F - I . Immunofluorescence images of dpERK staining of midgut sections of the R4 region of 40 - day Drosophila ( F ) , 40 - day Drosophila with ALA administration ( G ) , 14 - day Drosophila carrying esgts - GAL4 - driven UAS - lacZ ( H ) , and Drosophila carrying esgts - GAL4 - driven Las RNAi ( I ) . dpERK ( red ) . J . Quantification of the fluorescence intensity of dpERK in experiments ( F - I ) . Each dot corresponds to one cell . n is indicated . The numbers of quantified guts from left to right are 11 , 12 , 12 , and 13 . K Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10 μm Error bars represent SDs . The statistical tests used in other panels were student ' s t - tests . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

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

F-I. Immunofluorescence images of dpERK staining of midgut sections of the R4 region of 40-day Drosophila (F), 40-day Drosophila with ALA administration (G), 14-day Drosophila carrying esgts-GAL4-driven UAS-lacZ (H), and Drosophila carrying esgts-GAL4-driven Las RNAi (I). dpERK (red). J. Quantification of the fluorescence intensity of dpERK in experiments (F-I). Each dot corresponds to one cell. n is indicated. The numbers of quantified guts from left to right are 11, 12, 12, and 13. K Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm Error bars represent SDs. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


K - M . Representative images of midgut sections from the R4 region of Drosophila carrying esgts - GAL4 - driven expression of lacZ cDNA ( K , control ) , Las RNAi ( L ) , or Las RNAi and dominant negative form of EGFR ( EGFR - DN , M ) . GFP ( green ) and Dl staining ( red ) was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10

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

K-M. Representative images of midgut sections from the R4 region of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA (K, control), Las RNAi (L), or Las RNAi and dominant negative form of EGFR (EGFR-DN, M). GFP (green) and Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm


( A ) ATG - 5 staining in non - cancerous gastric tissues scored 285 ( × 200 ) ; ( B ) ATG - 5 staining in GC tumor tissues scored 50 ( × 400 ) ; ( C ) ATG - 5 staining in GC tumor tissues scored 270 ( × 400 ) ; ( D ) ATG - 5 staining in GC tumor tissues scored 400 ( × 200 ) .

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

(A) ATG-5 staining in non-cancerous gastric tissues scored 285(×200); (B) ATG-5 staining in GC tumor tissues scored 50(×400); (C) ATG-5 staining in GC tumor tissues scored 270(×400); (D) ATG-5 staining in GC tumor tissues scored 400(×200).


( A and B ) Patients were divided into four following subgroups based on ATG - 5 immunostaining : scored 0 - 99 ( curve a ) , scored 100 - 199 ( curve b ) , scored 200 - 299 ( curve c ) and scored 300 - 400 ( curve d ) . The difference among different subgroups was statistically significant as evaluated by overall Log - rank comparisons ( OS : P < 0 . 001 , DFS : P = 0 . 003 ) . Pairwise Log - rank comparisons showed that the subgroup D exhibited the poorest survival rates as compared to other subgroups ( OS : P < 0 . 05 , DFS : P < 0 . 01 ) .

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

(A and B) Patients were divided into four following subgroups based on ATG-5 immunostaining: scored 0-99 (curve a), scored 100-199 (curve b), scored 200-299 (curve c) and scored 300-400 (curve d). The difference among different subgroups was statistically significant as evaluated by overall Log-rank comparisons (OS: P<0.001, DFS: P = 0.003). Pairwise Log-rank comparisons showed that the subgroup D exhibited the poorest survival rates as compared to other subgroups (OS: P<0.05, DFS: P<0.01).


( C and D ) Patients were divided into four following subgroups based on MRP - 1 immunostaining : scored 0 - 99 ( curve a ) , scored 100 - 199 ( curve b ) , scored 200 - 299 ( curve c ) and scored 300 - 400 ( curve d ) . The difference among various subgroups was statistically significant by overall Log - rank comparisons ( OS : P = 0 . 001 , DFS : P = 0 . 018 ) . Pairwise Log - rank comparisons showed that the subgroup A had the most favorable prognosis among the four subgroups ( OS : P < 0 . 05 , DFS : P < 0 . 001 ) .

O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and D) Patients were divided into four following subgroups based on MRP-1 immunostaining: scored 0-99 (curve a), scored 100-199 (curve b), scored 200-299 (curve c) and scored 300-400 (curve d). The difference among various subgroups was statistically significant by overall Log-rank comparisons (OS: P = 0.001, DFS: P = 0.018). Pairwise Log-rank comparisons showed that the subgroup A had the most favorable prognosis among the four subgroups (OS: P<0.05, DFS: P<0.001).


( E and F ) Patients were divided into four subgroups according to different TNM stages . The difference among different subgroups was statistically significant by overall Log - rank comparisons ( OS : P < 0 . 001 , DFS : P < 0 . 001 ) . Pairwise Log - rank comparisons showed that subgroups III or IV exhibited poorer survival rates than that of subgroups IB and II ( OS : P < 0 . 01 , DFS : P < 0 . 001 ) .

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

(E and F) Patients were divided into four subgroups according to different TNM stages. The difference among different subgroups was statistically significant by overall Log-rank comparisons (OS: P<0.001, DFS: P<0.001). Pairwise Log-rank comparisons showed that subgroups III or IV exhibited poorer survival rates than that of subgroups IB and II (OS: P<0.01, DFS: P<0.001).


( A ) The level of ATG5 was detected in cell lines using western blot analysis . β - actin was used as internal controls .

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

(A) The level of ATG5 was detected in cell lines using western blot analysis. β -actin was used as internal controls.


( B ) The IC 25 , IC50 and IC75 of SGC - 7901 and SGC - 7901 / DDP cells were tested using the MTT assays after DPP treatment .

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

(B) The IC 25, IC50 and IC75 of SGC-7901 and SGC-7901/DDP cells were tested using the MTT assays after DPP treatment.


( A ) The mRNA level of ATG - 5 was detected by real time PCR after treatment with siRNAs . GAPDH was used as internal controls .

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

(A) The mRNA level of ATG-5 was detected by real time PCR after treatment with siRNAs. GAPDH was used as internal controls.


( B ) The protein level of ATG - 5 was detected by western blot after treatment with siRNAs . β - actin was used as internal controls .

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

(B) The protein level of ATG-5 was detected by western blot after treatment with siRNAs. β -actin was used as internal controls.


( C ) The proliferation ability was tested using MTT assay 48 hours or 72 hours after different treatment .

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

(C) The proliferation ability was tested using MTT assay 48 hours or 72 hours after different treatment.


( A ) The autophagy was detected by immunofluorescence assay of LC3B in the cells 48 hours after different treatment .

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

(A) The autophagy was detected by immunofluorescence assay of LC3B in the cells 48 hours after different treatment.


( B ) The protein levels of LC3A and LC3B were tested by western blot . β - actin was used as internal controls .

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

(B) The protein levels of LC3A and LC3B were tested by western blot. β-actin was used as internal controls.


( c ) Intravascular endothelial cell interactions and transmigration of neutrophils ( Ly - 6G + CX3CR - 1 - ; red ) and cMOs ( Ly - 6G - CX3CR - 1low ; green ) to the perivascular tissue as assessed 6 h after intrascrotal stimulation with recombinant murine uPA , PAI - 1 , or uPA - PAI - 1 in postcapillary venules of the cremaster of CX3CR - 1GFP / + mice by multi - channel in vivo microscopy . Representative still images ( scale bar : 50 : µm ) and quantitative data are shown ( mean ± SEM for n = 4 mice per group ; # p < 0 . 05 vs . saline ; * p < 0 . 05 vs . uPA ; § p < 0 . 05 vs . PAI - 1 ; One - way ANOVA ) .

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

(c) Intravascular endothelial cell interactions and transmigration of neutrophils (Ly-6G+ CX3CR-1-; red) and cMOs (Ly-6G- CX3CR-1low; green) to the perivascular tissue as assessed 6 h after intrascrotal stimulation with recombinant murine uPA, PAI-1, or uPA-PAI-1 in postcapillary venules of the cremaster of CX3CR-1GFP/+ mice by multi-channel in vivo microscopy. Representative still images (scale bar: 50: µm) and quantitative data are shown (mean±SEM for n=4 mice per group; #p<0.05 vs. saline; *p<0.05 vs. uPA; §p<0.05 vs. PAI-1; One-way ANOVA).


( b ) Expression of PECAM - 1 / CD31 ( red ) and ICAM - 1 / CD54 or VCAM - 1 / CD106 ( green ) in the cremaster muscle of WT mice as assessed ex vivo by confocal laser scanning microscopy 6 h after intrascrotal injection of recombinant murine uPA - PAI - 1 or saline , representative images ( scale bar : 50 µm ) and quantitative data are shown ( mean ± SEM for n = 4 mice per group ; # p < 0 . 05 vs . saline ; t test ) .

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

(b) Expression of PECAM-1/CD31 (red) and ICAM-1/CD54 or VCAM-1/CD106 (green) in the cremaster muscle of WT mice as assessed ex vivo by confocal laser scanning microscopy 6 h after intrascrotal injection of recombinant murine uPA-PAI-1 or saline, representative images (scale bar: 50 µm) and quantitative data are shown (mean±SEM for n=4 mice per group; #p<0.05 vs. saline; t test).


( g ) Quantitative analysis of LFA - 1 / CD11a clustering on the surface of mouse neutrophils under increasing flow conditions in vitro in the presence of either E - selectin / CD62E + ICAM - 1 / CD54 , E - selectin / CD62E + ICAM - 1 / CD54 + KC , or E - selectin / CD62E + ICAM - 1 / CD54 + KC + uPA - PAI - 1 as assessed by spinning disk confocal microscopy , representative images and quantitative data are shown ( scale bar : 10 μm ; mean ± SD ; n = 3 mice ; n = 10 - 13 flow chambers , n = 122 - 222 cells ) ; * p < 0 . 05 vs . E - selectin / CD62E + ICAM - 1 / CD54 ; One - way ANOVA ) .

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

(g) Quantitative analysis of LFA-1/CD11a clustering on the surface of mouse neutrophils under increasing flow conditions in vitro in the presence of either E-selectin/CD62E + ICAM-1/CD54, E-selectin/CD62E + ICAM-1/CD54 + KC, or E-selectin/CD62E + ICAM-1/CD54 + KC + uPA-PAI-1 as assessed by spinning disk confocal microscopy, representative images and quantitative data are shown (scale bar: 10 μm; mean±SD; n=3 mice; n=10-13 flow chambers, n =122-222 cells); *p<0.05 vs. E-selectin/CD62E + ICAM-1/CD54; One-way ANOVA).


( b ) Correlation analyses of RNA expression levels of uPA ( PLAU gene ) , PAI - 1 ( SERPINE1 gene ) , and the neutrophil marker gene FPR1 , and overall survival of breast cancer patients ( stages 0 and 1 ) with high and low PLAU or SERPINE1 gene expression levels ( cut - off z ≥ 2 . 0 ) in the METABRIC breast cancer cohort .

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

(b) Correlation analyses of RNA expression levels of uPA (PLAU gene), PAI-1 (SERPINE1 gene), and the neutrophil marker gene FPR1, and overall survival of breast cancer patients (stages 0 and 1) with high and low PLAU or SERPINE1 gene expression levels (cut-off z≥ 2.0) in the METABRIC breast cancer cohort.


( c ) Surface expression of MMP - 9 , VEGF , and NE as assessed on circulating neutrophils isolated from the peripheral blood of WT mice ( saline ) or from the peritoneal cavity of WT mice 6 h after intraperitoneal stimulation with uPA - PAI - 1 ( uPA - PAI - 1 ) by multi - channel flow cytometry , quantitative data are shown ( mean ± SEM for n = 4 - 6 mice per group ; # p < 0 . 05 vs . saline ; t test ) .

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

(c) Surface expression of MMP-9, VEGF, and NE as assessed on circulating neutrophils isolated from the peripheral blood of WT mice (saline) or from the peritoneal cavity of WT mice 6 h after intraperitoneal stimulation with uPA-PAI-1 (uPA-PAI-1) by multi-channel flow cytometry, quantitative data are shown (mean±SEM for n=4-6 mice per group; #p<0.05 vs. saline; t test).


( f ) Formation of NETs ( histone H3 + ; green ) as assessed ex vivo by confocal microscopy in the cremaster muscle of WT mice 6 h after intra - scrotal injection of uPA - PAI - 1 , TNF , or saline , PECAM - 1 / CD31 + postcapillary venules ( blue ) and Ly - 6G + neutrophils ( red ) are depicted . Representative images are shown ( scale bar : 50 µm ) .

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

(f) Formation of NETs (histone H3+; green) as assessed ex vivo by confocal microscopy in the cremaster muscle of WT mice 6 h after intra-scrotal injection of uPA-PAI-1, TNF, or saline, PECAM-1/CD31+ postcapillary venules (blue) and Ly-6G+ neutrophils (red) are depicted. Representative images are shown (scale bar: 50 µm).


( a ) Effect of compound WX - 340 on binding of recombinant murine uPA to PAI - 1 protein as assessed by ELISA , quantitative data are shown ( mean ± SEM for n = 3 experiments per group ; # p < 0 . 05 vs . drug vehicle ; One - way ANOVA ) .

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

(a) Effect of compound WX-340 on binding of recombinant murine uPA to PAI-1 protein as assessed by ELISA, quantitative data are shown (mean±SEM for n=3 experiments per group; #p<0.05 vs. drug vehicle; One-way ANOVA).


( d ) Relative development rates and tumor weight in animals treated with WX - 340 a priori or therapeutically after 1 week after tumor cell injection on a daily basis as assessed in an orthotopic model of 4T1 breast cancer in WT mice ( mean ± SEM for n = 4 - 6 mice per group ; * p < 0 . 05 vs . drug vehicle ; One - way ANOVA ) .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 B-CELL_LINE B-DISEASE I-DISEASE O O B-ORGANISM 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

(d) Relative development rates and tumor weight in animals treated with WX-340 a priori or therapeutically after 1 week after tumor cell injection on a daily basis as assessed in an orthotopic model of 4T1 breast cancer in WT mice (mean±SEM for n=4-6 mice per group; *p<0.05 vs. drug vehicle; One-way ANOVA).


( f ) Numbers of neutrophils and 4T1 tumor cells in lungs and brain of WT mice receiving WX - 340 or vehicle therapeutically after 1 week after tumor cell injection on a daily basis as assessed 14 days after intravenous injection of 4T1 tumor cells by multi - channel flow cytometry , quantitative data are shown ( mean ± SEM for n = 4 - 6 mice per group ; * p < 0 . 05 vs . drug vehicle ; t test ) .

O O O O O B-CELL_TYPE O B-CELL_LINE O O O B-TISSUE O B-TISSUE O O B-ORGANISM 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 B-CELL_LINE 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 B-ORGANISM O O O O O O O O O O O O O O O O O O

(f) Numbers of neutrophils and 4T1 tumor cells in lungs and brain of WT mice receiving WX-340 or vehicle therapeutically after 1 week after tumor cell injection on a daily basis as assessed 14 days after intravenous injection of 4T1 tumor cells by multi-channel flow cytometry, quantitative data are shown (mean±SEM for n=4-6 mice per group; *p<0.05 vs. drug vehicle; t test).


( A ) Representative EPSP and mEPSP electrophysiological traces from larval NMJs at Muscle 6 in which a motoneuron specific driver was used to overexpress either a control transgene ( DVGlut - GAL4 > UAS - RFP ) , dNmnat ( DVGlut - Gal4 > UAS - dNmnat ) , or Wnd ( DVGlut - Gal4 > UAS - Wnd ) .

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

(A) Representative EPSP and mEPSP electrophysiological traces from larval NMJs at Muscle 6 in which a motoneuron specific driver was used to overexpress either a control transgene (DVGlut-GAL4 > UAS-RFP), dNmnat (DVGlut-Gal4 > UAS-dNmnat), or Wnd (DVGlut-Gal4 > UAS-Wnd).


( B ) Quantification of mean ( ± SEM ) EPSP amplitudes , in which 75 consecutive evoked events were averaged per cell , and then cell amplitudes were averaged per genotype . [ UAS - RFP n = 13 cells , UAS - dNmnat n = 14 cells , UAS - Wnd n = 10 cells . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 36 , F = 12 . 66 , p < 0 . 0001 . UAS - RFP vs UAS - dNmnat p < 0 . 0001 ( * * * * ) , UAS - RFP vs UAS - Wnd p = 0 . 0093 ( * * ) ] .

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

(B) Quantification of mean (±SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then cell amplitudes were averaged per genotype. [UAS-RFP n = 13 cells, UAS-dNmnat n = 14 cells, UAS-Wnd n= 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=36, F=12.66, p<0.0001. UAS-RFP vs UAS-dNmnat p < 0.0001 (****), UAS-RFP vs UAS-Wnd p = 0.0093 (**)].


] . ( C ) Quantification of mean ( ± SEM ) mEPSP amplitudes , in which 75 consecutive spontaneous events were averaged per cell , and then cell amplitudes were averaged per genotype . [ UAS - RFP n = 15 cells , UAS - dNmnat n = 14 cells , UAS - Wnd n = 10 cells . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 38 , F = 11 . 66 , p = 0 . 0001 . UAS - RFP vs UAS - dNmnat p = 0 . 994 ( NS ) , UAS - RFP vs UAS - Wnd p = 0 . 0003 ( * * * ) ] .

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

]. (C) Quantification of mean (±SEM) mEPSP amplitudes, in which 75 consecutive spontaneous events were averaged per cell, and then cell amplitudes were averaged per genotype. [UAS-RFP n = 15 cells, UAS-dNmnat n = 14 cells, UAS-Wnd n= 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=38, F=11.66, p = 0.0001. UAS-RFP vs UAS-dNmnat p=0.994 (NS), UAS-RFP vs UAS-Wnd p = 0.0003 (***)].


( D ) Quantification of quantal content , which was calculated individually per cell by dividing the mean EPSP amplitude by the mean mEPSP amplitude , and then averaged per genotype . [ UAS - RFP n = 15 cells , UAS - dNmnat n = 14 cells , UAS - Wnd n = 10 cells . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 38 , F = 9 . 286 , p = 0 . 0006 . UAS - RFP vs UAS - dNmnat p = 0 . 0015 ( * * ) , UAS - RFP vs UAS - Wnd p = 0 . 0003 ( * * * ) ] .

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

(D) Quantification of quantal content, which was calculated individually per cell by dividing the mean EPSP amplitude by the mean mEPSP amplitude, and then averaged per genotype. [UAS-RFP n = 15 cells, UAS-dNmnat n = 14 cells, UAS-Wnd n= 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=38, F=9.286, p = 0.0006. UAS-RFP vs UAS-dNmnat p=0.0015 (**), UAS-RFP vs UAS-Wnd p = 0.0003 (***)].


] . ( E ) Representative images of the NMJ synaptic terminal at muscle 4 in third - instar larvae . The genotypes in this experiment were identical to those tested in the electrophysiology experiments in A - D . NMJs were stained for the presynaptic bouton marker DVGLUT ( green ) and nerve membrane marker HRP ( red ) .

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

]. (E) Representative images of the NMJ synaptic terminal at muscle 4 in third-instar larvae. The genotypes in this experiment were identical to those tested in the electrophysiology experiments in A-D. NMJs were stained for the presynaptic bouton marker DVGLUT (green) and nerve membrane marker HRP (red).


( F ) Quantification of the mean ( ± SEM ) number of DVGlut + boutons per muscle 4 NMJ in each genotype . [ UAS - RFP n = 23 , UAS - dNmnat n = 21 , UAS - Wnd n = 20 NMJs . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 63 , F = 68 . 31 , p < 0 . 0001 . UAS - RFP vs UAS - dNmnat p = 0 . 9818 ( NS ) , UAS - RFP vs UAS - Wnd p < 0 . 0001 ( * * * * ) ] .

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

(F) Quantification of the mean (±SEM) number of DVGlut+ boutons per muscle 4 NMJ in each genotype. [UAS-RFP n = 23, UAS-dNmnat n = 21, UAS-Wnd n= 20 NMJs. 1-way ANOVA w/Tukey's multiple comparisons, DF=63, F=68.31, p < 0.0001. UAS-RFP vs UAS-dNmnat p=0.9818 (NS), UAS-RFP vs UAS-Wnd p < 0.0001(****)].


( A ) Representative EPSP and mEPSP physiological traces from larval NMJs at Muscle 6 . Recordings were taken from WT control larvae ( Elav - Gal4 > UAS - RFP ) , hiw mutants driving a control transgene ( hiwND8 ; Elav - Gal4 > UAS - RFP ) , and hiw mutants expressing RNAis to knockdown either dNmnat ( hiwND8 ; Elav - Gal4 > UAS - dNmnat - RNAi [ # 29402 ] ) or Wnd ( hiwND8 ; Elav - Gal4 > UAS - Wnd - RNAi [ # 25396 ] ) .

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

(A) Representative EPSP and mEPSP physiological traces from larval NMJs at Muscle 6. Recordings were taken from WT control larvae (Elav-Gal4>UAS-RFP), hiw mutants driving a control transgene (hiwND8;Elav-Gal4 > UAS-RFP), and hiw mutants expressing RNAis to knockdown either dNmnat (hiwND8;Elav-Gal4 > UAS-dNmnat-RNAi [#29402] ) or Wnd (hiwND8;Elav-Gal4 > UAS-Wnd-RNAi [#25396]).


( B ) Quantification of mean ( ± SEM ) EPSP amplitudes , in which 75 consecutive evoked events were averaged per cell , and then cell amplitudes were averaged per genotype . [ WT n = 10 cells , hiw n = 10 cells , hiw > Wnd - RNAi n = 10 cells , hiw > dNmnat - RNAi n = 10 cells . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 39 , F = 14 . 02 , p < 0 . 0001 . WT vs hiw p = 0 . 0012 ( * * ) , WT vs hiw > Wnd - RNAi p < 0 . 0001 ( * * * * ) , WT vs hiw > dNmnat - RNAi p = 0 . 9880 ( NS ) ] .

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

(B) Quantification of mean (±SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then cell amplitudes were averaged per genotype. [WT n = 10 cells, hiw n = 10 cells, hiw > Wnd-RNAi n= 10 cells, hiw > dNmnat-RNAi n = 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=39, F=14.02, p<0.0001. WT vs hiw p = 0.0012 (**), WT vs hiw > Wnd-RNAi p < 0.0001 (****), WT vs hiw > dNmnat-RNAi p = 0.9880 (NS)].


( C ) Quantification of quantal content ( ± SEM ) . [ WT n = 9 cells , hiw n = 9 cells , hiw > Wnd - RNAi n = 10 cells , hiw > dNmnat - RNAi n = 10 cells . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 38 , F = 2 . 508 , p < 0 . 0001 . WT vs hiw p = 0 . 0361 ( * ) , WT vs hiw > Wnd - RNAi p = 0 . 0001 ( * * * ) , WT vs hiw > dNmnat - RNAi p = 0 . 9889 ( NS ) ] .

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

(C) Quantification of quantal content (±SEM). [WT n = 9 cells, hiw n = 9 cells, hiw > Wnd-RNAi n= 10 cells, hiw > dNmnat-RNAi n = 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=38, F= 2.508, p<0.0001. WT vs hiw p = 0.0361 (*), WT vs hiw > Wnd-RNAi p = 0.0001 (***), WT vs hiw > dNmnat-RNAi p = 0.9889 (NS)].


( D ) Representative images of the NMJ synaptic terminal at muscle 4 in third - instar larvae . The genotypes in this experiment were identical to those tested in panels A - C . NMJs were stained for the presynaptic bouton marker DVGLUT ( green ) and nerve membrane marker HRP ( red ) .

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

(D) Representative images of the NMJ synaptic terminal at muscle 4 in third-instar larvae. The genotypes in this experiment were identical to those tested in panels A-C. NMJs were stained for the presynaptic bouton marker DVGLUT (green) and nerve membrane marker HRP (red).


( E ) Quantification of the mean ( ± SEM ) number of DVGlut + boutons per muscle 4 NMJ in each genotype . [ WT n = 23 , hiw n = 21 , hiw > Wnd - RNAi n = 16 , hiw > dNmnat - RNAi n = 19 . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 78 , F = 169 . 1 , p < 0 . 0001 . WT vs hiw p < 0 . 0001 ( * * * * ) , WT vs hiw > Wnd - RNAi p = 0 . 9988 ( NS ) , WT vs hiw > dNmnat - RNAi p < 0 . 0001 ( * * * * ) , hiw vs hiw > dNmnat - RNAi p = 0 . 9695 ( NS ) ] .

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

(E) Quantification of the mean (±SEM) number of DVGlut+ boutons per muscle 4 NMJ in each genotype. [WT n = 23, hiw n = 21 , hiw > Wnd-RNAi n= 16, hiw > dNmnat-RNAi n=19. 1-way ANOVA w/Tukey's multiple comparisons, DF=78, F=169.1, p < 0.0001. WT vs hiw p < 0.0001 (****), WT vs hiw > Wnd-RNAi p = 0.9988 (NS), WT vs hiw > dNmnat-RNAi p < 0.0001 (****), hiw vs hiw > dNmnat-RNAi p = 0.9695 (NS)].


( A ) Representative images of immunostained larval NMJs and active zones from WT , hiw mutant , and hiw mutant driving an RNAi against dNmnat [ # 29402 ] . In the top 3 panels , greyscale images of entire terminal were acquired by staining for nerve marker HRP . Presynaptic Brp puncta ( green ) and postsynaptic dGluRIII clusters ( magenta ) were imaged to quantify N in each genotype .

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

(A) Representative images of immunostained larval NMJs and active zones from WT, hiw mutant, and hiw mutant driving an RNAi against dNmnat [#29402]. In the top 3 panels, greyscale images of entire terminal were acquired by staining for nerve marker HRP. Presynaptic Brp puncta (green) and postsynaptic dGluRIII clusters (magenta) were imaged to quantify N in each genotype.


( B ) Quantification of NMJ synaptic terminal surface area in each genotype . [ WT n = 14 , hiw n = 12 , hiw > dNmnat - RNAi n = 12 . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 37 , F = 22 . 427 , p < 0 . 0001 . WT vs hiw p < 0 . 0001 ( * * * * ) , WT vs hiw > dNmnat - RNAi p < 0 . 0001 ( * * * * ) , hiw vs hiw > dNmnat - RNAi p = 0 . 2907 ( NS ) ] .

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

(B) Quantification of NMJ synaptic terminal surface area in each genotype. [WT n = 14, hiw n = 12 , hiw > dNmnat-RNAi n=12. 1-way ANOVA w/Tukey's multiple comparisons, DF=37, F=22.427, p < 0.0001. WT vs hiw p < 0.0001 (****), WT vs hiw > dNmnat-RNAi p < 0.0001 (****), hiw vs hiw > dNmnat-RNAi p = 0.2907 (NS)].


( C ) Quantification of BRP puncta per NMJ [ WT n = 14 , hiw n = 12 , hiw > dNmnat - RNAi n = 12 . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 37 , F = 2 . 854 , p = 0 . 0711 . WT vs hiw p = 0 . 0573 ( NS ) , WT vs hiw > dNmnat - RNAi p = 0 . 4529 ( NS ) , hiw vs hiw > dNmnat - RNAi p = 0 . 5030 ( NS ) ] .

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

(C) Quantification of BRP puncta per NMJ [WT n = 14, hiw n = 12, hiw > dNmnat-RNAi n=12. 1-way ANOVA w/Tukey's multiple comparisons, DF=37, F=2.854, p = 0.0711. WT vs hiw p = 0.0573 (NS), WT vs hiw > dNmnat-RNAi p = 0.4529 (NS), hiw vs hiw > dNmnat-RNAi p = 0.5030 (NS)].


( D ) Quantification of percentage of Brp puncta that were apposed to a dGluRIII clusters at each NMJ [ WT n = 14 , hiw n = 12 , hiw > dNmnat - RNAi n = 12 . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 37 , F = 2 . 093 , p = 0 . 4716 . WT vs hiw p = 0 . 2168 ( NS ) , WT vs hiw > dNmnat - RNAi p = 0 . 1882 ( NS ) , hiw vs hiw > dNmnat - RNAi p = 0 . 9966 ( NS ) ] .

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

(D) Quantification of percentage of Brp puncta that were apposed to a dGluRIII clusters at each NMJ [WT n = 14, hiw n = 12, hiw > dNmnat-RNAi n=12. 1-way ANOVA w/Tukey's multiple comparisons, DF=37, F=2.093, p = 0.4716. WT vs hiw p = 0.2168 (NS), WT vs hiw > dNmnat-RNAi p = 0.1882 (NS), hiw vs hiw > dNmnat-RNAi p = 0.9966 (NS)].


( E ) Representative electrophysiological traces from a synaptic facilitation experiment in which trains of 5 pulses were used to evoke consecutive action potentials at 100 ms ISI . Genotypes for these experiments were : WT ( elav > UAS - RFP ) , hiw mutant ( hiw ; elav > UAS - RFP ) , or hiw depleted of dNmnat ( hiw ; elav > dNmnat - RNAi ) . ( F ) Quantification of the facilitation index , in which the amplitude of the 5th pulse is divided by the amplitude of the 1st pulse . [ 100 ms ISI : WT n = 11 , hiw n = 9 , hiw > dNmnat - RNAi n = 12 . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 31 , F = 7 . 546 , p = 0 . 0023 . WT vs hiw p = 0 . 0020 ( * * ) , hiw vs hiw > dNmnat - RNAi p = 0 . 0229 ( * ) , WT vs hiw > dNmnat - RNAi p = 0 . 5141 ( NS ) . 50 ms ISI : WT n = 11 , hiw n = 9 , hiw > dNmnat - RNAi n = 12 . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 31 , F = 5 . 587 , p = 0 . 0089 . WT vs hiw p = 0 . 0129 ( * ) , hiw vs hiw > dNmnat - RNAi p = 0 . 0217 ( * ) , WT vs hiw > dNmnat - RNAi p = 0 . 9546 ( NS ) . ]

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

(E) Representative electrophysiological traces from a synaptic facilitation experiment in which trains of 5 pulses were used to evoke consecutive action potentials at 100 ms ISI. Genotypes for these experiments were: WT (elav>UAS-RFP), hiw mutant (hiw; elav>UAS-RFP), or hiw depleted of dNmnat (hiw; elav>dNmnat-RNAi). (F) Quantification of the facilitation index, in which the amplitude of the 5th pulse is divided by the amplitude of the 1st pulse. [100 ms ISI: WT n=11, hiw n= 9, hiw>dNmnat-RNAi n= 12. 1-way ANOVA w/Tukey's multiple comparisons, DF = 31, F= 7.546, p =0.0023. WT vs hiw p =0.0020 (**), hiw vs hiw> dNmnat-RNAi p= 0.0229 (*), WT vs hiw> dNmnat-RNAi p= 0.5141 (NS). 50 ms ISI: WT n=11, hiw n= 9, hiw>dNmnat-RNAi n= 12. 1-way ANOVA w/Tukey's multiple comparisons, DF = 31, F= 5.587, p =0.0089. WT vs hiw p =0.0129 (*), hiw vs hiw> dNmnat-RNAi p= 0.0217 (*), WT vs hiw> dNmnat-RNAi p= 0.9546 (NS).]


( A ) Representative images of NMJ synaptic terminals in control ( DVGlut - Gal4 > UAS - HA - dNmnat ) and hiw mutant ( hiwND9 , DVGlut - Gal4 > UAS - HA - dNmnat ) larvae . NMJs at larval muscle 4 were identified using HRP ( blue in top panel ) immunostaining . Insets highlight active zones within single boutons , which were identified by staining for the presynaptic active zone protein Brp ( red ) . An anti - HA antibody was used to stain for an HA - tagged dNmnat ( green ) transgene to measure protein levels in control and hiw mutant active zones .

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

(A) Representative images of NMJ synaptic terminals in control (DVGlut-Gal4 > UAS-HA-dNmnat) and hiw mutant (hiwND9,DVGlut-Gal4 > UAS-HA-dNmnat) larvae. NMJs at larval muscle 4 were identified using HRP (blue in top panel) immunostaining. Insets highlight active zones within single boutons, which were identified by staining for the presynaptic active zone protein Brp (red). An anti-HA antibody was used to stain for an HA-tagged dNmnat (green) transgene to measure protein levels in control and hiw mutant active zones.


( B ) Quantification of HA - dNmnat levels across the entire NMJ synaptic terminal , normalized to total terminal area and presented as fold change over WT . [ WT > HA - dNmnat n = 10 terminals , hiw > HA - dNmnat n = 10 terminals . Unpaired T - test , DF = 18 , p = 0 . 0028 ( * * ) ] .

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

(B) Quantification of HA-dNmnat levels across the entire NMJ synaptic terminal, normalized to total terminal area and presented as fold change over WT. [WT > HA-dNmnat n = 10 terminals, hiw > HA-dNmnat n = 10 terminals. Unpaired T-test, DF = 18, p = 0.0028 (**)].


( C ) Quantification of HA - dNmnat levels per unit area in WT and hiw for synaptic terminals excluding active zones ( herein referred to as " terminals " ) versus active zones ( AZs ) only . [ WT n = 10 terminals , hiw n = 9 terminals , each parsed into terminal minus AZs and AZs only . 2 - way ANOVA with Tukey ' s multiple comparisons , DF = 34 , p = 0 . 0005 for row factor ( terminal versus AZs ) and p < 0 . 0001 for column factor ( WT vs hiw ) . WT terminal vs hiw terminal p = 0 . 9830 ( NS ) . Hiw terminal vs hiw AZs p < 0 . 0001 ( * * * * ) . WT AZs vs hiw AZs p < 0 . 0001 ( * * * * ) . ]

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

(C) Quantification of HA-dNmnat levels per unit area in WT and hiw for synaptic terminals excluding active zones (herein referred to as "terminals") versus active zones (AZs) only. [WT n = 10 terminals, hiw n = 9 terminals, each parsed into terminal minus AZs and AZs only. 2-way ANOVA with Tukey's multiple comparisons, DF = 34, p = 0.0005 for row factor (terminal versus AZs) and p < 0.0001 for column factor (WT vs hiw). WT terminal vs hiw terminal p = 0.9830 (NS). Hiw terminal vs hiw AZs p < 0.0001 (****). WT AZs vs hiw AZs p < 0.0001 (****).]


A ) Representative electron micrographs of entire boutons in wild type , hiw ; wnd , and hiw ; wnd , Elav - GAL4 > dNmnat RNAi NMJs .

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

A) Representative electron micrographs of entire boutons in wild type, hiw;wnd, and hiw;wnd, Elav-GAL4 > dNmnat RNAi NMJs.


B ) High magnification electron micrographs showing representative " T - bar " structures at active zones in NMJs of the indicated genotypes . Red arrows denote the beginning and end of the T - bar top , demonstrating changes in T - bar width in the genotypes tested . As illustrated , a significant reduction in T - bar size is observed in hiw ; wnd , and this defect is suppressed in hiw ; wnd , Elav - GAL4 > dNmnat RNAi .

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

B) High magnification electron micrographs showing representative "T-bar" structures at active zones in NMJs of the indicated genotypes. Red arrows denote the beginning and end of the T-bar top, demonstrating changes in T-bar width in the genotypes tested. As illustrated, a significant reduction in T-bar size is observed in hiw;wnd, and this defect is suppressed in hiw;wnd, Elav-GAL4 > dNmnat RNAi.


C ) Quantification of mean T - bar width [ WT n = 90 , hiw ; wnd n = 55 , hiw ; wnd , Elav - GAL4 > dNmnat RNAi n = 81 . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 225 , F = 13 . 829 , p < 0 . 0001 . WT vs hiw ; wnd p < 0 . 0001 ( * * * * ) , WT vs hiw ; wnd , Elav - GAL4 > dNmnat RNAi p = 0 . 9318 ( NS ) , hiw ; wnd vs hiw ; wnd , Elav - GAL4 > dNmnat RNAi p < 0 . 0001 ( * * * * ) ] . ( D ) Analysis of cumulative frequency distribution of T - bar width shows a left shift in distribution in hiw ; wnd but no change in hiw ; wnd , Elav - GAL4 > dNmnat RNAi compared with WT NMJs [ WT vs hiw ; wnd K - S test , D = 0 . 5636 , p < 0 . 0001 ( * * * * ) , WT vs hiw ; wnd , Elav - GAL4 > dNmnat RNAi K - S test , D = 0 . 1815 , p = 0 . 1240 ( NS ) , hiw ; wnd vs hiw ; wnd , Elav - GAL4 > dNmnat RNAi K - S test , D = 0 . 4121 , p < 0 . 0001 ( * * * * ) ] .

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

C) Quantification of mean T-bar width [WT n = 90, hiw;wnd n = 55, hiw;wnd, Elav-GAL4 > dNmnat RNAi n=81. 1-way ANOVA w/Tukey's multiple comparisons, DF=225, F=13.829, p < 0.0001. WT vs hiw;wnd p < 0.0001 (****), WT vs hiw;wnd, Elav-GAL4 > dNmnat RNAi p = 0.9318 (NS), hiw;wnd vs hiw;wnd, Elav-GAL4 > dNmnat RNAi p < 0.0001 (****)]. (D) Analysis of cumulative frequency distribution of T-bar width shows a left shift in distribution in hiw;wnd but no change in hiw;wnd, Elav-GAL4 > dNmnat RNAi compared with WT NMJs [WT vs hiw;wnd K-S test, D=0.5636, p < 0.0001 (****), WT vs hiw;wnd, Elav-GAL4 > dNmnat RNAi K-S test, D=0.1815, p = 0.1240 (NS), hiw;wnd vs hiw;wnd, Elav-GAL4 > dNmnat RNAi K-S test, D=0.4121, p < 0.0001 (****)].


] . ( E ) Quantification of mean T - bar height [ WT n = 90 , hiw ; wnd n = 55 , hiw ; wnd , Elav - GAL4 > dNmnat RNAi n = 81 . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 225 , F = 10 . 472 , p < 0 . 0001 . WT vs hiw ; wnd p = 0 . 0008 ( * * * ) , WT vs hiw ; wnd , Elav - GAL4 > dNmnat RNAi p = 0 . 6694 ( NS ) , hiw ; wnd vs hiw ; wnd , Elav - GAL4 > dNmnat RNAi p < 0 . 0001 ( * * * * ) ] ( F ) Analysis of cumulative frequency distribution of T - bar height shows a left shift in distribution in hiw ; wnd but no change in hiw ; wnd , Elav - GAL4 > dNmnat RNAi compared with WT NMJs [ WT vs hiw ; wnd K - S test , D = 0 . 3343 , p = 0 . 001 ( * * * ) , WT vs hiw ; wnd , Elav - GAL4 > dNmnat RNAi K - S test , D = 0 . 1012 , p = 0 . 7748 ( NS ) , hiw ; wnd vs hiw ; wnd , Elav - GAL4 > dNmnat RNAi K - S test , D = 0 . 3484 , p = 0 . 0008 ( * * * ) ] .

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

]. (E) Quantification of mean T-bar height [WT n = 90, hiw;wnd n = 55, hiw;wnd, Elav-GAL4 > dNmnat RNAi n=81. 1-way ANOVA w/Tukey's multiple comparisons, DF=225, F=10.472, p < 0.0001. WT vs hiw;wnd p = 0.0008 (***), WT vs hiw;wnd, Elav-GAL4 > dNmnat RNAi p = 0.6694 (NS), hiw;wnd vs hiw;wnd, Elav-GAL4 > dNmnat RNAi p < 0.0001 (****)] (F) Analysis of cumulative frequency distribution of T-bar height shows a left shift in distribution in hiw;wnd but no change in hiw;wnd, Elav-GAL4 > dNmnat RNAi compared with WT NMJs [WT vs hiw;wnd K-S test, D=0.3343, p = 0.001 (***), WT vs hiw;wnd, Elav-GAL4 > dNmnat RNAi K-S test, D=0.1012, p = 0.7748 (NS), hiw;wnd vs hiw;wnd, Elav-GAL4 > dNmnat RNAi K-S test, D=0.3484, p = 0.0008 (***)].


( B ) Representative EPSP and mEPSP electrophysiological traces from larval NMJs at Muscle 6 in which a motoneuron specific driver was used to overexpress either a control transgene ( DVGlut - GAL4 > UAS - RFP ) , or one of the catalytic dNmnat mutants ( DVGlut - Gal4 > UAS - WR - dNmnat , DVGlut - Gal4 > UAS - H30A - dNmnat ) .

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

(B) Representative EPSP and mEPSP electrophysiological traces from larval NMJs at Muscle 6 in which a motoneuron specific driver was used to overexpress either a control transgene (DVGlut-GAL4 > UAS-RFP), or one of the catalytic dNmnat mutants (DVGlut-Gal4 > UAS-WR-dNmnat, DVGlut-Gal4 > UAS-H30A-dNmnat).


( C ) Quantification of mean ( ± SEM ) EPSP amplitudes , in which 75 consecutive evoked events were averaged per cell , and then cell amplitudes were averaged per genotype . [ UAS - RFP n = 8 cells , UAS - WR - dNmnat n = 7 cells , UAS - H30A - dNmnat n = 7 cells . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 21 , F = 0 . 8596 , p = 0 . 4383 . UAS - RFP vs UAS - WR - dNmnat p = 0 . 8810 ( NS ) , UAS - RFP vs UAS - H30A - dNmnat p = 0 . 9997 ( NS ) ] .

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

(C) Quantification of mean (±SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then cell amplitudes were averaged per genotype. [UAS-RFP n = 8 cells, UAS-WR-dNmnat n = 7 cells, UAS-H30A-dNmnat n= 7 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=21, F=0.8596, p = 0.4383. UAS-RFP vs UAS-WR-dNmnat p = 0.8810 (NS), UAS-RFP vs UAS-H30A-dNmnat p = 0.9997 (NS)].


( D ) Quantification of quantal content ( ± SEM ) [ UAS - RFP n = 8 cells , UAS - WR - dNmnat n = 7 cells , UAS - H30A - dNmnat n = 7 cells . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 21 , F = 1 . 154 , p = 0 . 3365 . UAS - RFP vs UAS - WR - dNmnat p = 0 . 8696 ( NS ) , UAS - RFP vs UAS - H30A - dNmnat p = 0 . 9697 ( NS ) ] .

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

(D) Quantification of quantal content (±SEM) [UAS-RFP n = 8 cells, UAS-WR-dNmnat n = 7 cells, UAS-H30A-dNmnat n= 7 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=21, F=1.154, p = 0.3365. UAS-RFP vs UAS-WR-dNmnat p = 0.8696 (NS), UAS-RFP vs UAS-H30A-dNmnat p = 0.9697 (NS)].


] . ( E ) Representative EPSP and mEPSP electrophysiological traces from larval NMJs at Muscle 6 . Recordings were taken from WT control larvae ( Elav - Gal4 > UAS - RFP ) , hiw mutants driving a control transgene ( hiwND8 ; Elav - Gal4 > UAS - RFP ) , and hiw mutants expressing an RNAi to knockdown NAD + syn ( hiwND8 ; Elav - Gal4 > UAS - NAD + syn - RNAi ) .

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

]. (E) Representative EPSP and mEPSP electrophysiological traces from larval NMJs at Muscle 6. Recordings were taken from WT control larvae (Elav-Gal4>UAS-RFP), hiw mutants driving a control transgene (hiwND8;Elav-Gal4 > UAS-RFP), and hiw mutants expressing an RNAi to knockdown NAD+syn (hiwND8;Elav-Gal4 > UAS-NAD+syn-RNAi).


( F ) Quantification of mean ( ± SEM ) EPSP amplitudes , in which 75 consecutive evoked events were averaged per cell , and then cell amplitudes were averaged per genotype . [ WT n = 10 cells , hiw n = 10 cells , hiw > NADsyn - RNAi n = 10 cells . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 29 , F = 14 . 02 , p < 0 . 0001 . WT vs hiw p < 0 . 0001 ( * * * * ) , WT vs hiw > NADsyn - RNAi p = 0 . 5147 ( NS ) ] .

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

(F) Quantification of mean (±SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then cell amplitudes were averaged per genotype. [WT n = 10 cells, hiw n = 10 cells, hiw > NADsyn-RNAi n = 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=29, F=14.02, p<0.0001. WT vs hiw p <0.0001 (****), WT vs hiw > NADsyn-RNAi p = 0.5147 (NS)].


] . ( G ) Quantification of quantal content . [ WT n = 10 cells , hiw n = 10 cells , hiw > NADsyn - RNAi n = 10 cells . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 29 , F = 6 . 635 , p = 0 . 0047 . WT vs hiw p = 0 . 0063 ( * * ) , WT vs hiw > NADsyn - RNAi p = 0 . 8942 ( NS ) ] .

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

]. (G) Quantification of quantal content. [WT n = 10 cells, hiw n = 10 cells, hiw > NADsyn-RNAi n = 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=29, F= 6.635, p = 0.0047. WT vs hiw p = 0.0063 (**), WT vs hiw > NADsyn-RNAi p = 0.8942 (NS)].


( H ) Representative images of the NMJ synaptic terminal at muscle 4 in third - instar larvae . The genotypes in this experiment were identical to those tested in panels E - G . NMJs were stained for the presynaptic bouton marker DVGLUT ( green ) and nerve membrane marker HRP ( red ) .

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

(H) Representative images of the NMJ synaptic terminal at muscle 4 in third-instar larvae. The genotypes in this experiment were identical to those tested in panels E-G. NMJs were stained for the presynaptic bouton marker DVGLUT (green) and nerve membrane marker HRP (red).


( I ) Quantification of the mean ( ± SEM ) number of DVGlut + boutons per muscle 4 NMJ in each genotype . [ WT n = 25 , hiw n = 21 , hiw > NADsyn - RNAi n = 24 . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 67 , F = 141 . 3 , p < 0 . 0001 . WT vs hiw p < 0 . 0001 ( * * * * ) , WT vs hiw > Nadysn - RNAi p > 0 . 0001 ( * * * * ) ] .

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

(I) Quantification of the mean (±SEM) number of DVGlut+ boutons per muscle 4 NMJ in each genotype. [WT n = 25, hiw n = 21, hiw > NADsyn-RNAi n=24. 1-way ANOVA w/Tukey's multiple comparisons, DF=67, F=141.3, p < 0.0001. WT vs hiw p < 0.0001 (****), WT vs hiw > Nadysn-RNAi p > 0.0001 (****)].


Cross - sectional and longitudinal analysis of the association between amyloid - PET ( in centiloid ) , CSF p - tau181 , and CSF sTREM2 in early Aβ - accumulators ( i . e . Aβ CSF + / PET − ; n = 70 ) and late Aβ - accumulators ( i . e . Aβ CSF + / PET + ; n = 201 ) . Cross - sectional linear regressions between A ) centiloid and p - tau181 and B ) centiloid and sTREM2 . Standardized beta - estimates ( β ) , T - values , and p - values were derived from linear regressions controlling for age , sex , education , and clinical status .

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

Cross-sectional and longitudinal analysis of the association between amyloid-PET (in centiloid), CSF p-tau181, and CSF sTREM2 in early Aβ-accumulators (i.e. Aβ CSF+/PET−; n=70) and late Aβ-accumulators (i.e. Aβ CSF+/PET+; n=201). Cross-sectional linear regressions between A) centiloid and p-tau181 and B) centiloid and sTREM2. Standardized beta-estimates (β), T-values, and p-values were derived from linear regressions controlling for age, sex, education, and clinical status.


Cross - sectional and longitudinal analysis of the association between amyloid - PET ( in centiloid ) , CSF p - tau181 , and CSF sTREM2 in early Aβ - accumulators ( i . e . Aβ CSF + / PET − ; n = 70 ) and late Aβ - accumulators ( i . e . Aβ CSF + / PET + ; n = 201 ) . Longitudinal linear regressions between C ) centiloid and change in p - tau181 and D ) centiloid and change in sTREM2 . Standardized beta - estimates ( β ) , T - values , and p - values were derived from linear regressions controlling for age , sex , education , and clinical status .

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

Cross-sectional and longitudinal analysis of the association between amyloid-PET (in centiloid), CSF p-tau181, and CSF sTREM2 in early Aβ-accumulators (i.e. Aβ CSF+/PET−; n=70) and late Aβ-accumulators (i.e. Aβ CSF+/PET+; n=201). Longitudinal linear regressions between C) centiloid and change in p-tau181 and D) centiloid and change in sTREM2. Standardized beta-estimates (β), T-values, and p-values were derived from linear regressions controlling for age, sex, education, and clinical status.


B ) T - value projection of the association between sTREM2 on FDG - PET , stratified by group . FDG - PET z - scores were derived by referencing FDG - PET SUVRs to cognitively normal controls ( i . e . n = 131 ; Aβ CSF − / PET − ) .

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

B) T-value projection of the association between sTREM2 on FDG-PET, stratified by group. FDG-PET z-scores were derived by referencing FDG-PET SUVRs to cognitively normal controls (i.e. n=131; Aβ CSF−/PET−).


Bright field ( BF ) , red ( mCherry ) and green ( Atoh1 - nGFP ) fluorescent images of organoid cultures after 10 days of expansion . Scale bars = 400µm .

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

Bright field (BF), red (mCherry) and green (Atoh1-nGFP) fluorescent images of organoid cultures after 10 days of expansion. Scale bars = 400µm.


High power single plane confocal images of EdU labeled control and TRIM71 - expressing organoids . An EdU pulse was given on day 8 and EdU incorporation ( green ) was analyzed 1 . 5 hours later . JAG1 immunostaining ( magenta ) marks supporting cells / prosensory cells , mCherry ( red ) marks transduced cells , Hoechst labels cell nuclei ( blue ) . Scale bar = 25µm . Percentage of EdU + mCherry + cells per organoids in ( I ) ; ( n = 5 , two independent experiments ) . P - values were calculated using two - tailed , unpaired t test . * * P < 0 . 01 . Data information : Plotted are individual data points , representing the average value per animal and treatment , and mean ± standard deviation ( SD ) of biological replicates .

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

High power single plane confocal images of EdU labeled control and TRIM71-expressing organoids. An EdU pulse was given on day 8 and EdU incorporation (green) was analyzed 1.5 hours later. JAG1 immunostaining (magenta) marks supporting cells/prosensory cells, mCherry (red) marks transduced cells, Hoechst labels cell nuclei (blue). Scale bar= 25µm. Percentage of EdU+ mCherry+ cells per organoids in (I); (n = 5, two independent experiments). P-values were calculated using two-tailed, unpaired t test. **P < 0.01. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ±standard deviation (SD) of biological replicates.


Bright field ( BF ) , red ( mCherry ) and green ( Atoh1 - nGFP ) fluorescent images of organoid cultures after 2 days of differentiation . Scale bar = 400µm .

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

Bright field (BF), red (mCherry) and green (Atoh1-nGFP) fluorescent images of organoid cultures after 2 days of differentiation. Scale bar = 400µm.


RT - qPCR of Atoh1 mRNA in organoids ( n = 3 , two independent experiments ) . P - values were calculated using one - way ANOVA with Tukey ' s correction . * * * * P < 0 . 0001 . Data information : Plotted are individual data points , representing the average value per animal and treatment , and mean ± SD of biological replicates .

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

RT-qPCR of Atoh1 mRNA in organoids (n=3, two independent experiments). P-values were calculated using one-way ANOVA with Tukey's correction. ****P < 0.0001. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.


High power single plane confocal images of MYO7A immuno - stained ( magenta ) organoids . Atoh1 - GFP ( green ) and MYO7A ( magenta ) mark nascent hair cells . MCherry ( red ) marks transduced cells . Scale bar = 20µm . Quantification of MYO7A + mCherry + cells per organoid in ( E ) ( n = 3 , two independent experiments ) . P - values were calculated using one - way ANOVA with Tukey ' s correction . P > 0 . 5 deemed not significant ( n . s . ) . Data information : Plotted are individual data points , representing the average value per animal and treatment , and mean ± SD of biological replicates .

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

High power single plane confocal images of MYO7A immuno-stained (magenta) organoids. Atoh1-GFP (green) and MYO7A (magenta) mark nascent hair cells. MCherry (red) marks transduced cells. Scale bar = 20µm. Quantification of MYO7A+ mCherry+ cells per organoid in (E) (n=3, two independent experiments). P-values were calculated using one-way ANOVA with Tukey's correction. P>0.5 deemed not significant (n.s.). Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.


RT - PCR of Myo7a mRNA expression in organoids ( n = 3 , two independent experiments ) . P - values were calculated using one - way ANOVA with Tukey ' s correction . * P ≤ 0 . 05 . Data information : Plotted are individual data points , representing the average value per animal and treatment , and mean ± SD of biological replicates .

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RT-PCR of Myo7a mRNA expression in organoids (n=3, two independent experiments). P-values were calculated using one-way ANOVA with Tukey's correction. *P ≤ 0.05. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.


Colony forming efficiency ( n = 4 in WT , n = 5 in Trim71 KO , two independent experiments ) . A two - tailed , unpaired t test was used to calculate P values . n . s , not significant . Organoid diameter ( n = 4 in WT , n = 5 in Trim71 KO , two independent experiments ) . A two - tailed , unpaired t test was used to calculate P values . n . s , not significant . Data information : Plotted are individual data points , representing the average value per animal and treatment , and mean ± SD of biological replicates .

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Colony forming efficiency (n=4 in WT, n=5 in Trim71 KO, two independent experiments). A two-tailed, unpaired t test was used to calculate P values. n.s, not significant. Organoid diameter (n=4 in WT, n=5 in Trim71 KO, two independent experiments). A two-tailed, unpaired t test was used to calculate P values. n.s, not significant. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.


High power single plane confocal images of EdU ( red ) and Hoechst ( blue ) labeled WT and TRIM71 KO organoids . A single EdU pulse was given on day 7 and EdU incorporation was analyzed 1 . 5 hours later . Scale bars = 25µm . Percentage of EdU + cells in ( F ) ( n = 15 in WT , n = 11 in Trim71 KO , three independent experiments ) . Two - tailed , unpaired t test was used to calculate P values , n . s . not significant . Data information : Plotted are individual data points , representing the average value per animal and treatment , and mean ± SD of biological replicates .

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

High power single plane confocal images of EdU (red) and Hoechst (blue) labeled WT and TRIM71 KO organoids. A single EdU pulse was given on day 7 and EdU incorporation was analyzed 1.5 hours later. Scale bars = 25µm. Percentage of EdU+ cells in (F) (n=15 in WT, n=11 in Trim71 KO, three independent experiments). Two-tailed, unpaired t test was used to calculate P values, n.s. not significant. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.


RT - qPCR of Trim71 and hair cell - specific ( Atoh1 , Gfi1 and Pou4f3 ) mRNAs in WT and Trim71 KO organoids after 2 days of differentiation ( n = 3 , two independent experiments ) . A two - tailed , unpaired t test was used to calculate P values . * P < 0 . 05 . Data information : Plotted are individual data points , representing the average value per animal and treatment , and mean ± SD of biological replicates .

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

RT-qPCR of Trim71 and hair cell-specific (Atoh1, Gfi1 and Pou4f3) mRNAs in WT and Trim71 KO organoids after 2 days of differentiation (n=3, two independent experiments). A two-tailed, unpaired t test was used to calculate P values. *P < 0.05. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.


Cochlear epithelial cells isolated from P5 wild type mice were transduced with control ( Ctrl ) or TRIM71 - expressing lentivirus and bulk RNA sequencing was used to analyze differential gene expression after 10 days of expansion . Volcano plot of RNA - seq data . Plotted is beta - value ( x - axis ) versus − log10 q - value ( y - axis ) . Transcripts that are significantly upregulated in response to TRIM71 expression are marked in red circles , and transcripts that are significantly downregulated are marked in blue circles .

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

Cochlear epithelial cells isolated from P5 wild type mice were transduced with control (Ctrl) or TRIM71-expressing lentivirus and bulk RNA sequencing was used to analyze differential gene expression after 10 days of expansion. Volcano plot of RNA-seq data. Plotted is beta-value (x-axis) versus −log10 q-value (y-axis). Transcripts that are significantly upregulated in response to TRIM71 expression are marked in red circles, and transcripts that are significantly downregulated are marked in blue circles.


Immunoblots showing LIN28B , AGO2 , HMGA2 , P - SMAD1 / 5 / 9 and β - actin protein levels in P5 control , TRIM71 and △ NHL expressing cochlear organoids at 10 days of expansion . Note β - actin was used to normalize protein levels .

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Immunoblots showing LIN28B, AGO2, HMGA2, P-SMAD1/5/9 and β-actin protein levels in P5 control, TRIM71 and △NHL expressing cochlear organoids at 10 days of expansion. Note β-actin was used to normalize protein levels.


Cochlear epithelial cells isolated from P5 wild type were transduced with control ( Ctrl ) or TRIM71 - expressing lentivirus and expanded as organoids . MCherry ( red ) marks transduced cells . Hoechst staining ( blue ) labels cell nuclei . High power single plane confocal images of control ( Ctrl ) and TRIM71 - expressing organoids immuno - stained for progenitor / supporting cell marker JAG1 ( magenta ) and progenitor marker HMGA2 ( green ) after 10 days of expansion . Scale bars = 25µm . Quantification of JAG1 + mCherry + HMGA2 + cells per organoid in ( A ) ( n = 3 , two independent experiments ) . A two - tailed , unpaired t test was used to calculate P values . * * * P < 0 . 001 . Data information : Plotted are individual data points , representing the average value per animal and treatment , and mean ± SD of biological replicates .

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

Cochlear epithelial cells isolated from P5 wild type were transduced with control (Ctrl) or TRIM71-expressing lentivirus and expanded as organoids. MCherry (red) marks transduced cells. Hoechst staining (blue) labels cell nuclei. High power single plane confocal images of control (Ctrl) and TRIM71-expressing organoids immuno-stained for progenitor/supporting cell marker JAG1 (magenta) and progenitor marker HMGA2 (green) after 10 days of expansion. Scale bars=25µm. Quantification of JAG1+ mCherry+ HMGA2+ cells per organoid in (A) (n=3, two independent experiments). A two-tailed, unpaired t test was used to calculate P values. ***P < 0.001. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.


Cochlear epithelial cells isolated from P5 wild type were transduced with control ( Ctrl ) or TRIM71 - expressing lentivirus and expanded as organoids . MCherry ( red ) marks transduced cells . Hoechst staining ( blue ) labels cell nuclei . High power confocal images of control ( Ctrl ) or TRIM71 - expressing organoids immuno - stained for progenitor / supporting cell marker JAG1 ( magenta ) and supporting cell marker S100A1 ( green ) after 10 days of expansion . Scale bars = 25µm . Quantification of JAG1 + mCherry + S100A1 + cells per organoid in ( C ) ( n = 3 , two independent experiments ) . A two - tailed , unpaired t test was used to calculate P values . * * P < 0 . 01 . Data information : Plotted are individual data points , representing the average value per animal and treatment , and mean ± SD of biological replicates .

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

Cochlear epithelial cells isolated from P5 wild type were transduced with control (Ctrl) or TRIM71-expressing lentivirus and expanded as organoids. MCherry (red) marks transduced cells. Hoechst staining (blue) labels cell nuclei. High power confocal images of control (Ctrl) or TRIM71-expressing organoids immuno-stained for progenitor/supporting cell marker JAG1 (magenta) and supporting cell marker S100A1 (green) after 10 days of expansion. Scale bars=25µm. Quantification of JAG1+ mCherry+ S100A1+ cells per organoid in (C) (n=3, two independent experiments). A two-tailed, unpaired t test was used to calculate P values. **P < 0.01. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.


Cochlear epithelial cells isolated from Atoh1 - nGFP transgenic mice ( E , F ) were transduced with control ( Ctrl ) or TRIM71 - expressing lentivirus and expanded as organoids . MCherry ( red ) marks transduced cells . Hoechst staining ( blue ) labels cell nuclei . High power single plane confocal images of control ( Ctrl ) or TRIM71 - expressing organoids immuno - stained for transcription factor NFIB ( magenta ) after 2 days of differentiation . Atoh1 - nGFP ( green ) marks nascent hair cells . White arrowheads mark Atoh1 - GFP + cells with low NFIB expression ( NFIBlow ) . Scale bars = 25µm . Quantification of NFIBlow , NFIBhigh , NFIBlowAtoh1 - nGFP + , NFIBhigh Atoh1 - nGFP + cells in control ( Ctrl ) and TRIM71 - expressing organoids ( n = 4 , two independent experiments ) . Two - way ANOVA with Tukey ' s correction was used to calculate P values . * * * * P < 0 . 0001 . Data information : Plotted are individual data points , representing the average value per animal and treatment , and mean ± SD of biological replicates .

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

Cochlear epithelial cells isolated from Atoh1-nGFP transgenic mice (E, F) were transduced with control (Ctrl) or TRIM71-expressing lentivirus and expanded as organoids. MCherry (red) marks transduced cells. Hoechst staining (blue) labels cell nuclei. High power single plane confocal images of control (Ctrl) or TRIM71-expressing organoids immuno-stained for transcription factor NFIB (magenta) after 2 days of differentiation. Atoh1-nGFP (green) marks nascent hair cells. White arrowheads mark Atoh1-GFP+ cells with low NFIB expression (NFIBlow). Scale bars=25µm. Quantification of NFIBlow, NFIBhigh, NFIBlowAtoh1-nGFP+, NFIBhigh Atoh1-nGFP+ cells in control (Ctrl) and TRIM71-expressing organoids (n=4, two independent experiments). Two-way ANOVA with Tukey's correction was used to calculate P values. ****P < 0.0001. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.


Bright field ( BF ) and red and green fluorescent images of organoid cultures at 2 days of differentiation . MCherry ( red ) marks transduced cells and Atoh1 - nGFP ( green ) marks nascent hair cells . Scale bars = 400µm .

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Bright field (BF) and red and green fluorescent images of organoid cultures at 2 days of differentiation. MCherry (red) marks transduced cells and Atoh1-nGFP (green) marks nascent hair cells. Scale bars =400µm.


Percentage of mCherry + Atoh1 - nGFP + organoids in ( B ) ( n = 5 for Lin28b group , n = 6 for all the other groups ; two independent experiments ) . One - way ANOVA with Tukey ' s correction was used to calculate P values . * P ≤ 0 . 05 , * * P < 0 . 01 and * * * * P < 0 . 0001 . Data information : Plotted are individual data points , representing the average value per animal and treatment , and mean ± SD of biological replicates .

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Percentage of mCherry+Atoh1-nGFP+ organoids in (B) (n=5 for Lin28b group, n=6 for all the other groups; two independent experiments). One-way ANOVA with Tukey's correction was used to calculate P values. *P ≤ 0.05, **P < 0.01 and ****P < 0.0001. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.


RT - qPCR - analysis of progenitor ( Hmga2 ) and supporting cell - specific ( Zbtb20 , Nfib ) gene expression in control , Lin28b , TRIM71 or Lin28b + TRIM71 - expressing organoids after 10 days of expansion ( n = 3 , two independent experiments ) . One - way ANOVA with Tukey ' s correction was used to calculate P values . * P ≤ 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . Data information : Plotted are individual data points , representing the average value per animal and treatment , and mean ± SD of biological replicates .

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

RT-qPCR- analysis of progenitor (Hmga2) and supporting cell-specific (Zbtb20, Nfib) gene expression in control, Lin28b, TRIM71 or Lin28b+TRIM71-expressing organoids after 10 days of expansion (n=3, two independent experiments). One-way ANOVA with Tukey's correction was used to calculate P values. *P ≤ 0.05, **P < 0.01 and ***P < 0.001. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.


RT - qPCR analysis of hair cell ( Aoth1 and Pou4f3 ) and progenitor - specific ( Trim71 ) gene expression in Hmga2 knockdown and control cultures . Hmga2 expression was analyzed to confirm knockdown . Plotted are fold difference between control and Hmga2 shRNA group and mean ± SD ( n = 3 , biological replicates ; two independent experiments ) . A two - tailed , unpaired t test was used to calculate P values . * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 . Data information : Plotted are individual data points , representing the average value per animal and treatment , and mean ± SD of biological replicates .

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RT-qPCR analysis of hair cell (Aoth1 and Pou4f3) and progenitor-specific (Trim71) gene expression in Hmga2 knockdown and control cultures. Hmga2 expression was analyzed to confirm knockdown. Plotted are fold difference between control and Hmga2 shRNA group and mean ± SD (n=3, biological replicates; two independent experiments). A two-tailed, unpaired t test was used to calculate P values. **P < 0.01, ***P < 0.001 and ****P < 0.0001. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.


Low power bright field ( BF ) and green fluorescent images ( Atoh1 - nGFP ) of Hmga2 shRNA knockdown and control organoid cultures after 2 days of differentiation . Scale bars = 400 µm . Quantification of Atoh1 - nGFP + organoids in ( E ) ( n = 3 , two independent experiments ) . A two - tailed , unpaired t test was used to calculate P values . * P ≤ 0 . 05 . Data information : Plotted are individual data points , representing the average value per animal and treatment , and mean ± SD of biological replicates .

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

Low power bright field (BF) and green fluorescent images (Atoh1-nGFP) of Hmga2 shRNA knockdown and control organoid cultures after 2 days of differentiation. Scale bars=400 µm. Quantification of Atoh1-nGFP+ organoids in (E) (n=3, two independent experiments). A two-tailed, unpaired t test was used to calculate P values. *P ≤ 0.05. Data information: Plotted are individual data points, representing the average value per animal and treatment, and mean ± SD of biological replicates.


B , RMD assays performed after DSBs have been generated by sgRNAs / Cas9 . The distance between the sgRNA / Cas9 cleavage sites and the repeat sequences is indicated . U2OS cells carrying a single copy of the EGFP - SSA reporter ( 3427 - 17 ) were transfected with sgRNA / Cas9 plasmids , and the percentage of GFP - positive cells was determined ( left ) . U2OS cells transfected with the indicated reporters EGFP - SSA - 17 / 6 bp , EGFP - SSA - 0 . 3 kb , EGFP - SSA - 0 . 6 kb and EGFP - SSA - 1 . 0 kb were pooled as a population after selection of the drug marker on the reporters , followed by sgRNA / Cas9 cleavage ( right ) .

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

B, RMD assays performed after DSBs have been generated by sgRNAs/Cas9. The distance between the sgRNA/Cas9 cleavage sites and the repeat sequences is indicated. U2OS cells carrying a single copy of the EGFP-SSA reporter (3427-17) were transfected with sgRNA/Cas9 plasmids, and the percentage of GFP-positive cells was determined (left). U2OS cells transfected with the indicated reporters EGFP-SSA-17/6 bp, EGFP-SSA-0.3 kb, EGFP-SSA-0.6 kb and EGFP-SSA-1.0 kb were pooled as a population after selection of the drug marker on the reporters, followed by sgRNA/Cas9 cleavage (right).


C , RMD is strongly induced when a DSB is generated close to one repeat . The U2OS ( EGFP - SSA - 3427 - 17 ) cell line carrying a single copy of the reporter was transfected with sgRNA / Cas9 plasmids as indicated , and the percentage of GFP - positive cells was determined .

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

C, RMD is strongly induced when a DSB is generated close to one repeat. The U2OS (EGFP-SSA-3427-17) cell line carrying a single copy of the reporter was transfected with sgRNA/Cas9 plasmids as indicated, and the percentage of GFP-positive cells was determined.


Determination of RMD frequency in U2OS ( EGFP - SSA - 3427 - 17 ) cells when POLD3 is depleted by lentiviral expression of shRNA . Cells infected with a lentiviral empty vector were used as control . The knockdown level of the indicated proteins was shown by Western blot , using KU70 as the loading control .

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

Determination of RMD frequency in U2OS (EGFP-SSA-3427-17) cells when POLD3 is depleted by lentiviral expression of shRNA. Cells infected with a lentiviral empty vector were used as control. The knockdown level of the indicated proteins was shown by Western blot, using KU70 as the loading control.


Determination of RMD frequency in U2OS ( EGFP - SSA - 3427 - 17 ) cells when RAD52 is depleted by lentiviral expression of shRNA . Cells infected with a lentiviral empty vector were used as control . The knockdown level of the indicated proteins was shown by Western blot , using KU70 as the loading control .

Determination of RMD frequency in U2OS (EGFP-SSA-3427-17) cells when RAD52 is depleted by lentiviral expression of shRNA. Cells infected with a lentiviral empty vector were used as control. The knockdown level of the indicated proteins was shown by Western blot, using KU70 as the loading control.


G , Determination of RMD frequency in U2OS ( EGFP - SSA - 3427 - 17 ) cells when RAD51 is depleted by lentiviral expression of shRNA . Cells infected with a lentiviral empty vector were used as control . The knockdown level of the indicated proteins was shown by Western blot , using KU70 as the loading control .

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

G, Determination of RMD frequency in U2OS (EGFP-SSA-3427-17) cells when RAD51 is depleted by lentiviral expression of shRNA. Cells infected with a lentiviral empty vector were used as control. The knockdown level of the indicated proteins was shown by Western blot, using KU70 as the loading control.


B , RMD efficiency is decreased when the distance from a DSB to one repeat is increased . RMD assays were performed in U2OS ( EGFP - SSA - 3427 - 17 ) cells using the indicated sgRNAs / Cas9 . The distance between the sgRNA / Cas9 cleavage sites and the repeat sequences is shown .

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

B, RMD efficiency is decreased when the distance from a DSB to one repeat is increased. RMD assays were performed in U2OS (EGFP-SSA-3427-17) cells using the indicated sgRNAs/Cas9. The distance between the sgRNA/Cas9 cleavage sites and the repeat sequences is shown.


C and D , Knockdown of POLD3 impairs RMD when a DSB is close to one repeat . RMD frequency was determined in U2OS ( EGFP - SSA - 3427 - 17 ) cells using the indicated sgRNAs / Cas9 with or without depletion of POLD3 by lentivirus - expressed shRNA . Cells infected with a lentiviral empty vector were used as control . The distance between the sgRNA / Cas9 cleavage sites and the repeat sequences is indicated . The knockdown level of POLD3 was shown by Western blot , and KU70 was used as the loading control .

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

C and D, Knockdown of POLD3 impairs RMD when a DSB is close to one repeat. RMD frequency was determined in U2OS (EGFP-SSA-3427-17) cells using the indicated sgRNAs/Cas9 with or without depletion of POLD3 by lentivirus-expressed shRNA. Cells infected with a lentiviral empty vector were used as control. The distance between the sgRNA/Cas9 cleavage sites and the repeat sequences is indicated. The knockdown level of POLD3 was shown by Western blot, and KU70 was used as the loading control.


A , Cyclin E overexpression results in increased RMD when the DSB is close to one repeat . The U2OS ( EGFP - SSA - 3427 - 3 ) cell line carrying Tet - on - cyclin E plasmid was used , and RMD frequency was determined with or without induction of cyclin E expression by doxycycline ( DOX , 0 . 5 µg / mL ) , which was added 24 hours before transfection of the indicated sgRNAs / Cas9 . Cyclin E expression levels were validated by Western blot , using KU70 as the loading control .

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

A, Cyclin E overexpression results in increased RMD when the DSB is close to one repeat. The U2OS (EGFP-SSA-3427-3) cell line carrying Tet-on-cyclin E plasmid was used, and RMD frequency was determined with or without induction of cyclin E expression by doxycycline (DOX, 0.5 µg/mL), which was added 24 hours before transfection of the indicated sgRNAs/Cas9. Cyclin E expression levels were validated by Western blot, using KU70 as the loading control.


B , ATR is required for BIR / RMD upon cyclin E overexpression . RMD frequency was determined in U2OS ( EGFP - SSA - 3427 - 3 ) cells infected with lentivirus - expressed ATR shRNA or the control vector ( Ctrl ) with and without induction of cyclin E expression by DOX ( 0 . 5 µg / ml ) after R29 sgRNA / Cas9 cleavage . ATR knockdown was shown by Western blot with KU70 as the loading control .

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

B, ATR is required for BIR/RMD upon cyclin E overexpression. RMD frequency was determined in U2OS (EGFP-SSA-3427-3) cells infected with lentivirus-expressed ATR shRNA or the control vector (Ctrl) with and without induction of cyclin E expression by DOX (0.5 µg/ml) after R29 sgRNA/Cas9 cleavage. ATR knockdown was shown by Western blot with KU70 as the loading control.


D , RMD is stimulated when Cas9 - D10A is used to create a nick near the repeat . The repair frequencies of the U2OS ( MMEJ ) reporter cell line ( left ) and U2OS ( EGFP - SSA - 3427 - 17 ) cell line ( right ) were determined after expression of the indicated sgRNAs / Cas9 and sgRNAs / Cas9 - D10A . The distance between the sgRNA cleavage sites and the left and right repeat sequences is indicated ( right bottom ) .

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

D, RMD is stimulated when Cas9-D10A is used to create a nick near the repeat. The repair frequencies of the U2OS (MMEJ) reporter cell line (left) and U2OS (EGFP-SSA-3427-17) cell line (right) were determined after expression of the indicated sgRNAs/Cas9 and sgRNAs/Cas9-D10A. The distance between the sgRNA cleavage sites and the left and right repeat sequences is indicated (right bottom).


E , RMD induced by Cas9 - D10A is dependent on POLD3 . U2OS ( EGFP - SSA - 3427 - 17 ) cells were infected with lentiviruses expressing shRNA for POLD3 or vector control and RMD was determined after cleavage by the indicated sgRNAs / Cas9 - D10A . The distance between sgRNA / Cas9 - D10A cleavage sites and the left and right repeat sequences is indicated at the bottom . The knockdown level of POLD3 was shown by Western blot , and KU70 was used as the loading control .

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

E, RMD induced by Cas9-D10A is dependent on POLD3. U2OS (EGFP-SSA-3427-17) cells were infected with lentiviruses expressing shRNA for POLD3 or vector control and RMD was determined after cleavage by the indicated sgRNAs/Cas9-D10A. The distance between sgRNA/Cas9-D10A cleavage sites and the left and right repeat sequences is indicated at the bottom. The knockdown level of POLD3 was shown by Western blot, and KU70 was used as the loading control.