Arginine methylation of the C - terminal IDR enhances the RNA methylation activity of the METTL14 / METTL3 complex in vitro . In vitro RNA methylation assays were performed by incubating biotin - labeled RNA substrates with METTL3 / METTL14 methyltransferase complexes containing WT , hypomethylated ( MS023 ) , and arginine methylation - deficient ( RK ) mutant METTL14 in various concentrations ( 10 - 100 nM ) . The methylation status of the METTL3 / METTL14 complex was confirmed by Western blot analysis using an anti - ADMA antibody . Coomassie staining shows the purification of the enzyme complex . Enzymatic activity was measured in counts per minute ( c . p . m . ) using a scintillation counter . Data from three replicates were analyzed by Student ' s t - test and shown as mean ± SD . * , p < 0 . 05 ; * * , p < 0 . 01 ; ns , not significant .

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

Arginine methylation of the C-terminal IDR enhances the RNA methylation activity of the METTL14/METTL3 complex in vitro. In vitro RNA methylation assays were performed by incubating biotin-labeled RNA substrates with METTL3/METTL14 methyltransferase complexes containing WT, hypomethylated (MS023), and arginine methylation-deficient (RK) mutant METTL14 in various concentrations (10-100 nM). The methylation status of the METTL3/METTL14 complex was confirmed by Western blot analysis using an anti-ADMA antibody. Coomassie staining shows the purification of the enzyme complex. Enzymatic activity was measured in counts per minute (c.p.m.) using a scintillation counter. Data from three replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01; ns, not significant.


Co - IP assays were performed to compare the interactions between METTL14 and RNAPII in control and MS023 - treated HEK293 cells . Cells were treated with either DMSO or MS023 ( 1 μM ) for 48 h before they were lysed . IP was performed using control IgG and METTL14 antibodies , respectively . Western blot analysis was performed using anti - RNAPII , anti - ADMA , anti - METTL14 , and anti - METTL3 antibodies . Co - IP assays were performed to examine the involvement of RNA in the METTL14 - RNAPII interaction . Total cell lysates were either left untreated or treated with RNase A to remove the RNA component before IP . Western blot analysis was performed using anti - RNAPII , anti - METTL3 , and anti - METTL14 antibodies .

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

Co-IP assays were performed to compare the interactions between METTL14 and RNAPII in control and MS023-treated HEK293 cells. Cells were treated with either DMSO or MS023 (1 μM) for 48 h before they were lysed. IP was performed using control IgG and METTL14 antibodies, respectively. Western blot analysis was performed using anti-RNAPII, anti-ADMA, anti-METTL14, and anti-METTL3 antibodies.   Co-IP assays were performed to examine the involvement of RNA in the METTL14-RNAPII interaction. Total cell lysates were either left untreated or treated with RNase A to remove the RNA component before IP. Western blot analysis was performed using anti-RNAPII, anti-METTL3, and anti-METTL14 antibodies.


m6A levels are reduced in mESCs expressing arginine methylation - deficient ( RK ) mutant METTL14 . The mRNA purified from WT , Mettl14 KO , KO + WT , and KO + RK mESCs was subjected to LC - MS / MS analysis to quantify m6A levels ( presented as the m6A / A ratio ) . data from three independent replicates were analyzed by Student ' s t - test and shown as mean ± SD . * , p < 0 . 05 ; * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

m6A levels are reduced in mESCs expressing arginine methylation-deficient (RK) mutant METTL14. The mRNA purified from WT, Mettl14 KO, KO+WT, and KO+RK mESCs was subjected to LC-MS/MS analysis to quantify m6A levels (presented as the m6A/A ratio). data from three independent replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01, ***, p < 0.001.


MeRIP ( m6A - IP ) - qPCR assays were performed for WT , Mettl14 KO , KO + WT , and KO + RK mESCs to validate the MeRIP - seq results . m6A - negative regions of the transcripts ( blue ) were included as negative controls . : In ( B ) , data from three independent replicates were analyzed by Student ' s t - test and shown as mean ± SD . * , p < 0 . 05 ; * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

MeRIP (m6A-IP)-qPCR assays were performed for WT, Mettl14 KO, KO+WT, and KO+RK mESCs to validate the MeRIP-seq results. m6A-negative regions of the transcripts (blue) were included as negative controls. : In (B) , data from three independent replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01, ***, p < 0.001.


The expression of ICL repair genes is reduced in mESCs expressing arginine methylation - deficient mutant ( RK ) METTL14 . Total cell lysates from WT , Mettl14 KO , KO + WT , and KO + RK mESCs were subjected to Western blot analysis using the indicated antibodies .

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The expression of ICL repair genes is reduced in mESCs expressing arginine methylation-deficient mutant (RK) METTL14. Total cell lysates from WT, Mettl14 KO, KO+WT, and KO+RK mESCs were subjected to Western blot analysis using the indicated antibodies.


mESCs expressing arginine methylation - deficient mutant ( RK ) METTL14 are sensitive to ICL damage induced by MMC . WT , Mettl14 KO , KO + WT , and KO + RK mESCs were treated with various concentrations of MMC for 4 days before cell viability was measured . Similar to ( A ) , except that mESCs were treated with cisplatin , another ICL - inducing chemical , at various concentrations . In both ( A ) and ( B ) , each point represents the average of three independent replicates and error bars represent standard deviation ( SD ) . Statistical analysis was performed using Student ' s t - test . * , p < 0 . 05 .

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mESCs expressing arginine methylation-deficient mutant (RK) METTL14 are sensitive to ICL damage induced by MMC. WT, Mettl14 KO, KO+WT, and KO+RK mESCs were treated with various concentrations of MMC for 4 days before cell viability was measured.   Similar to (A), except that mESCs were treated with cisplatin, another ICL-inducing chemical, at various concentrations.   In both (A) and (B), each point represents the average of three independent replicates and error bars represent standard deviation (SD). Statistical analysis was performed using Student's t-test. *, p < 0.05.


Human Aβ exhibits a biphasic profile in CSF of APP transgenic miceA , B Aβ40 and Aβ42 concentrations in CSF of male APP23 mice ( heterozygous ; 3 ( n = 14 ) , 6 ( n = 12 ) , 8 ( n = 10 ) , 12 ( n = 11 ) , 19 ( n = 9 ) , and 25 ( n = 6 ) months of age ) . CSF Aβ40 ( F ( 1 , 56 ) = 22 . 351 , P < 0 . 001 ) as well as CSFAβ42 ( F ( 1 , 56 ) = 38 . 597 , P < 0 . 001 ) followed a significant quadratic trend .

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

Human Aβ exhibits a biphasic profile in CSF of APP transgenic miceA, B Aβ40 and Aβ42 concentrations in CSF of male APP23 mice (heterozygous; 3 (n = 14), 6 (n = 12), 8 (n = 10), 12 (n = 11), 19 (n = 9), and 25 (n = 6) months of age). CSF Aβ40 (F(1, 56) = 22.351, P < 0.001) as well as CSFAβ42 (F(1, 56) = 38.597, P < 0.001) followed a significant quadratic trend.


C Aβ42 / 40 ratio in CSF of APP23 mice showed a delayed decrease with age ( F ( 1 , 56 ) = 53 . 894 , P < 0 . 001 ) .

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C Aβ42/40 ratio in CSF of APP23 mice showed a delayed decrease with age (F(1, 56) = 53.894, P < 0.001).


D , E Aβ40 and Aβ42 concentrations in CSF of male and female APP24 mice ( homozygous ; 2 ( n = 13 ) , 3 - 4 ( n = 16 ) , 7 - 8 ( n = 15 ) , 18 - 19 ( n = 14 ) , 24 ( n = 16 ) , and 30 ( n = 18 ) months of age ) . CSF Aβ40 followed a significant quadratic trend ( F ( 1 , 86 ) = 6 . 678 , P = 0 . 011 ) and CSF Aβ42 best fitted a cubic trend ( F ( 1 , 86 ) = 30 . 599 , P < 0 . 001 ) .

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D, E Aβ40 and Aβ42 concentrations in CSF of male and female APP24 mice (homozygous; 2 (n = 13), 3-4 (n = 16), 7-8 (n = 15), 18-19 (n = 14), 24 (n = 16), and 30 (n = 18) months of age). CSF Aβ40 followed a significant quadratic trend (F(1, 86) = 6.678, P = 0.011) and CSF Aβ42 best fitted a cubic trend (F(1, 86) = 30.599, P < 0.001).


F Aβ42 / 40 ratio in CSF of APP24 mice showed a delayed decrease with age ( F ( 1 , 86 ) = 64 . 936 , P < 0 . 001 ) .

F Aβ42/40 ratio in CSF of APP24 mice showed a delayed decrease with age (F(1, 86) = 64.936, P < 0.001).


G , H Aβ40 and Aβ42 in the CSF of female APP51 mice ( heterozygous ; 3 ( n = 6 ) , 15 ( n = 8 ) , and 24 ( n = 8 ) months of age ; 22 mice in total ) . CSF Aβ40 ( F ( 1 , 19 ) = 37 . 349 , P < 0 . 001 ) as well as CSFAβ42 ( F ( 1 , 19 ) = 107 . 670 , P < 0 . 001 ) followed a significant quadratic trend .

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G, H Aβ40 and Aβ42 in the CSF of female APP51 mice (heterozygous; 3 (n = 6), 15 (n = 8), and 24 (n = 8) months of age; 22 mice in total). CSF Aβ40 (F(1, 19) = 37.349, P < 0.001) as well as CSFAβ42 (F(1, 19) = 107.670, P < 0.001) followed a significant quadratic trend.


I Aβ42 / 40 ratio in CSF of APP51 mice showed a delayed decrease with age ( F ( 1 , 19 ) = 26 . 367 , P < 0 . 001 ) .

I Aβ42/40 ratio in CSF of APP51 mice showed a delayed decrease with age (F(1, 19) = 26.367, P < 0.001).


HumanAβ in CSF and brain of APP transgenic miceA APP23CSFAβ40 and Aβ42 in the same animals as shown in Fig1 . CSFAβ42 and Aβ40 are expressed as percentages of levels measured in the youngest age group .

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HumanAβ in CSF and brain of APP transgenic miceA APP23CSFAβ40 and Aβ42 in the same animals as shown in Fig1. CSFAβ42 and Aβ40 are expressed as percentages of levels measured in the youngest age group.


B , C Aβ40 and Aβ42 ( pmol / g wet brain ) in the FA - soluble brain extract from the same APP23mice showed a robust increase with age ; ANOVA revealed a significant cubic trend ( F ( 1 , 56 ) = 221 . 114 , P < 0 . 001 and F ( 1 , 56 ) = 370 . 947 , P < 0 . 001 , respectively ) .

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B, C Aβ40 and Aβ42 (pmol/g wet brain) in the FA-soluble brain extract from the same APP23mice showed a robust increase with age; ANOVA revealed a significant cubic trend (F(1, 56) = 221.114, P < 0.001 and F(1, 56) = 370.947, P < 0.001, respectively).


D APP24 CSF Aβ40 and Aβ42 in the same animals shown in Fig1 as percentage of the youngest age group .

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D APP24 CSF Aβ40 and Aβ42 in the same animals shown in Fig1 as percentage of the youngest age group.


E , F Aβ40 and Aβ42 ( pmol / g wet brain ) in the brain from the same APP24mice also showed a robust increase with age ; ANOVA revealed a significant cubic trend ( F ( 1 , 86 ) = 202 . 173 , P < 0 . 001 and F ( 1 , 86 ) = 139 . 941 , P < 0 . 001 , respectively ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F Aβ40 and Aβ42 (pmol/g wet brain) in the brain from the same APP24mice also showed a robust increase with age; ANOVA revealed a significant cubic trend (F(1, 86) = 202.173, P < 0.001 and F(1, 86) = 139.941, P < 0.001, respectively).


G APP51 CSF Aβ40 and Aβ42 in the same animals shown in Fig1 as percentages of levels in the youngest age group .

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G APP51 CSF Aβ40 and Aβ42 in the same animals shown in Fig1 as percentages of levels in the youngest age group.


H , I Aβ40 and Aβ42 ( pmol / g wet brain ) in the brain from the same APP51 mice showed a robust increase with age ; ANOVA revealed a significant quadratic trend ( F ( 1 , 19 ) = 12 . 960 , P = 0 . 002 and F ( 1 , 19 ) = 19 . 366 , P < 0 . 001 , respectively ) .

H, I Aβ40 and Aβ42 (pmol/g wet brain) in the brain from the same APP51 mice showed a robust increase with age; ANOVA revealed a significant quadratic trend (F(1, 19) = 12.960, P = 0.002 and F(1, 19) = 19.366, P < 0.001, respectively).


Aβplaque pathology in the brains of APP23miceAβimmunostaining ( CN3 antibody , dark blue ) in 25 - μm sagittal brain sections shows only sparse Aβ deposits primarily in the frontal cortex of 6 - to 8 - month - old APP23mice . At 12 months and thereafter , there is a progressive increase in plaque number and size and a progressive involvement of different brain regions . Insets highlight the plaque characteristics at the different ages . Scale bar , 100 μm . The mean number of Aβ plaques per section per hemibrain increased with age in 3 - to 12 - month - old mice . Only four mice were analyzed in the 3 - month - old age group , as APP23mice do not develop plaques at this age ( Sturchler - Pierrat et al , 1997 ) . The 6 - , 8 - , and 12 - month - old age groups included 12 , 10 , and 11 mice , respectively ( these are the same mice that were used for CSF and brainAβ measurements ) . Note that the Aβ plaques became too numerous and often could no longer be individually distinguished in the age groups > 12 months of age . Data are represented as group means ± SEM .

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

Aβplaque pathology in the brains of APP23miceAβimmunostaining (CN3 antibody, dark blue) in 25-μm sagittal brain sections shows only sparse Aβ deposits primarily in the frontal cortex of 6- to 8-month-old APP23mice. At 12 months and thereafter, there is a progressive increase in plaque number and size and a progressive involvement of different brain regions. Insets highlight the plaque characteristics at the different ages. Scale bar, 100 μm.The mean number of Aβ plaques per section per hemibrain increased with age in 3- to 12-month-old mice. Only four mice were analyzed in the 3-month-old age group, as APP23mice do not develop plaques at this age (Sturchler-Pierrat et al, 1997). The 6-, 8-, and 12-month-old age groups included 12, 10, and 11 mice, respectively (these are the same mice that were used for CSF and brainAβ measurements). Note that the Aβ plaques became too numerous and often could no longer be individually distinguished in the age groups > 12 months of age. Data are represented as group means ± SEM.


Brain sAPPβ shows an age - related increase in APP23 , APP24 , and APP51 micesAPPβ was measured in Triton X - 100 brain extracts from largely the same mice as analyzed in Figs1 and2 and is expressed as percentages of levels measured in the youngest age group . Swedish sAPPβ showed an age - dependent increase in APP23 mice following a linear trend ( F ( 1 , 83 ) = 52 . 914 , P < 0 . 001 ) ; APP23 from two independent batches were included in this analysis ( see Materials and Methods and Supplementary Fig S2 for details ) . Swedish sAPPβ showed an age - dependent increase in APP24mice following a linear trend ( F ( 1 , 84 ) = 11 . 264 , P = 0 . 001 ) . Human wild - type sAPPβ showed an age - dependent increase in APP51 following a quadratic trend ( F ( 1 , 18 ) = 68 . 980 , P < 0 . 001 ) . Data information : Post hoc Dunnett ' s test group comparisons were always conducted between the youngest group and all other groups . All data are represented as group means ± SEM ; * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 . For absolute values , see Supplementary Fig S2 .

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

Brain sAPPβ shows an age-related increase in APP23, APP24, and APP51 micesAPPβ was measured in Triton X-100 brain extracts from largely the same mice as analyzed in Figs1 and2 and is expressed as percentages of levels measured in the youngest age group.Swedish sAPPβ showed an age-dependent increase in APP23 mice following a linear trend (F(1, 83) = 52.914, P < 0.001); APP23 from two independent batches were included in this analysis (see Materials and Methods and Supplementary Fig S2 for details).Swedish sAPPβ showed an age-dependent increase in APP24mice following a linear trend (F(1, 84) = 11.264, P = 0.001).Human wild-type sAPPβ showed an age-dependent increase in APP51 following a quadratic trend (F(1, 18) = 68.980, P < 0.001).Data information: Post hoc Dunnett's test group comparisons were always conducted between the youngest group and all other groups. All data are represented as group means ± SEM; *P < 0.05; **P < 0.01; and ***P < 0.001. For absolute values, see Supplementary Fig S2.


B - D BSP amyloid aggregation in vitro is pH - dependent in 10 mM EDTA ( B ) , protein concentration - dependent in 10 mM EDTA and at pH 4 . 5 ( C ) and strongly inhibited by millimolar Ca2 + at various protein concentrations ( D ) .

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B-D BSP amyloid aggregation in vitro is pH-dependent in 10 mM EDTA (B), protein concentration-dependent in 10 mM EDTA and at pH 4.5 (C) and strongly inhibited by millimolar Ca2+ at various protein concentrations (D).


E - H TEM images showing fibril morphology of BSP12 ( E ) , BSP123 ( F ) , BSP3 ( G ) , BSP45 ( H ) .

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E-H TEM images showing fibril morphology of BSP12 (E), BSP123 (F), BSP3 (G), BSP45 (H).


I - J Amyloid aggregation by BSP subconstructs . BSP12 exhibits more prominent aggregation propensity than BSP3 and BSP45 in 10 mM EDTA and at pH 4 . 5 ( I ) . The conjunction of module 3 with modules 1 and 2 promotes aggregation ( J ) .

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I-J Amyloid aggregation by BSP subconstructs. BSP12 exhibits more prominent aggregation propensity than BSP3 and BSP45 in 10 mM EDTA and at pH 4.5 (I). The conjunction of module 3 with modules 1 and 2 promotes aggregation (J).


B Representative fluorescence microscopy images of BSP and its subconstructs ( 20 μM ) at varying pHs . PEG 8K concentration is 2 % . Scale bar , 20 μm .

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B Representative fluorescence microscopy images of BSP and its subconstructs (20 μM) at varying pHs. PEG 8K concentration is 2 %. Scale bar, 20 μm.


E Quantification of droplet area is shown for each protein under various Ca2 + concentration conditions . Each sample is quantified by total area ( left ) and mean area ( right ) . Each experiment was repeated three times , data shown as mean ± SD .

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E Quantification of droplet area is shown for each protein under various Ca2+ concentration conditions. Each sample is quantified by total area (left) and mean area (right). Each experiment was repeated three times, data shown as mean ± SD.


( A ) Immunofluorescence staining against Iba - 1 in cortex ( Cx ) , hippocampus ( Hip ) and cerebellum ( Cb ) of NPA affected and control 3 year - old children . Scale bars , 50 μm . ( B ) Magnified image showing ramified ( left ) versus amoeboid ( right ) morphology in control and NPA microglia . Scale bars , 10 μm . ( C ) Mean ± SEM area of Iba - 1 positive cells in different brain regions from a NPA affected and a control child ( n = 20 cells , Student ´ s t - test ) .

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(A) Immunofluorescence staining against Iba-1 in cortex (Cx), hippocampus (Hip) and cerebellum (Cb) of NPA affected and control 3 year-old children. Scale bars, 50 μm. (B) Magnified image showing ramified (left) versus amoeboid (right) morphology in control and NPA microglia. Scale bars, 10 μm. (C) Mean ± SEM area of Iba-1 positive cells in different brain regions from a NPA affected and a control child (n = 20 cells, Student´s t-test).


( D ) Immunofluorescence staining against Iba - 1 in Cx , Hip and Cb of 3 month - old ASMko and wt mice . DAPI staining shows cell nuclei . Scale bars , 50 μm .

(D) Immunofluorescence staining against Iba-1 in Cx, Hip and Cb of 3 month-old ASMko and wt mice. DAPI staining shows cell nuclei. Scale bars, 50 μm.


( E ) Mean ± SEM number of Iba - 1 positive cells in different brain regions from ASMko and wt mice ( n = 5 mice per group , Student ´ s t - test ) .

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(E) Mean ± SEM number of Iba-1 positive cells in different brain regions from ASMko and wt mice (n = 5 mice per group, Student´s t-test).


( F ) 3D rendered images obtained with Imaris Surface software from high magnification images of immunofluorescence staining against Iba - 1 showing representative microglial morphology in Cx , Hip and Cb of ASMko and wt mice . Scale bars , 10 μm .

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(F) 3D rendered images obtained with Imaris Surface software from high magnification images of immunofluorescence staining against Iba-1 showing representative microglial morphology in Cx, Hip and Cb of ASMko and wt mice. Scale bars, 10 μm.


( G ) Mean ± SEM area of Iba - 1 positive cells in the different brain regions from ASMko and wt mice ( n = 5 mice per group , > 10 cells per mouse , Student ´ s t - test ) . Scale bars , 15 μm .

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(G) Mean ± SEM area of Iba-1 positive cells in the different brain regions from ASMko and wt mice (n = 5 mice per group, >10 cells per mouse, Student´s t-test). Scale bars, 15 μm.


( H ) Example of projection detection by the Filament Imaris software . ( I ) Mean ± SEM branch point number per process and process length in iba1 + cells obtained with the Imaris Filament software ( Student ´ s t - test ) .

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(H) Example of projection detection by the Filament Imaris software. (I) Mean ± SEM branch point number per process and process length in iba1+ cells obtained with the Imaris Filament software (Student´s t-test).


( A ) Immunofluorescence staining against Iba - 1 in Cb of ASMko and wt mice treated or not with PLX for 2 months . DAPI staining shows cell nuclei . Scale bar , 100 μm . ( B ) Magnified images ( from A ) showing ramified ( left ) versus amoeboid ( right ) morphology in wt and ASMko microglia treated or not with PLX . Scale bars , 30 μm . ( C ) Mean ± SEM number of Iba - 1 positive cells in the Cb of the different mouse groups ( n = 7 mice per group , Two - way Anova , Bonferroni post hoc ) .

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(A) Immunofluorescence staining against Iba-1 in Cb of ASMko and wt mice treated or not with PLX for 2 months. DAPI staining shows cell nuclei. Scale bar, 100 μm. (B) Magnified images (from A) showing ramified (left) versus amoeboid (right) morphology in wt and ASMko microglia treated or not with PLX. Scale bars, 30 μm. (C) Mean ± SEM number of Iba-1 positive cells in the Cb of the different mouse groups (n = 7 mice per group, Two-way Anova, Bonferroni post hoc).


( D , E ) Performance in the Rotarod test of ASMko and wt mice treated or not with PLX for 2 months expressed as the mean ± SEM time spent in the rotating rod on each of four trials ( D ) or the mean ± SEM time of the four trials ( E ) ( n = 7 mice per group , Two - way Anova , Games - Howell ( D ) and Bonferroni ( E ) post hoc ) .

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(D, E) Performance in the Rotarod test of ASMko and wt mice treated or not with PLX for 2 months expressed as the mean ± SEM time spent in the rotating rod on each of four trials (D) or the mean ± SEM time of the four trials (E) (n = 7 mice per group, Two-way Anova, Games-Howell (D) and Bonferroni (E) post hoc).


( F ) Mean ± SEM body weight of ASMko and wt mice treated or not with PLX at the indicated time during long - term treatment ( n = 7 mice per group , Two - way Anova , Bonferroni post hoc ) .

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(F) Mean ± SEM body weight of ASMko and wt mice treated or not with PLX at the indicated time during long-term treatment (n= 7 mice per group, Two-way Anova, Bonferroni post hoc).


( G ) Survival curve of ASMko and wt mice with or without long - term PLX treatment ( n = 7 mice per group ) .

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(G) Survival curve of ASMko and wt mice with or without long-term PLX treatment (n = 7 mice per group).


( H ) Immunofluorescence staining against the PC marker Calbindin in mid and posterior lobules of Cb in ASMko and wt mice treated or not with PLX for 2 months . DAPI staining shows cell nuclei . Scale bar , 500 μm . ( I ) Mean ± SEM number of Calbindin positive cells in the anterior ( lobules I - V ) , middle ( lobules VI - VIII ) and posterior zone ( lobules IX - X ) of the Cb from the different mouse groups ( n = 7 mouse per group , Two - way Anova , Bonferroni post hoc ) .

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(H) Immunofluorescence staining against the PC marker Calbindin in mid and posterior lobules of Cb in ASMko and wt mice treated or not with PLX for 2 months. DAPI staining shows cell nuclei. Scale bar, 500 μm. (I) Mean ± SEM number of Calbindin positive cells in the anterior (lobules I-V), middle (lobules VI-VIII) and posterior zone (lobules IX-X) of the Cb from the different mouse groups (n = 7 mouse per group, Two-way Anova, Bonferroni post hoc).


( A ) Immunofluorescence staining against CD16 / CD32 or Arg - 1 in Cb of ASMko mice ( red ) . Microglia were identified by F4 / 80 or Iba - 1 staining ( green ) . DAPI staining shows cell nuclei . Scale bar , 50 μm . ( B ) Mean ± SEM number of Arg - 1 and CD16 / CD32 positive microglia per area in Cb from ASMko and wt mice ( n = 6 mice per group ) .

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(A) Immunofluorescence staining against CD16/CD32 or Arg-1 in Cb of ASMko mice (red). Microglia were identified by F4/80 or Iba-1 staining (green). DAPI staining shows cell nuclei. Scale bar, 50 μm. (B) Mean ± SEM number of Arg-1 and CD16/CD32 positive microglia per area in Cb from ASMko and wt mice (n = 6 mice per group).


( C ) Mean ± SEM levels of TNFa protein in the Cb from ASMko and wt mice measured by ELISA ( n = 7 mice per group , Student ´ s t - test ) .

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(C) Mean ± SEM levels of TNFa protein in the Cb from ASMko and wt mice measured by ELISA (n = 7 mice per group, Student´s t-test).


( D ) Mean ± SEM levels of TNFa mRNA in Cb of ASMko and wt mice determined by qRT - PCR ( n = 7 mice per group , Student ´ s t - test ) .

(D) Mean ± SEM levels of TNFa mRNA in Cb of ASMko and wt mice determined by qRT-PCR (n = 7 mice per group, Student´s t-test).


( E ) Survival after LPS challenge in ASMko and wt mice treated or not with dexamethasone ( n = 8 mice per group ) .

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(E) Survival after LPS challenge in ASMko and wt mice treated or not with dexamethasone (n = 8 mice per group).


( F ) Immunofluorescence staining against MBP in Cb of ASMko and wt mice treated or not with PLX for 2 months . Scale bar , 50 μm .

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(F) Immunofluorescence staining against MBP in Cb of ASMko and wt mice treated or not with PLX for 2 months. Scale bar, 50 μm.


( G ) Mean ± SEM MBP intensity in the different mouse groups ( n = 7 mice per group , Two - way Anova , Bonferroni post hoc ) .

(G) Mean ± SEM MBP intensity in the different mouse groups (n = 7 mice per group, Two-way Anova, Bonferroni post hoc).


( H ) High magnification of immunofluorescence staining against MBP in ASMko mice treated or not with PLX for 2 months . White arrows show MBP aggregates indicative of myelin debris . Scale bar , 50 μm . ( I ) Mean ± SEM number of MBP aggregates per cell area in the different mouse groups ( n = 7 mice per group , Student ´ s t - test ) .

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(H) High magnification of immunofluorescence staining against MBP in ASMko mice treated or not with PLX for 2 months. White arrows show MBP aggregates indicative of myelin debris. Scale bar, 50 μm. (I) Mean ± SEM number of MBP aggregates per cell area in the different mouse groups (n = 7 mice per group, Student´s t-test).


( J ) High magnification images and their 3D rendered replicates from cerebellar wt and ASMko microglia identified by Iba - 1 and of the myelin marker MBP . Scale bars , 10 μm .

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(J) High magnification images and their 3D rendered replicates from cerebellar wt and ASMko microglia identified by Iba-1 and of the myelin marker MBP. Scale bars, 10 μm.


( K ) Amplified image and its 3D rendered replicate of amoeboid microglia containing myelin debris ( arrowheads ) in the ASMko cerebellum . The orthogonal projection of this cell in the lower panels confirms the presence of the myelin debris inside the cell and underneath the plasma membrane ( arrowhead ) . DAPI staining shows cell nuclei . Scale bars , 10 μm . ( L ) Mean ± SEM percentage of microglia showing internalised myelin debris in wt and ASMko mice ( n = 7 mice per group ) .

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(K) Amplified image and its 3D rendered replicate of amoeboid microglia containing myelin debris (arrowheads) in the ASMko cerebellum. The orthogonal projection of this cell in the lower panels confirms the presence of the myelin debris inside the cell and underneath the plasma membrane (arrowhead). DAPI staining shows cell nuclei. Scale bars, 10 μm. (L) Mean ± SEM percentage of microglia showing internalised myelin debris in wt and ASMko mice (n = 7 mice per group).


( A ) Transmission electron microscopy image of microglia in the Cb of ASMko mice showing accumulation of multillamelar vacuoles and other lysosome related vesicles filling the whole cytoplasm of these cells . Arrowhead identifies the nuclei . Scale bar , 5 μm . On the right , higher magnification showing a multillamelar body ( arrowhead ) with the characteristic concentric lamellar inclusions . Scale bar , 0 . 5 μm .

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(A) Transmission electron microscopy image of microglia in the Cb of ASMko mice showing accumulation of multillamelar vacuoles and other lysosome related vesicles filling the whole cytoplasm of these cells. Arrowhead identifies the nuclei. Scale bar, 5 μm. On the right, higher magnification showing a multillamelar body (arrowhead) with the characteristic concentric lamellar inclusions. Scale bar, 0.5 μm.


Immunofluorescence staining against Galectin3 - GFP in BV2 microglial cells treated with or without SM ( B ) Scale bar , 10 μm .

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Immunofluorescence staining against Galectin3-GFP in BV2 microglial cells treated with or without SM (B) Scale bar, 10 μm.


Immunofluorescence staining against Galectin3 - GFP in BV2 microglial cells treated with or without Siramesine ( C ) . Scale bar , 10 μm .

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Immunofluorescence staining against Galectin3-GFP in BV2 microglial cells treated with or without Siramesine (C). Scale bar, 10 μm.


( D ) Mean ± SEM number of Galectin3 - GFP positive vesicles in BV2 microglial cells in the different conditions ( n = 4 independent cultures , > 50 cells per culture , Student ´ s t - test ) .

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(D) Mean ± SEM number of Galectin3-GFP positive vesicles in BV2 microglial cells in the different conditions (n = 4 independent cultures, >50 cells per culture, Student´s t-test).


( E ) Lysotracker - Red staining in BMDMs from wt and ASMko mice . Scale bar , 10 μm .

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(E) Lysotracker-Red staining in BMDMs from wt and ASMko mice. Scale bar, 10 μm.


( F ) Western blot of CathB levels and the cytosolic protein GAPDH in the cellular and cytosolic fractions of wt and ASMko BMDMs extracted with digitonin ( left ) . Mean ± SEM pro - CathB and CathB levels in the cytosolic fractions ( right ) ( n = 2 cultures per genotype , Student ´ s t - test ) .

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(F) Western blot of CathB levels and the cytosolic protein GAPDH in the cellular and cytosolic fractions of wt and ASMko BMDMs extracted with digitonin (left). Mean ± SEM pro-CathB and CathB levels in the cytosolic fractions (right) (n=2 cultures per genotype, Student´s t-test).


( G ) Immunofluorescence staining against Iba - 1 ( green ) and CathB ( red ) in Cb of ASMko and wt mice . Arrowheads show double positive cells . Scale bar , 50 μm . Below , magnified images of the selected areas . Scale bar , 20 μm . ( H ) Immunofluorescence staining against CathB ( green ) , Arg - 1 ( red , anti - inflammatory microglia ) and F4 / 80 ( yellow , all microglia ) in Cb of ASMko mice . DAPI staining shows cell nuclei . Scale bar , 50 μm . Below , magnified images of the selected areas . Scale bar , 20 μm . ( I ) Table showing mean ± SEM percentage of microglia presenting CathB staining ( CathB + / Iba - 1 + ) , and the number of CathB positive microglia co - expressing Arg - 1 ( CathB + / Arg - 1 + ) in Cb of ASMko and wt mice ( n = 5 mice , > 10 cells per mouse ) .

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(G) Immunofluorescence staining against Iba-1 (green) and CathB (red) in Cb of ASMko and wt mice. Arrowheads show double positive cells. Scale bar, 50 μm. Below, magnified images of the selected areas. Scale bar, 20 μm. (H) Immunofluorescence staining against CathB (green), Arg-1 (red, anti-inflammatory microglia) and F4/80 (yellow, all microglia) in Cb of ASMko mice. DAPI staining shows cell nuclei. Scale bar, 50 μm. Below, magnified images of the selected areas. Scale bar, 20 μm. (I) Table showing mean ± SEM percentage of microglia presenting CathB staining (CathB +/Iba-1+), and the number of CathB positive microglia co-expressing Arg-1 (CathB+/Arg-1+) in Cb of ASMko and wt mice (n = 5 mice, >10 cells per mouse).


( A ) Western blot of CathB levels ( for both the precursor ( pro - CathB ) and the cleaved - mature forms ) in the culture media from ASMko and wt BMDMs . Staining of Ponceau - S is shown as loading control . ( B ) Mean ± SEM fold increase in CathB levels in the culture media from wt and ASMko BMDMs ( n = 5 independent cultures , Student ´ s t - test ) .

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(A) Western blot of CathB levels (for both the precursor (pro-CathB) and the cleaved-mature forms) in the culture media from ASMko and wt BMDMs. Staining of Ponceau-S is shown as loading control. (B) Mean ± SEM fold increase in CathB levels in the culture media from wt and ASMko BMDMs (n = 5 independent cultures, Student´s t-test).


( C ) Immunofluorescence staining against LAMP - 1 ( yellow ) and Iba - 1 ( red ) in Cb of ASMko and wt mice . Scale bar , 50 μm , zoomed images 20 μm .

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(C) Immunofluorescence staining against LAMP-1 (yellow) and Iba-1 (red) in Cb of ASMko and wt mice. Scale bar, 50 μm, zoomed images 20 μm.


( D ) Immunofluorescence staining against LAMP - 1 in non - permeabilised BMDMs from ASMko and wt mice . Scale bar , 50 μm .

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(D) Immunofluorescence staining against LAMP-1 in non-permeabilised BMDMs from ASMko and wt mice. Scale bar, 50 μm.


( E ) Mean ± SEM of LAMP - 1 levels on the surface of ASMko and wt BMDMs ( n = 4 independent cultures , > 40 cells per culture , Student ´ s t - test ) .

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(E) Mean ± SEM of LAMP-1 levels on the surface of ASMko and wt BMDMs (n = 4 independent cultures, >40 cells per culture, Student´s t-test).


( F ) Mean ± SEM microvesicle and exosome numbers in the culture media from ASMko and wt BMDMs ( n = 6 independent cultures , Student ´ s t - test ) .

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(F) Mean ± SEM microvesicle and exosome numbers in the culture media from ASMko and wt BMDMs (n = 6 independent cultures, Student´s t-test).


( G ) Western blot of CathB and Flotillin1 levels in the secreted exosome fractions from ASMko and wt BMDMs . ( H ) Western blot of CathB levels in the exosome depleted culture media from wt and ASMko BMDMs .

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(G) Western blot of CathB and Flotillin1 levels in the secreted exosome fractions from ASMko and wt BMDMs. (H) Western blot of CathB levels in the exosome depleted culture media from wt and ASMko BMDMs.


( I ) Mean ± SEM MTT absorbance reflecting cell viability in primary neurons from wt mice treated with conditioned media from ASMko and wt BMDMs in the presence or absence of the CathB inhibitor Ca074 ( n = 4 independent cultures Two - way Anova , Bonferroni post hoc ) .

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(I) Mean ± SEM MTT absorbance reflecting cell viability in primary neurons from wt mice treated with conditioned media from ASMko and wt BMDMs in the presence or absence of the CathB inhibitor Ca074 (n = 4 independent cultures Two-way Anova, Bonferroni post hoc).


( J ) Immunofluorescence staining against Iba - 1 ( green ) and CathB ( red ) in lobes of the Cb sensitive or resistant to Purkinje cell death . Asterisks indicate Purkinje cells in the resistant lobes . Scale bar , 50 μm .

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(J) Immunofluorescence staining against Iba-1 (green) and CathB (red) in lobes of the Cb sensitive or resistant to Purkinje cell death. Asterisks indicate Purkinje cells in the resistant lobes. Scale bar, 50 μm.


( K ) Mean ± SEM percentage of CathB positive microglia in resistant and sensitive Cb lobules of ASMko mice ( n = 5 mice , χ2 test ) .

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(K) Mean ± SEM percentage of CathB positive microglia in resistant and sensitive Cb lobules of ASMko mice (n = 5 mice, χ2 test).


( L ) Western blot of Cath B and β - Actin levels in cerebellar extracts of wt and ASMko mice treated or not with PLX . ( M ) Mean ± SEM of CathB levels normalized by β - Actin in cerebellar extracts of wt and ASMko mice treated or not with PLX ( n = 6 mice Two - way Anova , Bonferroni post hoc ) .

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(L) Western blot of Cath B and β-Actin levels in cerebellar extracts of wt and ASMko mice treated or not with PLX. (M) Mean ± SEM of CathB levels normalized by β-Actin in cerebellar extracts of wt and ASMko mice treated or not with PLX (n= 6 mice Two-way Anova, Bonferroni post hoc).


( O ) Western blot of CathB levels ( for both the precursor ( pro - CathB ) and the cleaved - mature forms ) in the culture media from ASMko and wt cerebellar organotypic cultures from mice fed with vehicle ( veh ) or PLX ( PLX ) . Below , Western blots of organotypic culture lysates against Iba - 1 , to confirm microglia depletion , and against GAPDH as loading control .

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(O) Western blot of CathB levels (for both the precursor (pro-CathB) and the cleaved-mature forms) in the culture media from ASMko and wt cerebellar organotypic cultures from mice fed with vehicle (veh) or PLX (PLX). Below, Western blots of organotypic culture lysates against Iba-1, to confirm microglia depletion, and against GAPDH as loading control.


( P ) Mean ± SEM increase in pro - CathB ( left ) and CathB ( right ) levels in the culture media from cerebellar organotypic cultures from wt and ASMko mice treated or not with PLX ( n = 3 mice per group Two - way Anova , Bonferroni post hoc ) .

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(P) Mean ± SEM increase in pro-CathB (left) and CathB (right) levels in the culture media from cerebellar organotypic cultures from wt and ASMko mice treated or not with PLX (n = 3 mice per group Two-way Anova, Bonferroni post hoc).


( A ) Mean ± SEM body weight of ASMko and wt mice treated or not with Ca074Me at the indicated time during treatment ( n = 7 mice per group ) .

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(A) Mean ± SEM body weight of ASMko and wt mice treated or not with Ca074Me at the indicated time during treatment (n = 7 mice per group).


Performance in the Rotarod test of ASMko and wt mice treated or not with Ca074Me for 1 month expressed as mean ± SEM time spent in the rotating rod on each of four trials ( B )

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Performance in the Rotarod test of ASMko and wt mice treated or not with Ca074Me for 1 month expressed as mean ± SEM time spent in the rotating rod on each of four trials (B)


Performance in the Rotarod test of ASMko and wt mice treated or not with Ca074Me for 1 month expressed as mean ± SEM time of the four trials ( C )

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Performance in the Rotarod test of ASMko and wt mice treated or not with Ca074Me for 1 month expressed as mean ± SEM time of the four trials (C)


( D ) Immunofluorescence staining against the PC marker Calbindin in the posterior lobules of the Cb in ASMko and wt mice treated or not with Ca074Me for 1 month . DAPI staining shows cell nuclei . Scale bar , 200 μm .

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(D) Immunofluorescence staining against the PC marker Calbindin in the posterior lobules of the Cb in ASMko and wt mice treated or not with Ca074Me for 1 month. DAPI staining shows cell nuclei. Scale bar, 200 μm.


( E ) Mean ± SEM number of Calbindin positive cells in the posterior zone ( lobules IX - X ) of the Cb from the different mouse groups ( n = 7 mice per group Two - way Anova , Bonferroni post hoc ) .

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(E) Mean ± SEM number of Calbindin positive cells in the posterior zone (lobules IX-X) of the Cb from the different mouse groups (n= 7 mice per group Two-way Anova, Bonferroni post hoc).


( A ) Immunofluorescence staining against Iba - 1 in Cx , Hip and Cb of 2 month - old Npc1nmf164 and wt mice . DAPI staining shows cell nuclei . Scale bars , 30 μm .

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(A) Immunofluorescence staining against Iba-1 in Cx, Hip and Cb of 2 month-old Npc1nmf164 and wt mice. DAPI staining shows cell nuclei. Scale bars, 30 μm.


( B ) 3D rendered images obtained with Imaris Surface software from high magnification z - stack images of immunofluorescence staining against Iba - 1 showing representative microglial morphology in Cx , Hip and Cb of Npc1nmf164 and wt mice . Scale bars , 10 μm .

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(B) 3D rendered images obtained with Imaris Surface software from high magnification z-stack images of immunofluorescence staining against Iba-1 showing representative microglial morphology in Cx, Hip and Cb of Npc1nmf164 and wt mice. Scale bars, 10 μm.


( C ) Mean ± SEM number ( upper panel ) and area ( lower panel ) of Iba - 1 positive cells in different brain regions from Npc1nmf164 and wt mice ( n = 5 mice per group , > 30 cells per mouse , Student ´ s t - test ) .

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(C) Mean ± SEM number (upper panel) and area (lower panel) of Iba-1 positive cells in different brain regions from Npc1nmf164 and wt mice (n = 5 mice per group, >30 cells per mouse, Student´s t-test).


( D ) Immunofluorescence staining against Iba - 1 in the Cb of a NPC affected and a control child . Scale bars , 50 μm . Graph shows mean ± SEM area of Iba - 1 positive cells ( n = 30 cells , Student ´ s t - test ) .

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(D) Immunofluorescence staining against Iba-1 in the Cb of a NPC affected and a control child. Scale bars, 50 μm. Graph shows mean ± SEM area of Iba-1 positive cells (n = 30 cells, Student´s t-test).


( E ) Immunofluorescence staining against MBP in Cb of Npc1nmf164 and wt mice . Scale bar , 50 μm . Graph shows mean ± SEM number of MBP aggregates per area ( n = 5 mice per group , Student ´ s t - test ) .

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(E) Immunofluorescence staining against MBP in Cb of Npc1nmf164 and wt mice. Scale bar, 50 μm. Graph shows mean ± SEM number of MBP aggregates per area (n = 5 mice per group, Student´s t-test).


( F ) High magnification images and their 3D rendered replicates from cerebellar Npc1nmf164 microglia stained against Iba - 1 and MBP containing myelin debris ( arrowheads ) . Scale bars , 10 μm ( up ) , 2 μm ( down ) . The orthogonal projection of the cells in the right panels confirms the presence of myelin debris inside the cell and underneath the plasma membrane . DAPI staining shows cell nuclei . Scale bars , 10 μm .

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(F) High magnification images and their 3D rendered replicates from cerebellar Npc1nmf164 microglia stained against Iba-1 and MBP containing myelin debris (arrowheads). Scale bars, 10 μm (up), 2 μm (down). The orthogonal projection of the cells in the right panels confirms the presence of myelin debris inside the cell and underneath the plasma membrane. DAPI staining shows cell nuclei. Scale bars, 10 μm.


( G ) Mean ± SEM percentage of microglia showing internalised myelin debris in wt and Npc1nmf164 mice ( n = 5 mice per group ) .

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(G) Mean ± SEM percentage of microglia showing internalised myelin debris in wt and Npc1nmf164 mice (n = 5 mice per group).


( H ) Immunofluorescence staining against F4 / 80 and CathB in Cb of Npc1nmf164 mice . Scale bar , 30 μm . The table shows mean ± SEM percentage of microglia presenting CathB staining in Npc1nmf164 and wt mice . Below , magnified images of the selected areas . Scale bar , 20 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence staining against F4/80 and CathB in Cb of Npc1nmf164 mice. Scale bar, 30 μm. The table shows mean ± SEM percentage of microglia presenting CathB staining in Npc1nmf164 and wt mice. Below, magnified images of the selected areas. Scale bar, 20 μm.


( I ) Immunofluorescence staining against Iba - 1 and CathB in Cb of a NPC patient . DAPI shows cell nuclei . Scale bar , 50 μm . A merged magnified image of the selected area is shown in the right down panel , scale bar 10 μm . Table shows mean ± SEM percentage of microglia presenting CathB staining in NPC and control children .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence staining against Iba-1 and CathB in Cb of a NPC patient. DAPI shows cell nuclei. Scale bar, 50 μm. A merged magnified image of the selected area is shown in the right down panel, scale bar 10 μm. Table shows mean ± SEM percentage of microglia presenting CathB staining in NPC and control children.


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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 O O O O O O O O O O O O O O O O O O O O O O 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, 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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

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

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

C 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

A 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Frequency of 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Frequency of 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.