 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 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 - 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 .

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

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 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

(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 - tailed unpaired t - test ,

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 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 ) 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 ,

O 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 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 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 - tailed unpaired t - test ,

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 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 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 -

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 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 - 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 ,

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

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 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 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 - 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 ,

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

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