( H ) qRT - PCR analysis of Acta1 and Nppa in NRVMs with or without NCoR1 overexpression . n = 4 : 3 . Data represent three independent experiments . Two - way ANOVA followed by Bonferoni post - tests was used for statistical analysis .

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

(H) qRT-PCR analysis of Acta1 and Nppa in NRVMs with or without NCoR1 overexpression. n=4:3. Data represent three independent experiments. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis.


( A ) Representative immunofluorescence staining of α - Actinin in NRVMs transfected with siRNA for 48h and then treated with vehicle ( H2O ) or PE for another 48h . Scale bar : 50μm . siMEF2a indicates MEF2a siRNA .

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

(A) Representative immunofluorescence staining of α-Actinin in NRVMs transfected with siRNA for 48h and then treated with vehicle (H2O) or PE for another 48h. Scale bar: 50μm. siMEF2a indicates MEF2a siRNA.


( B ) Quantification of the surface area of α - Actinin - positive NRVMs with or without knockdown of NCoR1 and / or MEF2a . A total of 15 - 20 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis .

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

(B) Quantification of the surface area of α-Actinin-positive NRVMs with or without knockdown of NCoR1 and/or MEF2a. A total of 15-20 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis.


( C ) Representative immunofluorescence staining of α - Actinin in NRVMs transfected with siRNA for 48h and then treated with vehicle or PE for another 48h . Scale bar : 50μm . siMEF2d indicates MEF2d siRNA .

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

(C) Representative immunofluorescence staining of α-Actinin in NRVMs transfected with siRNA for 48h and then treated with vehicle or PE for another 48h. Scale bar: 50μm. siMEF2d indicates MEF2d siRNA.


( D ) Quantification of the surface area of α - Actinin - positive NRVMs with or without knockdown of NCoR1 and / or MEF2d . A total of 15 - 19 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis .

(D) Quantification of the surface area of α-Actinin-positive NRVMs with or without knockdown of NCoR1 and/or MEF2d. A total of 15-19 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis.


( E ) qRT - PCR analysis of Acta1 and Nppa in NRVMs . n = 4 . Data represent three independent experiments . Student ' s t test was used for statistical analysis .

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

(E) qRT-PCR analysis of Acta1 and Nppa in NRVMs. n=4. Data represent three independent experiments. Student's t test was used for statistical analysis.


( A ) Luciferase assays of Acta1 and Nppa promoters in HEK293FT cells transfected with NCoR1 - Flag and / or MEF2a - HA or with empty plasmids . Student ' s t test was used for statistical

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

(A) Luciferase assays of Acta1 and Nppa promoters in HEK293FT cells transfected with NCoR1-Flag and/or MEF2a-HA or with empty plasmids. Student's t test was used for statistical analysis


( B ) Co - immunoprecipitation ( Co - IP ) analysis of NCoR1 and MEF2a in HEK293FT cells transfected with full - length NCoR1 - Flag and MEF2a - HA .

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

(B) Co-immunoprecipitation (Co-IP) analysis of NCoR1 and MEF2a in HEK293FT cells transfected with full-length NCoR1-Flag and MEF2a-HA.


( C ) Co - IP analysis of NCoR1 and truncated MEF2a in HEK293FT cells . Schematic illustration of MEF2a - HA construct is shown above the Co - IP results .

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

(C) Co-IP analysis of NCoR1 and truncated MEF2a in HEK293FT cells. Schematic illustration of MEF2a-HA construct is shown above the Co-IP results.


( D ) Co - IP analysis of MEF2a and truncated NCoR1 in HEK293FT cells . Schematic illustration of NCoR1 - Flag construct is shown above the Co - IP results .

(D) Co-IP analysis of MEF2a and truncated NCoR1 in HEK293FT cells. Schematic illustration of NCoR1-Flag construct is shown above the Co-IP results.


( E ) Luciferase assays of Acta1 and Nppa promoters in HEK293FT cells transfected with full - length MEF2a and domain - specific NCoR1 . Student ' s t test was used for statistical

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

(E) Luciferase assays of Acta1 and Nppa promoters in HEK293FT cells transfected with full-length MEF2a and domain-specific NCoR1. Student's t test was used for statistical analysis


( F ) Western blotting analysis of NCoR1 - flag in NRVMs infected by control lentivirus ( Control ) or NCoR1 ( 1939 - 2453 ) - flag lentivirus [ NCoR1 ( 1939 - 2453 ) - OV ] for 4 days .

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

(F) Western blotting analysis of NCoR1-flag in NRVMs infected by control lentivirus (Control) or NCoR1 (1939-2453)-flag lentivirus [NCoR1(1939-2453)-OV] for 4 days.


( G ) Representative immunofluorescence staining of α - Actinin in NRVMs infected with lentivirus for 48h and then treated with vehicle ( DMSO ) or PE for another 48h . Scale bar : 50μm .

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

(G) Representative immunofluorescence staining of α-Actinin in NRVMs infected with lentivirus for 48h and then treated with vehicle (DMSO) or PE for another 48h. Scale bar: 50μm.


( H ) Quantification of the surface area of α - Actinin - positive NRVMs . A total of 30 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis . Two - way ANOVA followed by Bonferoni post - tests was used for statistical

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

(H) Quantification of the surface area of α-Actinin-positive NRVMs. A total of 30 NRVMs was randomly chosen from 3 replicate coverslips for each group and used for statistical analysis. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis


( I ) qRT - PCR analysis of Acta1 and Nppa in NRVMs . n = 4 : 4 . Data represent three independent experiments . Two - way ANOVA followed by Bonferoni post - tests was used for statistical

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

(I) qRT-PCR analysis of Acta1 and Nppa in NRVMs. n=4:4. Data represent three independent experiments. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis


( A ) ChIP analysis showing enrichment of NCoR1 in MEF2 binding regions on the promoters of Acta1 and Nppa in ventricular samples .

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

(A) ChIP analysis showing enrichment of NCoR1 in MEF2 binding regions on the promoters of Acta1 and Nppa in ventricular samples.


( B ) ChIP analysis of enrichment of MEF2a on promoters of Acta1 and Nppa in ventricular samples . n = 3 : 3 .

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

(B) ChIP analysis of enrichment of MEF2a on promoters of Acta1 and Nppa in ventricular samples. n=3:3.


ChIP analysis of enrichment of HDAC4 ( C ) on promoters of Acta1 and Nppa in ventricular samples . n = 4 : 4 for ( C )

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

ChIP analysis of enrichment of HDAC4 (C) on promoters of Acta1 and Nppa in ventricular samples. n=4:4 for (C)


ChIP analysis of enrichment of HDAC5 ( D ) on promoters of Acta1 and Nppa in ventricular samples . n = 3 : 3 for (

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

ChIP analysis of enrichment of HDAC5 (D) on promoters of Acta1 and Nppa in ventricular samples. n=3:3 for (D


ChIP analysis of enrichment of , acetylated - Histone 4 ( ac - H4 ) ( E ) on promoters of Acta1 and Nppa in ventricular samples . , n = 3 :

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

ChIP analysis of enrichment of , acetylated-Histone 4 (ac-H4) (E) on promoters of Acta1 and Nppa in ventricular samples. , n=3:3


ChIP analysis of enrichment of ac - H3 ( F ) on promoters of Acta1 and Nppa in ventricular samples . n = 3 :

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

ChIP analysis of enrichment of ac-H3 (F) on promoters of Acta1 and Nppa in ventricular samples. n=3:3


( G ) ChIP analysis of RNA Polymerase II enrichment on the transcriptional regions of Acta1 and Nppa in ventricular samples . n = 3 : 3 .

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

(G) ChIP analysis of RNA Polymerase II enrichment on the transcriptional regions of Acta1 and Nppa in ventricular samples. n=3:3.


( A ) Western blotting analysis of RIDs of NCoR1 in left ventricular samples from mice infected with AAV9 GFP or AAV9 NCoR1 - RIDs ( 1939 - 2453 ) - Flag .

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

(A) Western blotting analysis of RIDs of NCoR1 in left ventricular samples from mice infected with AAV9 GFP or AAV9 NCoR1-RIDs (1939-2453)-Flag.


( B ) VW / BW of mice 2 weeks after sham operation or AAC . n = 7 : 7 : 12 : 12 .

O O O O O O O B-ORGANISM O O O O O O B-DISEASE O O O O O O O O O O O

(B) VW/BW of mice 2 weeks after sham operation or AAC. n=7:7:12:12.


( C ) Representative H & E staining of cross sections of left ventricles . Scale bar : 10μm .

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

(C) Representative H&E staining of cross sections of left ventricles. Scale bar: 10μm.


( D ) Representative WGA staining . Scale bar : 10μm .

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

(D) Representative WGA staining. Scale bar: 10μm.


( E ) Quantification of cardiomyocyte size . n = 6 : 6 : 7 : 8 .

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

(E) Quantification of cardiomyocyte size. n=6:6:7:8.


( F ) qRT - PCR analysis of hypertrophy - related genes in left ventricles . n = 8 : 8 : 11 : 11 .

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

(F) qRT-PCR analysis of hypertrophy-related genes in left ventricles. n=8:8:11:11.


( G ) Representative picrosirius red staining of cross sections of left ventricles . Scale bar : 20μm .

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

(G) Representative picrosirius red staining of cross sections of left ventricles. Scale bar: 20μm.


( H ) Quantification of fibrotic areas . n = 5 : 5 : 7 : 7 .

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

(H) Quantification of fibrotic areas. n=5:5:7:7.


( I ) qRT - PCR analysis of fibrosis - related genes in left ventricles . n = 8 : 8 : 11 : 11 .

(I) qRT-PCR analysis of fibrosis-related genes in left ventricles. n=8:8:11:11.


( J ) Ejection fraction measured before , 2 weeks and 4 weeks after AAC . n = 6 : 6 . Two - way ANOVA followed by Bonferoni post - tests was used for statistical analysis .

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

(J) Ejection fraction measured before, 2 weeks and 4 weeks after AAC. n=6:6. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis.


( K ) Fractional shortening measured before , 2 weeks and 4 weeks after AAC . n = 6 : 6 . Two - way ANOVA followed by Bonferoni post - tests was used for statistical analysis .

(K) Fractional shortening measured before, 2 weeks and 4 weeks after AAC. n=6:6. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis.


A TUNEL assay comparing apoptotic cells in Miwi + / - and Miwi - / - testis during diakinesis . Blue , DAPI ; Green , TUNEL labeling . Asterisk indicates TUNEL - positive stained cells in the meiotic metaphase stage . Scale bars , 10 μm .

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

A TUNEL assay comparing apoptotic cells in Miwi+/- and Miwi-/- testis during diakinesis. Blue, DAPI; Green, TUNEL labeling. Asterisk indicates TUNEL-positive stained cells in the meiotic metaphase stage. Scale bars, 10 μm.


B , C Meiotic metaphase I plate in FACS - sorted Miwi heterozygous ( B ) and knockout ( C ) spermatocytes . Green : phospho - histone H3 ; red : α / β - tubulin . Asterisk indicates misaligned chromosome . Scale bars , 10 μm . D Frequency of abnormal arrangement in the meiotic metaphase I plate . White , Miwi heterozygous ; Light gray , Miwi knockout . Three mice were used for each genotype , total numbers of counted metaphase I plate in Miwi heterozygous and knockout are 113 and 181 , respectively .

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

B, C Meiotic metaphase I plate in FACS-sorted Miwi heterozygous (B) and knockout (C) spermatocytes. Green: phospho-histone H3; red: α/β-tubulin. Asterisk indicates misaligned chromosome. Scale bars, 10 μm. D Frequency of abnormal arrangement in the meiotic metaphase I plate. White, Miwi heterozygous; Light gray, Miwi knockout. Three mice were used for each genotype, total numbers of counted metaphase I plate in Miwi heterozygous and knockout are 113 and 181, respectively.


B , C Meiotic metaphase I plate in FACS - sorted Miwi heterozygous ( B ) and knockout ( C ) spermatocytes . Green : phospho - histone H3 ; red : α / β - tubulin . Asterisk indicates misaligned chromosome . Scale bars , 10 μm .

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

B, C Meiotic metaphase I plate in FACS-sorted Miwi heterozygous (B) and knockout (C) spermatocytes. Green: phospho-histone H3; red: α/β-tubulin. Asterisk indicates misaligned chromosome. Scale bars, 10 μm.


E , F Meiotic metaphase II spread preparations of Miwi heterozygous ( E ) and knockout ( F ) spermatocytes . Scale bars , 5 μm . G Frequency of meiotic metaphase II ( MII ) cells with chromosomal abnormalities . Chromosome counts were defined as the number of paired sister chromatids per MII metaphase cell . The frequency was calculated as the number of cells with given pairs of sister chromatids ( 20 , < 20 , or > 20 ) divided by the total number of cells . Three mice were used for each genotype , total numbers of counted MII cells in Miwi heterozygous and knockout are 204 and 302 , respectively .

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

E, F Meiotic metaphase II spread preparations of Miwi heterozygous (E) and knockout (F) spermatocytes. Scale bars, 5 μm. G Frequency of meiotic metaphase II (MII) cells with chromosomal abnormalities. Chromosome counts were defined as the number of paired sister chromatids per MII metaphase cell. The frequency was calculated as the number of cells with given pairs of sister chromatids (20, <20, or >20) divided by the total number of cells. Three mice were used for each genotype, total numbers of counted MII cells in Miwi heterozygous and knockout are 204 and 302, respectively.


E , F Meiotic metaphase II spread preparations of Miwi heterozygous ( E ) and knockout ( F ) spermatocytes . Scale bars , 5 μm .

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

E, F Meiotic metaphase II spread preparations of Miwi heterozygous (E) and knockout (F) spermatocytes. Scale bars, 5 μm.


H , I Meiotic metaphase I spread preparations of Miwi heterozygous ( H ) and knockout ( I ) spermatocytes . Scale bars , 5 μm . J Frequency of meiotic metaphase I ( MI ) cells with chromosomal abnormalities . Chromosome counts were defined as the number of paired homologous chromosomes per MI metaphase cells . The frequency was calculated in the same way as in ( G ) , except for that the number of paired chromosomes was counted instead . Three mice were used for each genotype , total numbers of counted MI cells in Miwi heterozygous and knockout are 265 and 358 , respectively .

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

H, I Meiotic metaphase I spread preparations of Miwi heterozygous (H) and knockout (I) spermatocytes. Scale bars, 5 μm. J Frequency of meiotic metaphase I (MI) cells with chromosomal abnormalities. Chromosome counts were defined as the number of paired homologous chromosomes per MI metaphase cells. The frequency was calculated in the same way as in (G), except for that the number of paired chromosomes was counted instead. Three mice were used for each genotype, total numbers of counted MI cells in Miwi heterozygous and knockout are 265 and 358, respectively.


H , I Meiotic metaphase I spread preparations of Miwi heterozygous ( H ) and knockout ( I ) spermatocytes . Scale bars , 5 μm .

H, I Meiotic metaphase I spread preparations of Miwi heterozygous (H) and knockout (I) spermatocytes. Scale bars, 5 μm.


K , L Mitotic metaphase spread preparations of Miwi heterozygous ( K ) and knockout ( L ) testes . Scale bars , 5 μm . M Frequency of mitotic metaphase cells with chromosomal abnormalities . Chromosome counts were defined as the number of chromosomes per mitotic metaphase cell . Three mice were used for each genotype , total numbers of counted mitotic metaphase cells in Miwi heterozygous and knockout are 52 and 71 , respectively .

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

K, L Mitotic metaphase spread preparations of Miwi heterozygous (K) and knockout (L) testes. Scale bars, 5 μm. M Frequency of mitotic metaphase cells with chromosomal abnormalities. Chromosome counts were defined as the number of chromosomes per mitotic metaphase cell. Three mice were used for each genotype, total numbers of counted mitotic metaphase cells in Miwi heterozygous and knockout are 52 and 71, respectively.


A Equal amounts of total RNA isolated from Miwi knockout ( - / - ) , heterozygote ( + / - ) and slicer activity - deficient mutant ( - / ADH ) spermatocytes , were resolved on a 1 % agarose gel . The gel was subjected to northern blot analysis using probes specific for the sense ( S ) and antisense ( As ) strands of major ( Maj ) and minor ( Min ) satellite RNA transcripts . The blot was re - hybridized with a 28S - complementary probe to demonstrate equal loading ( bottom on the left ) . The relative abundance was quantified using ImageJ software , normalized to the 28S signal from the blot .

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

A Equal amounts of total RNA isolated from Miwi knockout (-/-), heterozygote (+/-) and slicer activity-deficient mutant (-/ADH) spermatocytes, were resolved on a 1% agarose gel. The gel was subjected to northern blot analysis using probes specific for the sense (S) and antisense (As) strands of major (Maj) and minor (Min) satellite RNA transcripts. The blot was re-hybridized with a 28S-complementary probe to demonstrate equal loading (bottom on the left). The relative abundance was quantified using ImageJ software, normalized to the 28S signal from the blot.


B 100ng of total RNA was isolated from Miwi heterozygous ( + / - ) , knockout ( - / - ) and slicer activity - deficient mutant ( - / ADH ) spermatocytes and was subjected to qRT - PCR with primers specific to major satellite RNA , minor satellite RNA , 5S , 5 . 8S and Gapdh . Data were normalized to 5S rRNA . 5 . 8S rRNA and Gapdh were used as negative controls .

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

B 100ng of total RNA was isolated from Miwi heterozygous (+/-), knockout (-/-) and slicer activity-deficient mutant (-/ADH) spermatocytes and was subjected to qRT-PCR with primers specific to major satellite RNA, minor satellite RNA, 5S, 5.8S and Gapdh. Data were normalized to 5S rRNA. 5.8S rRNA and Gapdh were used as negative controls.


B 100ng of total RNA was isolated from Miwi heterozygous ( + / - ) , knockout ( - / - ) and slicer activity - deficient mutant ( - / ADH ) spermatocytes and was subjected to qRT - PCR with primers specific to major satellite RNA , minor satellite RNA , 5S , 5 .

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

B 100ng of total RNA was isolated from Miwi heterozygous (+/-), knockout (-/-) and slicer activity-deficient mutant (-/ADH) spermatocytes and was subjected to qRT-PCR with primers specific to major satellite RNA, minor satellite RNA, 5S, 5.8S


Sertoli - spermatogenic cell co - cultures were transfected with vectors ectopically expressing both strands of major satellite RNA ( Maj , dark gray bar ) , both strands of minor satellite RNA ( Min , black bar ) , full - length 18S ( 18S , light gray bar ) or empty vector ( Ctrl , white bar ) . Total RNA isolated from equal numbers of FACS - sorted MIWI - expressing spermatocytes were subjected to qRT - PCR with primers specific to major satellite RNA ( C ) , minor satellite RNA ( D ) , 18S ( E )

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

Sertoli-spermatogenic cell co-cultures were transfected with vectors ectopically expressing both strands of major satellite RNA (Maj, dark gray bar), both strands of minor satellite RNA (Min, black bar), full-length 18S (18S, light gray bar) or empty vector (Ctrl, white bar). Total RNA isolated from equal numbers of FACS-sorted MIWI-expressing spermatocytes were subjected to qRT-PCR with primers specific to major satellite RNA (C), minor satellite RNA (D), 18S (E)


Sertoli - spermatogenic cell co - cultures were transfected with vectors ectopically expressing both strands of major satellite RNA ( Maj , dark gray bar ) , both strands of minor satellite RNA ( Min , black bar ) , full - length 18S ( 18S , light gray bar ) or empty vector ( Ctrl , white bar ) . Total RNA isolated from equal numbers of FACS - sorted MIWI - expressing spermatocytes were subjected to qRT - PCR with primers specific to 5 . 8S ( F ) and Gapdh ( G ) . Data presented were normalized to 5S rRNA . 5 . 8S rRNA ( F ) and Gapdh ( G ) were used as negative controls .

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

Sertoli-spermatogenic cell co-cultures were transfected with vectors ectopically expressing both strands of major satellite RNA (Maj, dark gray bar), both strands of minor satellite RNA (Min, black bar), full-length 18S (18S, light gray bar) or empty vector (Ctrl, white bar). Total RNA isolated from equal numbers of FACS-sorted MIWI-expressing spermatocytes were subjected to qRT-PCR with primers specific to 5.8S (F) and Gapdh (G). Data presented were normalized to 5S rRNA. 5.8S rRNA (F) and Gapdh (G) were used as negative controls.


H Immunofluorescence images of Meiotic metaphase I plate in Sertoli - spermatogenic cell co - cultures . Shown were spermatocytes over - expressing both strands of major satellite RNA ( Maj ) , both strands of minor satellite RNA ( Min ) , full - length 18S ( 18S ) or empty vector ( Ctrl ) . Green : phospho - histone H3 ; red : α / β - tubulin . Asterisk indicates misaligned chromosome . Scale bar , 5 μm .

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

H Immunofluorescence images of Meiotic metaphase I plate in Sertoli-spermatogenic cell co-cultures. Shown were spermatocytes over-expressing both strands of major satellite RNA (Maj), both strands of minor satellite RNA (Min), full-length 18S (18S) or empty vector (Ctrl). Green: phospho-histone H3; red: α/β-tubulin. Asterisk indicates misaligned chromosome. Scale bar, 5 μm.


I Frequency of mis - arrangement of the meiotic metaphase I plate in Sertoli - spermatogenic cell cocultures using Miwi + / - ; Stra8 - Cre testes . Spermatocytes transfected with vectors expressing both strands of major satellite RNA ( Maj , dark gray ) , both strands of minor satellite RNA ( Min , black ) , full - length 18S ( 18S , light gray ) or empty vector ( Ctrl , white ) . n = 3 cultures for each group . The total numbers of counted meiotic metaphase plate in Ctrl , 18S , Maj and Min were 153 , 144 , 130 and 141 , respectively .

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

I Frequency of mis-arrangement of the meiotic metaphase I plate in Sertoli-spermatogenic cell cocultures using Miwi+/-;Stra8-Cre testes. Spermatocytes transfected with vectors expressing both strands of major satellite RNA (Maj, dark gray), both strands of minor satellite RNA (Min, black), full-length 18S (18S, light gray) or empty vector (Ctrl, white). n=3 cultures for each group. The total numbers of counted meiotic metaphase plate in Ctrl, 18S, Maj and Min were 153, 144, 130 and 141, respectively.


J Combined RNA - DNA FISH analysis of major satellite transcripts and DNA regions in Miwi heterozygote ( Miwi + / - ) and knockout ( Miwi - / - ) diakinesis spermatocytes . Sense major satellite RNA transcripts were first detected using an antisense probe ( red signal ) , and then the major satellite DNA region was further detected using a sense probe ( green signal ) . For RNase A treatment , the slides were incubated with ( + RNase A ) or without ( - RNase A ) 100 μg / ml RNase A for 1 hour before adding antisense probe . Nuclear DNA was stained by DAPI ( blue ) . Arrows indicate the same pericentric heterochromatin in each channel per cell . Scale bars represent 2 µm .

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

J Combined RNA-DNA FISH analysis of major satellite transcripts and DNA regions in Miwi heterozygote (Miwi+/-) and knockout (Miwi-/-) diakinesis spermatocytes. Sense major satellite RNA transcripts were first detected using an antisense probe (red signal), and then the major satellite DNA region was further detected using a sense probe (green signal). For RNase A treatment, the slides were incubated with (+RNase A) or without (-RNase A) 100 μg/ml RNase A for 1 hour before adding antisense probe. Nuclear DNA was stained by DAPI (blue). Arrows indicate the same pericentric heterochromatin in each channel per cell. Scale bars represent 2 µm.


A Combined immunofluorescence and DNA FISH analysis of the kinetochore inner plate / centromere formation in Miwi heterozygous ( Miwi + / - ) and knockout ( Miwi - / - ) spermatocytes at diakinesis / metaphase I . For slicer activity - deficient mutant spermatocytes ( Miwi - / ADH ) , the images are presented in Fig EV1A . Chromosomes were stained with anti - centromere protein antibody ( CREST , purple signal ) , indicating the kinetochore inner plate / centromere . Major satellite DNA regions were detected using an antisense probe ( red signal ) which revealed the specific localization of pericentromeres in chromosomes . Nuclear DNA was stained by DAPI ( blue ) . Scale bars represent 2 µm .

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

A Combined immunofluorescence and DNA FISH analysis of the kinetochore inner plate/centromere formation in Miwi heterozygous (Miwi+/-) and knockout (Miwi-/-) spermatocytes at diakinesis/metaphase I. For slicer activity-deficient mutant spermatocytes (Miwi-/ADH), the images are presented in Fig EV1A. Chromosomes were stained with anti-centromere protein antibody (CREST, purple signal), indicating the kinetochore inner plate/centromere. Major satellite DNA regions were detected using an antisense probe (red signal) which revealed the specific localization of pericentromeres in chromosomes. Nuclear DNA was stained by DAPI (blue). Scale bars represent 2 µm.


B Left panel : illustration of kinetochore configurations in meiotic metaphase I . The kinetochores of sister chromatids cohere to form a single microtubule - binding interface . The inner - outer kinetochore axis connects the pericentric chromatin to the spindle microtubule . CREST ( centromere protein ) and BUBR1 ( budding uninhibited by benzimidazole - related 1 ) served as markers for the inner and outer regions of the kinetochore , respectively . A major satellite DNA ( Maj sat DNA ) probe was used to detect the pericentric chromatin . Right panel : categories of inner kinetochore configurations from A . Right panels : Inner kinetochore configurations in MI were classified as overlapping ( two sister kinetochores which overlap by more than their individual radii , also see yellow arrow in A ) , adjacent ( two sister kinetochores which overlap by less than 100 % of their individual radii , also see magenta arrow in A ) or separated ( two sister kinetochores without overlapping , also see light blue arrow in A ) kinetochores . The scale bar represents 1 µm . C Population of overlapping , adjacent , and separated inner kinetochores shown in B . The results represent the mean ± SD of three independent experiments . n = 3 mice for each genotype . The total numbers of counted cells in Miwi + / - , Miwi - / - and Miwi - / ADH spermatocytes were 88 , 118 and 125 , respectively . The total numbers of counted sister kinetochore pairs in Miwi + / - , Miwi - / - and Miwi - / ADH spermatocytes were 3 , 520 , 4 , 720 and 5 , 000 , respectively .

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

B Left panel: illustration of kinetochore configurations in meiotic metaphase I. The kinetochores of sister chromatids cohere to form a single microtubule-binding interface. The inner-outer kinetochore axis connects the pericentric chromatin to the spindle microtubule. CREST (centromere protein) and BUBR1 (budding uninhibited by benzimidazole-related 1) served as markers for the inner and outer regions of the kinetochore, respectively. A major satellite DNA (Maj sat DNA) probe was used to detect the pericentric chromatin. Right panel: categories of inner kinetochore configurations from A. Right panels: Inner kinetochore configurations in MI were classified as overlapping (two sister kinetochores which overlap by more than their individual radii, also see yellow arrow in A), adjacent (two sister kinetochores which overlap by less than 100% of their individual radii, also see magenta arrow in A) or separated (two sister kinetochores without overlapping, also see light blue arrow in A) kinetochores. The scale bar represents 1 µm. C Population of overlapping, adjacent, and separated inner kinetochores shown in B. The results represent the mean ± SD of three independent experiments. n=3 mice for each genotype. The total numbers of counted cells in Miwi+/-, Miwi-/- and Miwi-/ADH spermatocytes were 88, 118 and 125, respectively. The total numbers of counted sister kinetochore pairs in Miwi+/-, Miwi-/- and Miwi-/ADH spermatocytes were 3,520, 4,720 and 5,000, respectively.


B Left panel : illustration of kinetochore configurations in meiotic metaphase I . The kinetochores of sister chromatids cohere to form a single microtubule - binding interface . The inner - outer kinetochore axis connects the pericentric chromatin to the spindle microtubule . CREST ( centromere protein ) and BUBR1 ( budding uninhibited by benzimidazole - related 1 ) served as markers for the inner and outer regions of the kinetochore , respectively . A major satellite DNA ( Maj sat DNA ) probe was used to detect the pericentric chromatin . Right panel : categories of inner kinetochore configurations from A . Right panels : Inner kinetochore configurations in MI were classified as overlapping ( two sister kinetochores which overlap by more than their individual radii , also see yellow arrow in A ) , adjacent ( two sister kinetochores which overlap by less than 100 % of their individual radii , also see magenta arrow in A ) or separated ( two sister kinetochores without overlapping , also see light blue arrow in A ) kinetochores . The scale bar represents 1 µm .

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

B Left panel: illustration of kinetochore configurations in meiotic metaphase I. The kinetochores of sister chromatids cohere to form a single microtubule-binding interface. The inner-outer kinetochore axis connects the pericentric chromatin to the spindle microtubule. CREST (centromere protein) and BUBR1 (budding uninhibited by benzimidazole-related 1) served as markers for the inner and outer regions of the kinetochore, respectively. A major satellite DNA (Maj sat DNA) probe was used to detect the pericentric chromatin. Right panel: categories of inner kinetochore configurations from A. Right panels: Inner kinetochore configurations in MI were classified as overlapping (two sister kinetochores which overlap by more than their individual radii, also see yellow arrow in A), adjacent (two sister kinetochores which overlap by less than 100% of their individual radii, also see magenta arrow in A) or separated (two sister kinetochores without overlapping, also see light blue arrow in A) kinetochores. The scale bar represents 1 µm.


D Combined immunofluorescence and DNA FISH analysis of kinetochore inner and outer plate formation in Miwi heterozygous ( Miwi + / - ) and knockout ( Miwi - / - ) at diakinesis / metaphase I . For slicer activity - deficient mutant spermatocytes ( Miwi - / ADH ) , the images are presented in Fig EV1B . Chromosomes were stained with anti - BUBR1 ( green ) and anti - centromere - protein ( CREST , purple ) antibodies to mark the kinetochore outer and inner plate , respectively . In addition , major satellite DNA regions were further detected using an antisense probe ( red signal ) . Nuclear DNA was stained by DAPI ( blue ) . O - O kinetochore pairs : yellow arrow ; S - S , magenta arrow ; O - S , light blue arrow ; and S - O , light green arrow . Scale bars represent 2 µm . E Magnified images of categories of sister kinetochore configurations from D . One arrow indicates overlapping pair . Double arrows indicate split pair . The scale bar represents 1 µm . F Population of the sister kinetochores shown in E . n = 3 mice for each genotype . The total numbers of counted cells in Miwi + / - , Miwi - / - and Miwi - / ADH spermatocytes were 93 , 89 and 81 , respectively . The total numbers of counted sister kinetochore pairs in Miwi + / - , Miwi - / - and Miwi - / ADH spermatocytes were 3 , 720 , 3 , 560 and 3 , 240 , respectively .

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

D Combined immunofluorescence and DNA FISH analysis of kinetochore inner and outer plate formation in Miwi heterozygous (Miwi+/-) and knockout (Miwi-/-) at diakinesis/metaphase I. For slicer activity-deficient mutant spermatocytes (Miwi-/ADH), the images are presented in Fig EV1B. Chromosomes were stained with anti-BUBR1 (green) and anti-centromere-protein (CREST, purple) antibodies to mark the kinetochore outer and inner plate, respectively. In addition, major satellite DNA regions were further detected using an antisense probe (red signal). Nuclear DNA was stained by DAPI (blue). O-O kinetochore pairs: yellow arrow; S-S, magenta arrow; O-S, light blue arrow; and S-O, light green arrow. Scale bars represent 2 µm. E Magnified images of categories of sister kinetochore configurations from D. One arrow indicates overlapping pair. Double arrows indicate split pair. The scale bar represents 1 µm. F Population of the sister kinetochores shown in E. n=3 mice for each genotype. The total numbers of counted cells in Miwi+/-, Miwi-/- and Miwi-/ADH spermatocytes were 93, 89 and 81, respectively. The total numbers of counted sister kinetochore pairs in Miwi+/-, Miwi-/- and Miwi-/ADH spermatocytes were 3,720, 3,560 and 3,240, respectively.


D Combined immunofluorescence and DNA FISH analysis of kinetochore inner and outer plate formation in Miwi heterozygous ( Miwi + / - ) and knockout ( Miwi - / - ) at diakinesis / metaphase I . For slicer activity - deficient mutant spermatocytes ( Miwi - / ADH ) , the images are presented in Fig EV1B . Chromosomes were stained with anti - BUBR1 ( green ) and anti - centromere - protein ( CREST , purple ) antibodies to mark the kinetochore outer and inner plate , respectively . In addition , major satellite DNA regions were further detected using an antisense probe ( red signal ) . Nuclear DNA was stained by DAPI ( blue ) . O - O kinetochore pairs : yellow arrow ; S - S , magenta arrow ; O - S , light blue arrow ; and S - O , light green arrow . Scale bars represent 2 µm .

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

D Combined immunofluorescence and DNA FISH analysis of kinetochore inner and outer plate formation in Miwi heterozygous (Miwi+/-) and knockout (Miwi-/-) at diakinesis/metaphase I. For slicer activity-deficient mutant spermatocytes (Miwi-/ADH), the images are presented in Fig EV1B. Chromosomes were stained with anti-BUBR1 (green) and anti-centromere-protein (CREST, purple) antibodies to mark the kinetochore outer and inner plate, respectively. In addition, major satellite DNA regions were further detected using an antisense probe (red signal). Nuclear DNA was stained by DAPI (blue). O-O kinetochore pairs: yellow arrow; S-S, magenta arrow; O-S, light blue arrow; and S-O, light green arrow. Scale bars represent 2 µm.


B RNA pull - down assay using biotinylated major and minor satellite RNA transcripts and whole cell extracts from Miwi heterozygoous ( Miwi + / - ) , knockout ( Miwi - / - ) , slicer activity - deficient mutant ( Miwi - / ADH ) , and Mov10l1 germline knockout ( Mov10l1f / - ; Stra8 - Cre ) testes . Mov10l1f / - ; Stra8 - Cre mice represent a pachytene piRNA - deficient condition . Approximately one repeat of In vitro transcribed major and minor satellite RNAs corresponding to the sequences in A were used as baits . Precipitated proteins were visualized by Western blotting using antibodies against the indicated proteins ( MIWI and MILI ) . The input equals 1 / 10 to 1 / 80 of the protein lysate used in the pull - down . Transcripts corresponding to 300 nt and 162 nt of the 18S rRNA sequence served as controls for the antisense ( As ) and sense ( S ) sequence of major and minor satellite transcripts , respectively . The blot was re - probed with anti - GAPDH antibody serving as a negative control .

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

B RNA pull-down assay using biotinylated major and minor satellite RNA transcripts and whole cell extracts from Miwi heterozygoous (Miwi+/-), knockout (Miwi-/-), slicer activity-deficient mutant (Miwi-/ADH), and Mov10l1 germline knockout (Mov10l1f/-;Stra8-Cre) testes. Mov10l1f/-;Stra8-Cre mice represent a pachytene piRNA-deficient condition. Approximately one repeat of In vitro transcribed major and minor satellite RNAs corresponding to the sequences in A were used as baits. Precipitated proteins were visualized by Western blotting using antibodies against the indicated proteins (MIWI and MILI). The input equals 1/10 to 1/80 of the protein lysate used in the pull-down. Transcripts corresponding to 300 nt and 162 nt of the 18S rRNA sequence served as controls for the antisense (As) and sense (S) sequence of major and minor satellite transcripts, respectively. The blot was re-probed with anti-GAPDH antibody serving as a negative control.


C In vitro RNA processing assay with nuage - nuclear fraction from testicular lysates of Miwi knockout ( - / - ) , heterozygous ( + / - ) , and slicer activity - deficient ( - / ADH ) mutants , and 5 ' - end radioactively labelled RNA targets corresponding to approximately one and two repeats of major and minor satellite sequences ( see panel A for details ) . Transcripts corresponding to 162 , 300 , and 542 nt of the 18S rRNA sequence served as controls . Arrow indicates the cleavage products of In vitro transcribed sense major satellite RNAs after reactions .

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

C In vitro RNA processing assay with nuage-nuclear fraction from testicular lysates of Miwi knockout (-/-), heterozygous (+/-), and slicer activity-deficient (-/ADH) mutants, and 5'-end radioactively labelled RNA targets corresponding to approximately one and two repeats of major and minor satellite sequences (see panel A for details). Transcripts corresponding to 162, 300, and 542 nt of the 18S rRNA sequence served as controls. Arrow indicates the cleavage products of In vitro transcribed sense major satellite RNAs after reactions.


D In vitro RNA processing assay with nuage - nuclear fraction from Mov10l1 heterozygous ( Mov10l1f / + ; Stra8 - Cre ) and Mov10l1 germline knockout ( Mov10l1f / - ; Stra8 - Cre ) testis lysates , and 5 ' - end radioactively labelled RNA targets corresponding to approximately one and two repeats of sense and antisense major satellite sequences . Mov10l1f / - ; Stra8 - Cre mice represent a pachytene piRNA - deficient condition . The arrow indicates the cleavage product of the in vitro transcribed sense major satellite RNA .

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

D In vitro RNA processing assay with nuage-nuclear fraction from Mov10l1 heterozygous (Mov10l1f/+;Stra8-Cre) and Mov10l1 germline knockout (Mov10l1f/-;Stra8-Cre) testis lysates, and 5'-end radioactively labelled RNA targets corresponding to approximately one and two repeats of sense and antisense major satellite sequences. Mov10l1f/-;Stra8-Cre mice represent a pachytene piRNA-deficient condition. The arrow indicates the cleavage product of the in vitro transcribed sense major satellite RNA.


RNA - immunoprecipitation was performed using anti - MIWI antibody . RNA was extracted from anti - MIWI immunoprecipitates and subjected to Northern blot analysis using probes specific for potential piRNAs ( piR major - a and b ) , as indicated in panel 20 % of input RNA from each group was used as the control . The blot was re - hybridized with piR - A and piR - E complementary probes to demonstrate the efficiency of the RNA - IP . A miR - 16 complementary probe served as a negative control .

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

RNA-immunoprecipitation was performed using anti-MIWI antibody. RNA was extracted from anti-MIWI immunoprecipitates and subjected to Northern blot analysis using probes specific for potential piRNAs (piR major-a and b), as indicated in panel 20% of input RNA from each group was used as the control. The blot was re-hybridized with piR-A and piR-E complementary probes to demonstrate the efficiency of the RNA-IP. A miR-16 complementary probe served as a negative control.


A RNA pull - down was performed using in vitro transcribed biotinylated transcripts and whole cell extracts from Miwi + / - , Miwi - / - , Miwi - / ADH , and Mov10l1f / - ; Stra8 - Cre testes . Mov10l1f / - ; Stra8 - Cre mice represent a pachytene piRNA - deficient condition . In vitro transcribed RNAs corresponding to approximately one repeat of major and minor satellite sequences were used as baits . Precipitated protein was visualized by Western blotting using anti - Dicer antibody .

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

A RNA pull-down was performed using in vitro transcribed biotinylated transcripts and whole cell extracts from Miwi+/-, Miwi-/-, Miwi-/ADH, and Mov10l1f/-;Stra8-Cre testes. Mov10l1f/-;Stra8-Cre mice represent a pachytene piRNA-deficient condition. In vitro transcribed RNAs corresponding to approximately one repeat of major and minor satellite sequences were used as baits. Precipitated protein was visualized by Western blotting using anti-Dicer antibody.


B Equal amounts of total RNA isolated from spermatocytes of Miwi + / - ; Dicerf / + ; Stra8 - Cre , Miwi - / - ; Dicerf / + ; Stra8 - Cre , Miwi + / - ; Dicerf / - ; Stra8 - Cre and Miwi - / - ; Dicerf / - ; Stra8 - Cre mice , were resolved on a 1 % agarose gel . The gel was subjected to Northern blot analysis using probes specific for the sense and antisense sequences of major and minor satellites . The blot was re - hybridized with a 28S rRNA - complementary probe to demonstrate equal loading ( bottom on the left ) .

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

B Equal amounts of total RNA isolated from spermatocytes of Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, Miwi+/-;Dicerf/-;Stra8-Cre and Miwi-/-;Dicerf/-;Stra8-Cre mice, were resolved on a 1% agarose gel. The gel was subjected to Northern blot analysis using probes specific for the sense and antisense sequences of major and minor satellites. The blot was re-hybridized with a 28S rRNA-complementary probe to demonstrate equal loading (bottom on the left).


C in vitro RNA processing assay with nuage - enriched nuclear fraction from Miwi + / - ; Dicerf / + ; Stra8 - Cre , Miwi - / - ; Dicerf / + ; Stra8 - Cre , and Miwi + / - ; Dicerf / - ; Stra8 - Cre mice , and 5 ' - end radioactively labelled RNA targets corresponding to approximately one and two repeats of sense and antisense major satellite sequences . Arrow indicates the cleavage products of In vitro transcribed sense major satellite RNAs by MIWI - piRNA complex .

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

C in vitro RNA processing assay with nuage-enriched nuclear fraction from Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, and Miwi+/-;Dicerf/-;Stra8-Cre mice, and 5'-end radioactively labelled RNA targets corresponding to approximately one and two repeats of sense and antisense major satellite sequences. Arrow indicates the cleavage products of In vitro transcribed sense major satellite RNAs by MIWI-piRNA complex.


D Equal amounts of total RNA isolated from spermatocytes of Miwi + / - ; Dicerf / + ; Stra8 - Cre , Miwi - / - ; Dicerf / + ; Stra8 - Cre , Miwi + / - ; Dicerf / - ; Stra8 - Cre and Miwi - / - ; Dicerf / - ; Stra8 - Cre mice , were digested with RNase III ( RNase III ) or nuclease S1 ( S1 ) , and further resolved on a 1 % agarose gel . The gel was subjected to northern blot analysis using probes specific for the sense ( S ) and antisense ( As ) strands of major ( Maj ) and minor ( Min ) satellite transcripts . The blot was re - hybridized with 18S and 28S - complementary probes to demonstrate equal loading and the same efficiency of RNase III or nuclease S1 digestion .

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

D Equal amounts of total RNA isolated from spermatocytes of Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, Miwi+/-;Dicerf/-;Stra8-Cre and Miwi-/-;Dicerf/-;Stra8-Cre mice, were digested with RNase III (RNase III) or nuclease S1 (S1), and further resolved on a 1% agarose gel. The gel was subjected to northern blot analysis using probes specific for the sense (S) and antisense (As) strands of major (Maj) and minor (Min) satellite transcripts. The blot was re-hybridized with 18S and 28S-complementary probes to demonstrate equal loading and the same efficiency of RNase III or nuclease S1 digestion.


E Sequential in vitro RNA processing assay using 5 ' - end radioactively labelled RNA targets corresponding to approximately one repeat of sense and antisense major satellite sequences . The nuage - nuclear fraction from Miwi + / - testes is indicated as N . Total cellular extracts ( T ) from Miwi + / - testes were subjected to immunodepletion using anti - Dicer antibodies ( α - Dicer ) , with IgG ( IgG ) as a negative control . The incubation time ( Incub . t ) was 0 . 5 and 1 hour . The arrow indicates the product of MIWI - mediated cleavage of the in vitro transcribed sense major satellite RNA .

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

E Sequential in vitro RNA processing assay using 5'-end radioactively labelled RNA targets corresponding to approximately one repeat of sense and antisense major satellite sequences. The nuage-nuclear fraction from Miwi+/- testes is indicated as N. Total cellular extracts (T) from Miwi+/- testes were subjected to immunodepletion using anti-Dicer antibodies (α-Dicer), with IgG (IgG ) as a negative control. The incubation time (Incub. t) was 0.5 and 1 hour. The arrow indicates the product of MIWI-mediated cleavage of the in vitro transcribed sense major satellite RNA.


E Sequential in vitro RNA processing assay using 5 ' - end radioactively labelled RNA targets corresponding to approximately one repeat of sense and antisense major satellite sequences . The nuage - nuclear fraction from Miwi + / - testes is indicated as N . Total cellular extracts ( T ) from Miwi + / - testes were subjected to immunodepletion using anti - Dicer antibodies ( α - Dicer ) , with IgG (

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

E Sequential in vitro RNA processing assay using 5'-end radioactively labelled RNA targets corresponding to approximately one repeat of sense and antisense major satellite sequences. The nuage-nuclear fraction from Miwi+/- testes is indicated as N. Total cellular extracts (T) from Miwi+/- testes were subjected to immunodepletion using anti-Dicer antibodies (α-Dicer), with IgG (IgG


A Frequency of overlapping , adjacent , and separated inner kinetochores . Spermatocytes from Miwi + / - ; Dicerf / + ; Stra8 - Cre , Miwi - / - ; Dicerf / + ; Stra8 - Cre , Miwi + / - ; Dicerf / - ; Stra8 - Cre , and Miwi - / - ; Dicerf / - ; Stra8 - Cre mice were used to examine sister kinetochore configuration at meiosis I as shown in Fig 3A and 3B . n = 3 mice for each genotype . The total numbers of counted cells in the four types of spermatocytes were 101 , 117 , 129 and 115 , respectively . The total numbers of counted sister kinetochore pairs in the four types of spermatocytes were 4 , 040 , 4 , 680 , 5 , 160 and 4 , 600 , respectively . B Frequency of the different sister kinetochore configurations . The four types of spermatocytes in A were used to examine sister kinetochore configurations in meiosis I metaphase with O - O , S - S , O - S , or S - O as defined in the text . n = 3 mice for each genotype . The total numbers of counted cells in the four types of spermatocytes were 134 , 140 , 140 and 130 , respectively . The total numbers of counted sister kinetochore pairs in the four types of spermatocytes were 5 , 360 , 5 , 600 , 5 , 600 and 5 , 200 , respectively .

O O O O O O O O O O B-SUBCELLULAR O B-CELL_TYPE O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-CELL_TYPE O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O

A Frequency of overlapping, adjacent, and separated inner kinetochores. Spermatocytes from Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, Miwi+/-;Dicerf/-;Stra8-Cre, and Miwi-/-;Dicerf/-;Stra8-Cre mice were used to examine sister kinetochore configuration at meiosis I as shown in Fig 3A and 3B. n=3 mice for each genotype. The total numbers of counted cells in the four types of spermatocytes were 101, 117, 129 and 115, respectively. The total numbers of counted sister kinetochore pairs in the four types of spermatocytes were 4,040, 4,680, 5,160 and 4,600, respectively. B Frequency of the different sister kinetochore configurations. The four types of spermatocytes in A were used to examine sister kinetochore configurations in meiosis I metaphase with O-O, S-S, O-S, or S-O as defined in the text. n=3 mice for each genotype. The total numbers of counted cells in the four types of spermatocytes were 134, 140, 140 and 130, respectively. The total numbers of counted sister kinetochore pairs in the four types of spermatocytes were 5,360, 5,600, 5,600 and 5,200, respectively.


A Frequency of overlapping , adjacent , and separated inner kinetochores . Spermatocytes from Miwi + / - ; Dicerf / + ; Stra8 - Cre , Miwi - / - ; Dicerf / + ; Stra8 - Cre , Miwi + / - ; Dicerf / - ; Stra8 - Cre , and Miwi - / - ; Dicerf / - ; Stra8 - Cre mice were used to examine sister kinetochore configuration at meiosis I as shown in Fig 3A and 3B . n = 3 mice for each genotype . The total numbers of counted cells in the four types of spermatocytes were 101 , 117 , 129 and 115 , respectively . The total numbers of counted sister kinetochore pairs in the four types of spermatocytes were 4 , 040 , 4 , 680 , 5 , 160 and 4 , 600 , respectively .

O O O O O O O O O O B-SUBCELLULAR O B-CELL_TYPE O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O

A Frequency of overlapping, adjacent, and separated inner kinetochores. Spermatocytes from Miwi+/-;Dicerf/+;Stra8-Cre, Miwi-/-;Dicerf/+;Stra8-Cre, Miwi+/-;Dicerf/-;Stra8-Cre, and Miwi-/-;Dicerf/-;Stra8-Cre mice were used to examine sister kinetochore configuration at meiosis I as shown in Fig 3A and 3B. n=3 mice for each genotype. The total numbers of counted cells in the four types of spermatocytes were 101, 117, 129 and 115, respectively. The total numbers of counted sister kinetochore pairs in the four types of spermatocytes were 4,040, 4,680, 5,160 and 4,600, respectively.


C Frequency of abnormal arrangement in the meiotic metaphase I plate . The four types of spermatocytes in A and B were used to perform immunofluorescence as shown in Fig 1B and C . n = 3 mice for each genotype . The total numbers of counted metaphase I plates in the four types of spermatocytes were 221 , 181 , 421 and 240 , respectively . D Frequency of meiosis I metaphase cells with chromosomal abnormalities . The four types of spermatocytes in A - C were used to perform a metaphase spread as shown in Fig 1H and 1I . n = 3 mice for each genotype . The total numbers of counted MI cells in the four types of spermatocytes were 89 , 73 , 132 and 104 , respectively . E Frequency of meiosis II metaphase cells with chromosomal abnormalities . The four types of spermatocytes in A - D were used to perform a metaphase spread as shown in Fig 1K and L . n = 3 mice for each genotype . The total numbers of counted MII cells in the four types of spermatocytes were 74 , 87 , 85 and 94 , respectively .

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

C Frequency of abnormal arrangement in the meiotic metaphase I plate. The four types of spermatocytes in A and B were used to perform immunofluorescence as shown in Fig 1B and C. n=3 mice for each genotype. The total numbers of counted metaphase I plates in the four types of spermatocytes were 221, 181, 421 and 240, respectively. D Frequency of meiosis I metaphase cells with chromosomal abnormalities. The four types of spermatocytes in A-C were used to perform a metaphase spread as shown in Fig 1H and 1I. n=3 mice for each genotype. The total numbers of counted MI cells in the four types of spermatocytes were 89, 73, 132 and 104, respectively. E Frequency of meiosis II metaphase cells with chromosomal abnormalities. The four types of spermatocytes in A-D were used to perform a metaphase spread as shown in Fig 1K and L. n=3 mice for each genotype. The total numbers of counted MII cells in the four types of spermatocytes were 74, 87, 85 and 94, respectively.


C Frequency of abnormal arrangement in the meiotic metaphase I plate . The four types of spermatocytes in A and B were used to perform immunofluorescence as shown in Fig 1B and C . n = 3 mice for each genotype . The total numbers of counted metaphase I plates in the four types of spermatocytes were 221 , 181 , 421 and 240 , respectively .

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

C Frequency of abnormal arrangement in the meiotic metaphase I plate. The four types of spermatocytes in A and B were used to perform immunofluorescence as shown in Fig 1B and C. n=3 mice for each genotype. The total numbers of counted metaphase I plates in the four types of spermatocytes were 221, 181, 421 and 240, respectively.


A - F ' Upper panels ( A - F ) show morphology of c4da neurons of the indicated genotypes at the third instar larval stage , lower panels ( A ' - F ' ) show c4da neuron morphology at 18 h after puparium formation ( APF ) . Neurons were labeled by CD8 : : GFP expression under ppk - GAL4 or by tdtomato expression in MARCM clones . ( A , A ' ) C4da neurons expressing control Orco dsRNA . Scale bars are 100 μm in A ( for larval images ) and 50 μm in A ' ( for pupal images ) . ( B , B ' ) C4da neurons expressing PP2A - 29B dsRNA . ( C , C ' ) Homozygous PP2A - 29BGE16781 mutant c4da neuron MARCM clones . ( D , D ' ) Homozygous PP2A - 29BGE16781 mutant c4da neuron MARCM clones expressing UAS - HAPP2A - 29B . ( E , E ' ) C4da neurons expressing dominant - negative Mts . ( F , F ' ) Homozygous mtss5286 mutant c4da neuron MARCM clones . G Penetrance of pruning defects at 18 h APF in A ' - E ' . * * P < 0 . 005 , * * * P < 0 . 0005 , Fisher ' s exact test . Number of neurons for each genotype are given in the figure , the number of animals is given below in parentheses . H Severity of pruning defects at 18 h APF in A ' - E ' as assessed by number of primary and secondary dendrites attached to soma at 18 h APF . Data are mean + / - s . d . , * * P < 0 . 005 , * * * P < 0 . 0005 , Wilcoxon ' s test . Numbers of neurons ( animals ) for each genotype are given in the figure .

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

A - F' Upper panels (A - F) show morphology of c4da neurons of the indicated genotypes at the third instar larval stage, lower panels (A' - F') show c4da neuron morphology at 18 h after puparium formation (APF). Neurons were labeled by CD8::GFP expression under ppk-GAL4 or by tdtomato expression in MARCM clones. (A, A') C4da neurons expressing control Orco dsRNA. Scale bars are 100 μm in A (for larval images) and 50 μm in A' (for pupal images). (B, B') C4da neurons expressing PP2A-29B dsRNA. (C, C') Homozygous PP2A-29BGE16781 mutant c4da neuron MARCM clones. (D, D') Homozygous PP2A-29BGE16781 mutant c4da neuron MARCM clones expressing UAS-HAPP2A-29B. (E, E') C4da neurons expressing dominant-negative Mts. (F, F') Homozygous mtss5286 mutant c4da neuron MARCM clones. G Penetrance of pruning defects at 18 h APF in A' - E'. ** P<0.005, *** P<0.0005, Fisher's exact test. Number of neurons for each genotype are given in the figure, the number of animals is given below in parentheses. H Severity of pruning defects at 18 h APF in A' - E' as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., ** P<0.005, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.


A - J The indicated Sox14 or Mical constructs were tested for their ability to suppress pruning defects induced by PP2A - 29B dsRNA expression . C4da neurons were labeled by CD8 : : GFP expression under ppk - GAL4 , and dendrite pruning defects were assessed at 18 h APF . Panels A - E show effects of Sox14 / Mical pathway manipulations alone , panels F - J show neurons coexpressing PP2A - 29B dsRNA . The scale bar in A is 50 μm . ( A , F ) C4da neurons ( co - ) expressing lacZ . ( B , G ) C4da neurons ( co - ) overexpressing Sox14 . ( C , H ) C4da neurons ( co - ) overexpressing Mical . ( D , I ) C4da neurons ( co - ) expressing Mical ∆ redox . ( E , J ) C4da neurons ( co - ) expressing Mical ∆ CH . K Penetrance of pruning defects in panels A - J . * P < 0 . 05 , * * * P < 0 . 0005 , Fisher ' s exact test . Numbers of neurons ( animals ) for each genotype are given in the figure the numbers of animals are given below in parentheses . L Severity of pruning defects in panels A - J as assessed by number of primary and secondary dendrites attached to soma at 18 h APF . Data are mean + / - s . d . , * P < 0 . 05 , * * * P < 0 . 0005 , Wilcoxon ' s test . Numbers of neurons ( animals ) for each genotype are given in the figure .

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

A-J The indicated Sox14 or Mical constructs were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. C4da neurons were labeled by CD8::GFP expression under ppk-GAL4, and dendrite pruning defects were assessed at 18 h APF. Panels A - E show effects of Sox14/Mical pathway manipulations alone, panels F - J show neurons coexpressing PP2A-29B dsRNA. The scale bar in A is 50 μm. (A, F) C4da neurons (co-)expressing lacZ. (B, G) C4da neurons (co-)overexpressing Sox14. (C, H) C4da neurons (co-)overexpressing Mical. (D, I) C4da neurons (co-)expressing Mical∆redox. (E, J) C4da neurons (co-)expressing Mical∆CH. K Penetrance of pruning defects in panels A - J. * P<0.05, *** P<0.0005, Fisher's exact test. Numbers of neurons (animals) for each genotype are given in the figure the numbers of animals are given below in parentheses. L Severity of pruning defects in panels A - J as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., * P<0.05, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.


C4da neurons were labeled by CD8 : : GFP expression under ppk - GAL4 , and expression of Sox14 was assessed by immunofluorescence . A - C Sox14 stainings at 2 h APF . ( A ) Control c4da neurons . ( B ) C4da neurons expressing PP2A - 29B dsRNA . ( C ) C4da neuron expressing dominant - negative Mts . D Quantification of Sox14 expression in ( A - C ) . C4da neuron Sox14 expression was normalized to Sox14 expression in neighboring ddaE c1da neurons ( identified by characteristic cell body shape ) . Data are mean + / - s . d . . The numbers of neurons ( animals ) for each genotype are given in each graph ( data were not derived from independent experiments ) . n . s . , not significant , * * * P < 0 . 0005 , Wilcoxon ' s test .

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

C4da neurons were labeled by CD8::GFP expression under ppk-GAL4, and expression of Sox14 was assessed by immunofluorescence. A - C Sox14 stainings at 2 h APF. (A) Control c4da neurons. (B) C4da neurons expressing PP2A-29B dsRNA. (C) C4da neuron expressing dominant-negative Mts. D Quantification of Sox14 expression in (A - C). C4da neuron Sox14 expression was normalized to Sox14 expression in neighboring ddaE c1da neurons (identified by characteristic cell body shape). Data are mean +/- s. d.. The numbers of neurons (animals) for each genotype are given in each graph (data were not derived from independent experiments). n. s., not significant, *** P<0.0005, Wilcoxon's test.


C4da neurons were labeled by CD8 : : GFP expression under ppk - GAL4 , and expression of Mical was assessed by immunofluorescence . E - H Mical stainings at the indicated time points . ( E ) Mical expression in control c4da neurons at 2 h APF . ( F ) Mical expression in c4da neurons expressing dominant - negative Mts at 2 h APF . ( G ) Mical expression in c4da neurons expressing PP2A - 29B dsRNA at 2 h APF . ( H ) Mical expression in c4da neurons expressing Orco dsRNA at 0 h APF . ( I ) Mical expression in c4da neurons expressing PP2A - 29B dsRNA at 0 h APF . J Quantification of Mical expression in ( E - I ) . C4da neuron Mical expression was normalized to Mical expression in neighboring ddaE c1da neurons ( marked by asterisks ) . Data are mean + / - s . d . . The numbers of neurons ( animals ) for each genotype are given in each graph ( data were not derived from independent experiments ) . n . s . , not significant , * * P < 0 . 005 , * * * P < 0 . 0005 , Wilcoxon ' s test . Data information : Scale bars in A and E are 10 μm .

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

C4da neurons were labeled by CD8::GFP expression under ppk-GAL4, and expression of Mical was assessed by immunofluorescence. E - H Mical stainings at the indicated time points. (E) Mical expression in control c4da neurons at 2 h APF. (F) Mical expression in c4da neurons expressing dominant-negative Mts at 2 h APF. (G) Mical expression in c4da neurons expressing PP2A-29B dsRNA at 2 h APF. (H) Mical expression in c4da neurons expressing Orco dsRNA at 0 h APF. (I) Mical expression in c4da neurons expressing PP2A-29B dsRNA at 0 h APF. J Quantification of Mical expression in (E - I). C4da neuron Mical expression was normalized to Mical expression in neighboring ddaE c1da neurons (marked by asterisks). Data are mean +/- s. d.. The numbers of neurons (animals) for each genotype are given in each graph (data were not derived from independent experiments). n. s., not significant, ** P<0.005, *** P<0.0005, Wilcoxon's test. Data information: Scale bars in A and E are 10 μm.


A - G The indicated UAS - tsr constructs were tested for their ability to suppress pruning defects induced by PP2A - 29B dsRNA expression . Suppression tests were performed as in Figure 2 . Panels A - C show effects of the expression of wild type or phosphomutant tsr variants on dendrite pruning , panels D - G show neurons coexpressing PP2A - 29B dsRNA . ( A , E ) C4da neuron ( co - ) overexpressing wild type tsr . ( B , F ) C4da neuron ( co - ) overexpressing tsr S3A . ( C , G ) C4da neuron ( co - ) overexpressing tsr S3E . The arrow in panel G denotes a characteristic varicosity at the tip of an unpruned dendrite . ( D ) C4da neuron co - expressing PP2A - 29B dsRNA and lacZ as a UAS titration control . H Penetrance of pruning defects in panels A - G . * P < 0 . 05 , Fisher ' s exact test . Numbers of neurons ( animals ) for each genotype are given in the figure . I Severity of pruning defects in panels A - G as assessed by number of primary and secondary dendrites attached to soma at 18 h APF . Data are mean + / - s . d . , * P < 0 . 05 , Wilcoxon ' s test . Numbers of neurons ( animals ) for each genotype are given in the figure .

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

A - G The indicated UAS-tsr constructs were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. Suppression tests were performed as in Figure 2. Panels A - C show effects of the expression of wild type or phosphomutant tsr variants on dendrite pruning, panels D - G show neurons coexpressing PP2A-29B dsRNA. (A, E) C4da neuron (co-)overexpressing wild type tsr. (B, F) C4da neuron (co-)overexpressing tsr S3A. (C, G) C4da neuron (co-)overexpressing tsr S3E. The arrow in panel G denotes a characteristic varicosity at the tip of an unpruned dendrite. (D) C4da neuron co-expressing PP2A-29B dsRNA and lacZ as a UAS titration control. H Penetrance of pruning defects in panels A - G. * P<0.05, Fisher's exact test. Numbers of neurons (animals) for each genotype are given in the figure. I Severity of pruning defects in panels A - G as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., * P<0.05, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.


A - G The indicated UAS - tsr constructs were tested for their ability to suppress pruning defects induced by PP2A - 29B dsRNA expression . Suppression tests were performed as in Figure 2 . Panels A - C show effects of the expression of wild type or phosphomutant tsr variants on dendrite pruning , panels D - G show neurons coexpressing PP2A - 29B dsRNA . ( A , E ) C4da neuron ( co - ) overexpressing wild type tsr . ( B , F ) C4da neuron ( co - ) overexpressing tsr S3A . ( C , G ) C4da neuron ( co - ) overexpressing tsr S3E . The arrow in panel G denotes a characteristic varicosity at the tip of an unpruned dendrite . ( D ) C4da neuron co - expressing PP2A - 29B dsRNA and lacZ as a UAS titration control .

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

A - G The indicated UAS-tsr constructs were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. Suppression tests were performed as in Figure 2. Panels A - C show effects of the expression of wild type or phosphomutant tsr variants on dendrite pruning, panels D - G show neurons coexpressing PP2A-29B dsRNA. (A, E) C4da neuron (co-)overexpressing wild type tsr. (B, F) C4da neuron (co-)overexpressing tsr S3A. (C, G) C4da neuron (co-)overexpressing tsr S3E. The arrow in panel G denotes a characteristic varicosity at the tip of an unpruned dendrite. (D) C4da neuron co-expressing PP2A-29B dsRNA and lacZ as a UAS titration control.


J - L GFP - tagged Drosophila cofilin ( GFP : : tsr ) was expressed in c4da neurons under ppk - GAL4 , and its localization was assessed at the indicated developmental stages by live imaging . Panels J - L show GFP : : tsr localization in c4da neurons at the third instar stage , panels J ' - L ' show GFP : : tsr localization in c4da neurons at 6 h APF . ( J , J ' ) Control c4da neurons expressing Orco dsRNA . ( K , K ' ) C4da neurons expressing Mical dsRNA . ( L , L ' ) C4da neurons expressing PP2A - 29B dsRNA . M Quantification of GFP : : tsr punctae in samples J - L ' . Data are mean + / - s . d . , the numbers of neurons ( animals ) analyzed for each genotype are given in the figure ( data were not derived from independent experiments ) . * * P < 0 . 005 , * * * P < 0 . 0005 , n . s . , not significant , Wilcoxon ' s test .

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

J - L GFP-tagged Drosophila cofilin (GFP::tsr) was expressed in c4da neurons under ppk-GAL4, and its localization was assessed at the indicated developmental stages by live imaging. Panels J - L show GFP::tsr localization in c4da neurons at the third instar stage, panels J' - L' show GFP::tsr localization in c4da neurons at 6 h APF. (J, J') Control c4da neurons expressing Orco dsRNA. (K, K') C4da neurons expressing Mical dsRNA. (L, L') C4da neurons expressing PP2A-29B dsRNA. M Quantification of GFP::tsr punctae in samples J - L'. Data are mean +/- s. d., the numbers of neurons (animals) analyzed for each genotype are given in the figure (data were not derived from independent experiments). ** P<0.005, *** P<0.0005, n. s., not significant, Wilcoxon's test.


A - H Genetic interactions between PP2A - 29B and actin regulators during dendrite pruning . The indicated manipulations of actin regulators were tested for their ability to suppress pruning defects induced by PP2A - 29B dsRNA expression . Suppression tests were performed as in Figure 2 . ( A ) C4da neuron coexpressing PP2A - 29B dsRNA and tdtomato as suppression control . ( B ) C4da neuron coexpressing PP2A - 29B dsRNA and GFP : : tsr . ( C ) C4da neuron coexpressing PP2A - 29B dsRNA and RacN17 . ( D ) C4da neuron coexpressing PP2A - 29B dsRNA and RhoN19 . ( E ) C4da neuron expressing PP2A - 29B dsRNA in ena210 / + background . ( F ) C4da neuron coexpressing PP2A - 29B dsRNA and Orco dsRNA as suppression control . ( G ) C4da neuron coexpressing PP2A - 29B dsRNA and Chicadee dsRNA . ( H ) C4da neuron coexpressing PP2A - 29B dsRNA and moesin dsRNA . I Penetrance of pruning defects in panels E - L . n . s . , not significant , Fisher ' s exact test . Numbers of neurons ( animals ) for each genotype are given in the figure . J Severity of pruning defects in panels E - L as assessed by number of primary and secondary dendrites attached to soma at 18 h APF . Data are mean + / - s . d . , * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 0005 , Wilcoxon ' s test . Numbers of neurons ( animals ) for each genotype are given in the figure .

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

A - H Genetic interactions between PP2A-29B and actin regulators during dendrite pruning. The indicated manipulations of actin regulators were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. Suppression tests were performed as in Figure 2. (A) C4da neuron coexpressing PP2A-29B dsRNA and tdtomato as suppression control. (B) C4da neuron coexpressing PP2A-29B dsRNA and GFP::tsr. (C) C4da neuron coexpressing PP2A-29B dsRNA and RacN17. (D) C4da neuron coexpressing PP2A-29B dsRNA and RhoN19. (E) C4da neuron expressing PP2A-29B dsRNA in ena210/+ background. (F) C4da neuron coexpressing PP2A-29B dsRNA and Orco dsRNA as suppression control. (G) C4da neuron coexpressing PP2A-29B dsRNA and Chicadee dsRNA. (H) C4da neuron coexpressing PP2A-29B dsRNA and moesin dsRNA. I Penetrance of pruning defects in panels E - L. n. s., not significant, Fisher's exact test. Numbers of neurons (animals) for each genotype are given in the figure. J Severity of pruning defects in panels E - L as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., * P<0.05, ** P<0.005, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.


A . Immunohistochemical analysis of SHARPIN expression in the human mammary gland . Cross - section of a mammary duct ( upper panel ) and magnification of the marked area ( lower panel ) . SHARPIN - positive luminal ( grey arrow ) and stromal cells ( red arrow ) , and the approximate position of the basal lamina ( dashed red line ) are indicated . Scale bar represents 50 μm .

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

A. Immunohistochemical analysis of SHARPIN expression in the human mammary gland. Cross-section of a mammary duct (upper panel) and magnification of the marked area (lower panel). SHARPIN-positive luminal (grey arrow) and stromal cells (red arrow), and the approximate position of the basal lamina (dashed red line) are indicated. Scale bar represents 50 μm.


B . Western blot analysis of SHARPIN protein expression in isolated primary mammary epithelial cells ( MECs ) and mammary stromal fibroblasts ( MSFs ) . CDH1 and vimentin were used as markers of epithelial and stromal cell lineages , respectively . GAPDH served as a control for protein loading .

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

B. Western blot analysis of SHARPIN protein expression in isolated primary mammary epithelial cells (MECs) and mammary stromal fibroblasts (MSFs). CDH1 and vimentin were used as markers of epithelial and stromal cell lineages, respectively. GAPDH served as a control for protein loading.


C . FACS - based isolation of mouse mammary gland basal epithelial cells ( LinnegCD24intICAM - 1hi ) , mature luminal epithelial cells ( LinnegCD24hiICAM - 1neg ) , luminal progenitor cells ( LinnegCD24hiICAM - 1int ) , and stromal cells ( LinnegCD24neg ) .

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

C. FACS-based isolation of mouse mammary gland basal epithelial cells (LinnegCD24intICAM-1hi), mature luminal epithelial cells (LinnegCD24hiICAM-1neg), luminal progenitor cells (LinnegCD24hiICAM-1int), and stromal cells (LinnegCD24neg).


D . Quantitative PCR analysis of SHARPIN mRNA expression in cell populations isolated in ( C ) ( mean ± SEM , n = 3 - 5 ) .

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D. Quantitative PCR analysis of SHARPIN mRNA expression in cell populations isolated in (C) (mean ± SEM, n = 3 - 5).


A - G . Mammary ductal outgrowth in 3 - 7 weeks old wt or Sharpincpdm female mice . A . Representative carmine - alum stained mammary gland whole mounts . Arrow indicates the inguinal lymph node . Scale bars represent 2 mm . B . Quantification of mammary ductal outgrowth area ( n = 4 - 15 glands ) . C . Quantification of the number of ductal branch points per mammary gland ( n = 7 - 9 glands , 7 weeks old mice ) .

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A-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. A. Representative carmine-alum stained mammary gland whole mounts. Arrow indicates the inguinal lymph node. Scale bars represent 2 mm. B. Quantification of mammary ductal outgrowth area (n = 4 - 15 glands). C. Quantification of the number of ductal branch points per mammary gland (n = 7 - 9 glands, 7 weeks old mice).


A - G . Mammary ductal outgrowth in 3 - 7 weeks old wt or Sharpincpdm female mice . D . Cryosections from 7 weeks old wt or Sharpincpdm mouse mammary glands stained with HE ( upper panel ) or immunolabelled with the indicated antibodies . Scale bars represent 20

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A-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. D. Cryosections from 7 weeks old wt or Sharpincpdm mouse mammary glands stained with HE (upper panel) or immunolabelled with the indicated antibodies. Scale bars represent 20 μm


A - G . Mammary ductal outgrowth in 3 - 7 weeks old wt or Sharpincpdm female mice . E . Representative carmine - alum stained images highlighting terminal end buds ( TEBs ) in 7 weeks old wt and Sharpincpdm mouse mammary glands and F . quantification of the number of TEBs per gland ( n = 9 - 10 glands ) . Scale bar represents 500 μm .

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A-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. E. Representative carmine-alum stained images highlighting terminal end buds (TEBs) in 7 weeks old wt and Sharpincpdm mouse mammary glands and F. quantification of the number of TEBs per gland (n = 9 - 10 glands). Scale bar represents 500 μm.


A - G . Mammary ductal outgrowth in 3 - 7 weeks old wt or Sharpincpdm female mice . G . TEBs in paraffin sections from wt or Sharpincpdm mouse mammary glands stained with HE ( upper panel ) or immunolabelled with the indicated antibodies ( lower panel ) . Scale bars represent 50 µm . Mean ± SEM .

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A-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. G. TEBs in paraffin sections from wt or Sharpincpdm mouse mammary glands stained with HE (upper panel) or immunolabelled with the indicated antibodies (lower panel). Scale bars represent 50 µm. Mean ± SEM.


A - C . Monitoring of mammary gland development following transplantation of wt or Sharpincpdm mouse mammary epithelium in the mammary fat pads ( epithelium - free ) of virgin wt mice . A . Representative carmine - alum stained images of mouse mammary glands generated from wt or Sharpincpdm epithelial tissue transplants . B . Quantification of transplant growth take - on - rate ( n = 17 ) . C . Quantification of fat pad filling rate of grown transplants ( n = 7 - 10 ) in wt hosts .

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A-C. Monitoring of mammary gland development following transplantation of wt or Sharpincpdm mouse mammary epithelium in the mammary fat pads (epithelium-free) of virgin wt mice. A. Representative carmine-alum stained images of mouse mammary glands generated from wt or Sharpincpdm epithelial tissue transplants. B. Quantification of transplant growth take-on-rate (n = 17). C. Quantification of fat pad filling rate of grown transplants (n = 7 - 10) in wt hosts.