( 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 O O O O O O 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-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O 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-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O 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 O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O 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 O O B-MEASURED_VAR O O 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-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O 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 O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O 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 O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O 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 O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 O O O O O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O O O O 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-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR 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) 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 .

O O O O O O O O O O O O O B-MEASURED_VAR O O 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 .

O O O O O O O O O O O B-CONTROLLED_VAR O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR 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 .

O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O

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 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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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 .

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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 O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, 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 .

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-CONTROLLED_VAR O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O 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-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O 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-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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 O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O 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.


F , p27 suppresses BIR / RMD and SSA / RMD . U2OS ( EGFP - SSA - 3427 - 3 ) cells carrying the Tet - on - cyclin E allele were infected with and without p27 lentiviral viruses . RMD was determined before and after induction of cyclin E overexpression by DOX ( 0 . 5 µg / mL ) added 24 hours before transfection with the indicated sgRNAs / Cas9 . Cyclin E expression levels and cell cycle profiles are shown in Appendix Fig S6 .

O O B-CONTROLLED_VAR O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O

F, p27 suppresses BIR/RMD and SSA/RMD. U2OS (EGFP-SSA-3427-3) cells carrying the Tet-on-cyclin E allele were infected with and without p27 lentiviral viruses. RMD was determined before and after induction of cyclin E overexpression by DOX (0.5 µg/mL) added 24 hours before transfection with the indicated sgRNAs/Cas9. Cyclin E expression levels and cell cycle profiles are shown in Appendix Fig S6.


B , Impact of the 5 ' DSB / repeat distance on RMD frequency when the 3 ' DSB / repeat distance is fixed . RMD assays were performed using mES cells carrying the RMD - GFP reporter , and RMD frequency was determined by the number of GFP + cells normalized to transfection efficiency . The distance between the sgRNA targeting sites and the repeats is indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Impact of the 5' DSB/repeat distance on RMD frequency when the 3' DSB/repeat distance is fixed. RMD assays were performed using mES cells carrying the RMD-GFP reporter, and RMD frequency was determined by the number of GFP+ cells normalized to transfection efficiency. The distance between the sgRNA targeting sites and the repeats is indicated.


C , Long - range RMD is dependent on POLD3 when one DSB is close to a repeat . mES cells carrying the RMD - GFP reporter were transfected with nontargeting siRNA ( Ctrl ) or a pool of POLD3 siRNAs ( siPOLD3 ) 24 hours before for the first time , and with transfection of sgRNAs / Cas9 the second time . The graphs show the frequency of GFP + cells normalized to the transfection efficiency . The distance between the sgRNA targeting sites and the repeats is indicated .

O O O O O O O O O B-CONTROLLED_VAR O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Long-range RMD is dependent on POLD3 when one DSB is close to a repeat. mES cells carrying the RMD-GFP reporter were transfected with nontargeting siRNA (Ctrl) or a pool of POLD3 siRNAs (siPOLD3) 24 hours before for the first time, and with transfection of sgRNAs/Cas9 the second time. The graphs show the frequency of GFP+ cells normalized to the transfection efficiency. The distance between the sgRNA targeting sites and the repeats is indicated.


D and E , Impact of the 3 ' DSB / repeat distance on RMD frequency when the 5 ' DSB / repeat distance is fixed . RMD assays were performed using mES cells carrying the RMD - GFP reporter , and RMD frequency was determined by the number of GFP + cells normalized to transfection efficiency . The distance between the sgRNA targeting sites and the repeats is indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and E, Impact of the 3' DSB/repeat distance on RMD frequency when the 5' DSB/repeat distance is fixed. RMD assays were performed using mES cells carrying the RMD-GFP reporter, and RMD frequency was determined by the number of GFP+ cells normalized to transfection efficiency. The distance between the sgRNA targeting sites and the repeats is indicated.


F , RMD frequency of two far - apart repeats that are a short distance from the DSB . RMD was determined in mES cells carrying the RMD - GFP reporter using the indicated sgRNAs / Cas9 , with the frequency of GFP + cells normalized to transfection efficiency . The distance between the sgRNA targeting sites and the repeats is indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, RMD frequency of two far-apart repeats that are a short distance from the DSB. RMD was determined in mES cells carrying the RMD-GFP reporter using the indicated sgRNAs/Cas9, with the frequency of GFP+ cells normalized to transfection efficiency. The distance between the sgRNA targeting sites and the repeats is indicated.


G , The roles of POLD3 , RAD51 and RAD52 in RMD of two far - apart repeats that are a short distance from the DSB . RMD was determined in mES cells carrying the RMD - GFP reporter using the indicated sgRNAs / Cas9 after POLD3 ( left ) or RAD51 ( middle ) was depleted by siRNA or RAD52 was knocked out ( right ) . The distance between the sgRNA targeting sites and the repeats is indicated .

O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

G, The roles of POLD3, RAD51 and RAD52 in RMD of two far-apart repeats that are a short distance from the DSB. RMD was determined in mES cells carrying the RMD-GFP reporter using the indicated sgRNAs/Cas9 after POLD3 (left) or RAD51 (middle) was depleted by siRNA or RAD52 was knocked out (right). The distance between the sgRNA targeting sites and the repeats is indicated.


SSA / RMD and BIR / SSA are both dependent on ATM . SSA / RMD and BIR / SSA frequencies in U2OS ( EGFP - SSA - 3427 - 17 ) cells were determined after the addition of ATM inhibitor ( 20mM ku55933 ) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA /

O O O O O O O O O O O B-CONTROLLED_VAR O O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

SSA/RMD and BIR/SSA are both dependent on ATM. SSA/RMD and BIR/SSA frequencies in U2OS (EGFP-SSA-3427-17) cells were determined after the addition of ATM inhibitor (20mM ku55933) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA/Cas9


BIR / SSA are both dependent on ATM . BIR / RMD in mES cells carrying the RMD - GFP reporter were determined after the addition of ATM inhibitor ( 20mM ku55933 ) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA / Cas9 or in ATM KO mES cells carrying the RMD - GFP reporter ( right ) .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O

BIR/SSA are both dependent on ATM. BIR/RMD in mES cells carrying the RMD-GFP reporter were determined after the addition of ATM inhibitor (20mM ku55933) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA/Cas9 or in ATM KO mES cells carrying the RMD-GFP reporter (right).


Dependence of RMD on H2AX . SSA / RMD and BIR / RMD frequencies in U2OS ( EGFP - SSA - 3427 - 17 ) cells were determined using the indicated sgRNA / Cas9 after depletion of H2AX by lentivirus expressed shRNA Depletion of H2AX by shRNA in was determined by

O O O O B-CONTROLLED_VAR O O O O O O O O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O

Dependence of RMD on H2AX. SSA/RMD and BIR/RMD frequencies in U2OS (EGFP-SSA-3427-17) cells were determined using the indicated sgRNA/Cas9 after depletion of H2AX by lentivirus expressed shRNA Depletion of H2AX by shRNA in was determined by qPCR


D , Dependence of RMD on H2AX . BIR / RMD in mES cells carrying the RMD - GFP reporter , or by transfection with H2AX siRNA or in RAD52 KO

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O

D, Dependence of RMD on H2AX. BIR/RMD in mES cells carrying the RMD-GFP reporter , or by transfection with H2AX siRNA or in RAD52 KO cells


A , Frequency of long - range RMD is increased by an additional DSB downstream of the 3 ' repeat . Illustrations show the GFP - RMD reporter , with sgRNAs / Cas9 cleavage sites indicated as red arrows ( left ) . The frequency of BIR / SSA in mES cells carrying the RMD - GFP reporter was determined after cleavage of the reporter by the indicated sgRNAs / Cas9 ( right ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Frequency of long-range RMD is increased by an additional DSB downstream of the 3' repeat. Illustrations show the GFP-RMD reporter, with sgRNAs/Cas9 cleavage sites indicated as red arrows (left). The frequency of BIR/SSA in mES cells carrying the RMD-GFP reporter was determined after cleavage of the reporter by the indicated sgRNAs/Cas9 (right).


B and C , The effects of RAD52 , POLD3 , ATM and H2AX inactivation on BIR / RMD with cleavage at L17 / D10k . The frequency of BIR / RMD in mES cells carrying the RMD - GFP reporter was determined using the sgRNAs / Cas9 at L17 / D10k in RAD52 KO cells or after POLD3 depletion by siRNA ( B ) , or after treatment with ATM inhibitor ( ku55933 , 15mM ) or H2AX depletion by siRNA ( C ) .

O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O

B and C, The effects of RAD52, POLD3, ATM and H2AX inactivation on BIR/RMD with cleavage at L17/D10k. The frequency of BIR/RMD in mES cells carrying the RMD-GFP reporter was determined using the sgRNAs/Cas9 at L17/D10k in RAD52 KO cells or after POLD3 depletion by siRNA (B), or after treatment with ATM inhibitor (ku55933, 15mM) or H2AX depletion by siRNA (C).


A , Long - range BIR / RMD with identical repeats or divergent repeats . The frequency of BIR / RMD in mES cells carrying the RMD - GFP reporter with identical repeats ( 0 % ) or repeats with 1 % and 3 % divergence was determined after cleavage at L17 / R3 . 3k or L17 / R28 . 4k .

A, Long-range BIR/RMD with identical repeats or divergent repeats. The frequency of BIR/RMD in mES cells carrying the RMD-GFP reporter with identical repeats (0%) or repeats with 1% and 3% divergence was determined after cleavage at L17/R3.3k or L17/R28.4k.


B and C , The effect of MSH2 on BIR / RMD when repeat sequences are divergent . The frequency of BIR / RMD was determined after cleavage at L17 / R3 . 3k or L17 / R28 . 4k in mES cells carrying the RMD - GFP reporter with 1 % and 3 % divergent repeats , and with and without MSH2 KO ( B ) or with MSH2 KO and POLD3 depletion by siRNAs ( C ) . The MSH2 KO was validated by Western blot , using KU70 as the loading control as shown in ( B ) . Data information : Error bars represent standard deviation ( SD ) of at least three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , two - tailed non - paired t - test .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and C, The effect of MSH2 on BIR/RMD when repeat sequences are divergent. The frequency of BIR/RMD was determined after cleavage at L17/R3.3k or L17/R28.4k in mES cells carrying the RMD-GFP reporter with 1% and 3% divergent repeats, and with and without MSH2 KO (B) or with MSH2 KO and POLD3 depletion by siRNAs (C). The MSH2 KO was validated by Western blot, using KU70 as the loading control as shown in (B). Data information: Error bars represent standard deviation (SD) of at least three independent experiments. *P < 0.05, **P < 0.01, two-tailed non-paired t-test.


( A ) Kaplan Meier plot for survival in NeuT ; Apln + / + ( n = 11 ) and NeuT ; Apln - / - ( n = 10 ) mice with mammary cancer after tumor onset . * P = 0 . 0185 ; Log rank test .

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(A) Kaplan Meier plot for survival in NeuT;Apln+/+ (n=11) and NeuT;Apln-/- (n=10) mice with mammary cancer after tumor onset. *P=0.0185; Log rank test.


( B ) Tumor volumes , followed over time , of control mammary tumor E0771 cells ( shRenilla ) and Apelin - depleted ( shApln ) E0771 cells orthotopically - injected into both syngeneic C57BL / 6J Apln + / + and Apln - / - mice ( 5x105 cells / mouse ) , respectively . Tumor volumes were determined using calipers and are shown as mean tumor volumes ± S . E . M . Data shown is pooled from 2 independent experiments . Apln + / + ; shRenilla ( n = 18 ) , Apln + / + ; shApln ( n = 17 ) , Apln - / - ; shRenilla ( n = 14 ) , Apln - / - ; shApln ( n = 15 ) ; * * P < 0 . 01 , * * * P < 0 . 001 ; two - way ANOVA .

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(B) Tumor volumes, followed over time, of control mammary tumor E0771 cells (shRenilla) and Apelin-depleted (shApln) E0771 cells orthotopically-injected into both syngeneic C57BL/6J Apln+/+ and Apln-/- mice (5x105 cells/mouse), respectively. Tumor volumes were determined using calipers and are shown as mean tumor volumes ± S.E.M. Data shown is pooled from 2 independent experiments. Apln+/+;shRenilla (n=18), Apln+/+;shApln (n=17), Apln-/-;shRenilla (n=14), Apln-/-;shApln (n=15); ** P<0.01, *** P<0.001; two-way ANOVA.


( C ) Mean percentages ( ± S . E . M . ) of CD31 + area in E0771 shRenilla ( n = 3 ) or shApln ( n = 3 ) mammary tumors , assessed on day 23 post - orthotopic injection into C57BL / 6J Apln + / + or Apln - / - mice , respectively . * * P < 0 . 01 ; t test . ( D ) Mean percentages ( ± S . E . M . ) of extravasated Dextran in E0771 shRenilla ( n = 9 ) or shApln ( n = 12 ) mammary tumors , assessed on day 19 post - orthotopic injection into C57BL / 6J Apln + / + or Apln - / - mice , respectively . * * P < 0 . 01 ; t test . Right panel shows representative immunofluorescence of Dextran ( red ) , CD31 + vessels ( green ) , and DAPI ( blue ) . The white arrows indicate regions of Dextran extravasation . Scale bars = 100

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O 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-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O 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-MEASURED_VAR O O O O O O 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-CONTROLLED_VAR O O O O B-CONTROLLED_VAR 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-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O

(C) Mean percentages (± S.E.M.) of CD31+ area in E0771 shRenilla (n=3) or shApln (n=3) mammary tumors, assessed on day 23 post-orthotopic injection into C57BL/6J Apln+/+ or Apln-/- mice, respectively. **P<0.01; t test. (D) Mean percentages (± S.E.M.) of extravasated Dextran in E0771 shRenilla (n=9) or shApln (n=12) mammary tumors, assessed on day 19 post-orthotopic injection into C57BL/6J Apln+/+ or Apln-/- mice, respectively. **P<0.01; t test. Right panel shows representative immunofluorescence of Dextran (red), CD31+ vessels (green), and DAPI (blue). The white arrows indicate regions of Dextran extravasation. Scale bars = 100 μm


( E ) Mean counts ( ± S . E . M . ) of pimonidazole positive foci , assessed on day 26 post - orthotopic injection of E0771 shRenilla ( n = 6 ) or shApln ( n = 4 ) into C57BL / 6J wild - type mice ( 5x105 cells / mouse ) . * P < 0 . 05 ; t test . Right panels show representative immunohistochemical pimonidazole staining at two different magnifications ; Scale bars = 200 μm ( upper panels ) and 50 μm ( lower panels ) .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(E) Mean counts (± S.E.M.) of pimonidazole positive foci, assessed on day 26 post-orthotopic injection of E0771 shRenilla (n=6) or shApln (n=4) into C57BL/6J wild-type mice (5x105 cells/mouse). *P<0.05; t test. Right panels show representative immunohistochemical pimonidazole staining at two different magnifications; Scale bars = 200 μm (upper panels) and 50 μm (lower panels).


( F ) Mean percentage ( ± S . E . M . ) of tumor - infiltrating immune cells normalized to CD45 + . E0771 shRenilla ( n = 8 ) or shApln ( n = 6 ) were orthotopically injected into C57BL / 6J Apln + / + or Apln - / - mice ( 5x105 cells / mouse ) , respectively and tumors were harvested day 25 post - injection . * P < 0 . 05 , * * P < 0 . 01 ; t test . All immune cell populations were gated for viable CD45 + cells and then further defined as : CD8 T cells ( Thy1 . 2 + , CD8 + ) , CD4 T cells , ( Thy1 . 2 + , CD4 + ) , Inflammatory Monocytes ( Ly6C + , Cd11b + , Ly6G - ) , PMN - MDSCs ( Ly6G + , Cd11b + ) , natural killer cells ( Thy1 . 2 - , Ly6G - , NK1 . 1 + ) , natural killer T cells ( Thy1 . 2 + , CD4 - , CD8 - , NK1 . 1 + ) and peripheral dendritic cells ( Ly6G - , Ly6C + , Cd11b - , PDAC1 + , B220 + ) .

O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O B-MEASURED_VAR O O B-MEASURED_VAR O O B-MEASURED_VAR O O O

(F) Mean percentage (± S.E.M.) of tumor-infiltrating immune cells normalized to CD45+. E0771 shRenilla (n=8) or shApln (n=6) were orthotopically injected into C57BL/6J Apln+/+ or Apln-/- mice (5x105 cells/mouse), respectively and tumors were harvested day 25 post-injection. *P<0.05, **P<0.01; t test. All immune cell populations were gated for viable CD45+ cells and then further defined as: CD8 T cells (Thy1.2+, CD8+), CD4 T cells, (Thy1.2+, CD4+), Inflammatory Monocytes (Ly6C+, Cd11b+, Ly6G-), PMN-MDSCs (Ly6G+, Cd11b+), natural killer cells (Thy1.2-, Ly6G-, NK1.1+), natural killer T cells (Thy1.2+, CD4-, CD8-, NK1.1+) and peripheral dendritic cells (Ly6G- ,Ly6C+, Cd11b- , PDAC1+, B220+).


( A ) Left - Heatmap of RNA - Seq transcriptome analysis of CD31 + / CD105 + endothelial cells sorted from tumors established by E0771 shRenilla ( n = 6 ) or shApln ( n = 3 ) cells orthotopically injected into C57BL / 6J Apln + / + or Apln - / - mice , respectively . Tumors were harvested day 25 post - injection and genes displayed are significantly deregulated at the adjusted p - value cut - off of 0 . 05 . Right - Ingenuity Pathway Analysis for biological processes predicted to be decreased downstream of the differentially expressed genes .

O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left - Heatmap of RNA-Seq transcriptome analysis of CD31+/CD105+ endothelial cells sorted from tumors established by E0771 shRenilla (n=6) or shApln (n=3) cells orthotopically injected into C57BL/6J Apln+/+ or Apln-/- mice, respectively. Tumors were harvested day 25 post-injection and genes displayed are significantly deregulated at the adjusted p-value cut-off of 0.05. Right - Ingenuity Pathway Analysis for biological processes predicted to be decreased downstream of the differentially expressed genes.


( B ) Quantification of vessel sprouts ( mean values ± S . E . M . ) upon VEGF treatment ( 30ng / mL ) of embryoid bodies ( EB ) derived from murine ES cells ( mESCs ) with sense integrations in the Apelin gene ( Apln STOP ) of the splice acceptor described in Appendix Figure S1B or Cre - reverted antisense ( Apln GO ) sister cells . Apln GO ( Day 2 n = 6 ; Day 4 n = 40 , Day 6 n = 54 , Day 8 n = 32 , Day 10 n = 35 , Day 11 n = 31 ) , Apln STOP ( Day 2 n = 4 ; Day 4 n = 15 , Day 6 n = 31 , Day 8 n = 28 , Day 10 n = 32 , Day 11 n = 37 ) ; * * P < 0 . 01 , * * * P < 0 . 001 , two - way ANOVA . Representative brightfield images and automated analysis of vessel sprouts by Definiens software are shown in the bottom panels . Scale bars = 200

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O 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-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 vessel sprouts (mean values ± S.E.M.) upon VEGF treatment (30ng/mL) of embryoid bodies (EB) derived from murine ES cells (mESCs) with sense integrations in the Apelin gene (Apln STOP) of the splice acceptor described in Appendix Figure S1B or Cre-reverted antisense (Apln GO) sister cells. Apln GO (Day 2 n=6; Day 4 n=40, Day 6 n=54, Day 8 n= 32, Day 10 n=35, Day 11 n=31), Apln STOP (Day 2 n=4; Day 4 n=15, Day 6 n=31, Day 8 n= 28, Day 10 n=32, Day 11 n=37); ** P<0.01, *** P<0.001, two-way ANOVA. Representative brightfield images and automated analysis of vessel sprouts by Definiens software are shown in the bottom panels. Scale bars = 200 μm


( C ) Differentially expressed genes using RNAseq transcriptome analysis of CD31 + endothelial cells ( ECs ) sorted from sprouting EBs from Apelin STOP cells stimulated with VEGF ( 30ng / mL ) and DMSO ( - Apln ) and repaired Apelin GO sister cells stimulated with VEGF and Apelin ( 1000nM ) ( + Apln ) . VEGF target genes and angiogenesis - related genes , predicted by Ingenuity Pathway Analysis ( IPA ) software , are indicated by bars on the upper axis of the heatmap . GO terms were analyzed by DAVID on - line software .

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(C) Differentially expressed genes using RNAseq transcriptome analysis of CD31+ endothelial cells (ECs) sorted from sprouting EBs from Apelin STOP cells stimulated with VEGF (30ng/mL) and DMSO (-Apln) and repaired Apelin GO sister cells stimulated with VEGF and Apelin (1000nM) (+Apln). VEGF target genes and angiogenesis-related genes, predicted by Ingenuity Pathway Analysis (IPA) software, are indicated by bars on the upper axis of the heatmap. GO terms were analyzed by DAVID on-line software.


( A ) Experimental set up and ( right ) Kaplan Meier survival plot of NeuT ; Apln + / + and NeuT ; Apln - / - mice with mammary cancer , left untreated ( control ) or treated with the indicate dose of the broad VEGFR blocker sunitinib . NeuT ; Apln + / + Control ( n = 8 ) , NeuT ; Apln - / - Control ( n = 11 ) , NeuT ; Apln + / + Sunitinib ( n = 11 ) , NeuT ; Apln - / - Sunitinib ( n = 12 ) ; * P < 0 . 05 ; Log rank test . Mice were sacrificed when the tumor size reached 1cm3 , following ethical guidelines . The dotted line indicates 50 % of survival .

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(A) Experimental set up and (right) Kaplan Meier survival plot of NeuT;Apln+/+ and NeuT;Apln-/- mice with mammary cancer, left untreated (control) or treated with the indicate dose of the broad VEGFR blocker sunitinib. NeuT;Apln+/+ Control (n=8), NeuT;Apln-/- Control (n=11), NeuT;Apln+/+ Sunitinib (n=11), NeuT;Apln-/- Sunitinib (n=12); *P<0.05; Log rank test. Mice were sacrificed when the tumor size reached 1cm3, following ethical guidelines. The dotted line indicates 50% of survival.


( B ) Percentages ( mean ± S . E . M . ) of tumor burden in mammary glands of untreated ( control ) and sunitinib - treated NeuT ; Apln + / + and NeuT ; Apln - / - mice , assessed 4 weeks after tumor onset . NeuT ; Apln + / + Control ( n = 6 ) , NeuT ; Apln - / - Control ( n = 8 ) , NeuT ; Apln + / + Sunitinib ( n = 5 ) , NeuT ; Apln - / - Sunitinib ( n = 5 ) ; * P < 0 . 05 ; * * * P < 0 . 001 ; one - way ANOVA . Representative H & E images are shown for each genotype in the right panels . Scale bars = 1000 μm ( large panels ) and 50 μm ( insets ) .

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(B) Percentages (mean ± S.E.M.) of tumor burden in mammary glands of untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mice, assessed 4 weeks after tumor onset. NeuT;Apln+/+ Control (n=6), NeuT;Apln-/- Control (n=8), NeuT;Apln+/+ Sunitinib (n=5), NeuT;Apln-/- Sunitinib (n=5); *P<0.05; ***P<0.001; one-way ANOVA. Representative H&E images are shown for each genotype in the right panels. Scale bars = 1000 μm (large panels) and 50 μm (insets).


( C ) Tumor volumes of untreated ( control ) and sunitinib - treated NeuT ; Apln + / + and NeuT ; Apln - / - mammary tumors , size - matched at 20 - 70mm3 and followed over time by MRI analysis ; NeuT ; Apln + / + Control ( n = 7 ) , NeuT ; Apln - / - Control ( n = 5 ) , NeuT ; Apln + / + Sunitinib ( n = 5 ) , NeuT ; Apln - / - Sunitinib ( n = 6 ) mice per group ; Lines represent nonlinear fit of tumor growth . Box and arrow indicate the time point used for analysis in panel D . ( D ) Mitotic counts ( mean ± S . E . M . ) and representative H & E images of mammary tumors in untreated ( control ) and sunitinib - treated NeuT ; Apln + / + and NeuT ; Apln - / - mice , assessed 6 weeks after tumor onset . ; NeuT ; Apln + / + Control ( n = 7 ) , NeuT ; Apln - / - Control ( n = 6 ) , NeuT ; Apln + / + Sunitinib ( n = 9 ) , NeuT ; Apln - / - Sunitinib ( n = 4 ) tumors per group ; * P < 0 . 05 ; one - way ANOVA to sunitinib - treated NeuT ; Apln - / - . White arrows indicate mitotic figures . Scale bars = 50

O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

(C) Tumor volumes of untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mammary tumors, size-matched at 20-70mm3 and followed over time by MRI analysis; NeuT;Apln+/+ Control (n=7), NeuT;Apln-/- Control (n=5), NeuT;Apln+/+ Sunitinib (n=5), NeuT;Apln-/- Sunitinib (n=6) mice per group; Lines represent nonlinear fit of tumor growth. Box and arrow indicate the time point used for analysis in panel D. (D) Mitotic counts (mean ± S.E.M.) and representative H&E images of mammary tumors in untreated (control) and sunitinib-treated NeuT;Apln+/+ and NeuT;Apln-/- mice, assessed 6 weeks after tumor onset.; NeuT;Apln+/+ Control (n=7), NeuT;Apln-/- Control (n=6), NeuT;Apln+/+ Sunitinib (n=9), NeuT;Apln-/- Sunitinib (n=4) tumors per group; *P<0.05; one-way ANOVA to sunitinib-treated NeuT;Apln-/-. White arrows indicate mitotic figures. Scale bars = 50 μm


( E ) Tumor volumes , followed over the indicated time , of orthotopically injected E0771 shRenilla cells left untreated ( control ) or treated three times a week from day 8 after tumor injection with an Apelin antagonist alone ( MM54 , 0 . 4μg / g ) , sunitinib alone ( 60mg / kg ) or a combination of both . Tumor volumes were measured using calipers and are shown as mean tumor volumes ± S . E . M . E0771 shRenilla Control ( n = 6 ) , MM54 ( n = 5 ) , Sunitinib ( n = 5 ) , Sunitinib + MM54 ( n = 5 ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , two - way ANOVA .

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(E) Tumor volumes, followed over the indicated time, of orthotopically injected E0771 shRenilla cells left untreated (control) or treated three times a week from day 8 after tumor injection with an Apelin antagonist alone (MM54, 0.4μg/g), sunitinib alone (60mg/kg) or a combination of both. Tumor volumes were measured using calipers and are shown as mean tumor volumes ± S.E.M. E0771 shRenilla Control (n=6), MM54 (n=5), Sunitinib (n=5), Sunitinib+MM54 (n=5); *P<0.05, **P<0.01, ***P<0.001, two-way ANOVA.