Figure 1 ( 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 . ( 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 . ( 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.(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.(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).


Figure 2 ( 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 ) . ( 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 ) . ( 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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).(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).(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).


Figure 3 ( 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 ) . ( 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 ) . ( 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 ) . ( 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 . ( 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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).(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).(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).(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.(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).


Figure 4 ( 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 . 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 ) . ( 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 ) . ( 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.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).(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).(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).


Figure 5 ( 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 . ( 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 ) . ( 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 . ( 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 . ( 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 ) . ( 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.(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).(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.(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.(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). (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).


Figure 6 ( 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 ) 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 . ( 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 ) .

O O B-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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)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.(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).


Figure 7 ( 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 . ( 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 ) . ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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.(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).(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.(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.(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.


Figure 1 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 . 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 . 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 . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 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.  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.  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. 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.


Figure 2 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 . 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 . 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 . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 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.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.  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.  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.


Figure 3 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 . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 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.  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.


Figure 4B 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B 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.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.


Figure 5 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 ) . 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 . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 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).  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.  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.


Figure 6 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 . C Frequency of abnormal arrangement in the meiotic metaphase I plate . The four types of spermatocytes in A and B were used to perform immunofluorescence as shown in Fig 1B and C . n = 3 mice for each genotype . The total numbers of counted metaphase I plates in the four types of spermatocytes were 221 , 181 , 421 and 240 , respectively . D Frequency of meiosis I metaphase cells with chromosomal abnormalities . The four types of spermatocytes in A - C were used to perform a metaphase spread as shown in Fig 1H and 1I . n = 3 mice for each genotype . The total numbers of counted MI cells in the four types of spermatocytes were 89 , 73 , 132 and 104 , respectively . E Frequency of meiosis II metaphase cells with chromosomal abnormalities . The four types of spermatocytes in A - D were used to perform a metaphase spread as shown in Fig 1K and L . n = 3 mice for each genotype . The total numbers of counted MII cells in the four types of spermatocytes were 74 , 87 , 85 and 94 , respectively .

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

Figure 6 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.  C Frequency of abnormal arrangement in the meiotic metaphase I plate. The four types of spermatocytes in A and B were used to perform immunofluorescence as shown in Fig 1B and C. n=3 mice for each genotype. The total numbers of counted metaphase I plates in the four types of spermatocytes were 221, 181, 421 and 240, respectively. D Frequency of meiosis I metaphase cells with chromosomal abnormalities. The four types of spermatocytes in A-C were used to perform a metaphase spread as shown in Fig 1H and 1I. n=3 mice for each genotype. The total numbers of counted MI cells in the four types of spermatocytes were 89, 73, 132 and 104, respectively. E Frequency of meiosis II metaphase cells with chromosomal abnormalities. The four types of spermatocytes in A-D were used to perform a metaphase spread as shown in Fig 1K and L. n=3 mice for each genotype. The total numbers of counted MII cells in the four types of spermatocytes were 74, 87, 85 and 94, respectively.


Figure 1 : ( a ) Effect of steroids on MERS - CoV replication . Vero cells were infected with MERS - CoV at MOI = 0 . 01 in the presence of steroids for 24 h . The viral titer in the cell supernatant was quantified by standard plaque assay using Vero / TMPRSS2 cells . Cell viability in the presence of steroids was quantified by WST assay at 24 hours post - infection ( hpi ) . ( b ) Ciclesonide escape mutant . Vero cells treated with ciclesonide were infected with parental MERS - CoV or ciclesonide escape mutant . Viral titer was measured as described in panel a . ( c ) Vero cells were infected with the parental MERS - CoV / EMC strain ( Re - EMC / MERS ) or the recombinant mutant strain ( Re - Nsp15 - A25V ) with an amino acid substitution at A25V in NSP15 . Viral titer was measured as described in panel a .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1:(a) Effect of steroids on MERS-CoV replication. Vero cells were infected with MERS-CoV at MOI = 0.01 in the presence of steroids for 24 h. The viral titer in the cell supernatant was quantified by standard plaque assay using Vero/TMPRSS2 cells. Cell viability in the presence of steroids was quantified by WST assay at 24 hours post-infection (hpi).(b) Ciclesonide escape mutant. Vero cells treated with ciclesonide were infected with parental MERS-CoV or ciclesonide escape mutant. Viral titer was measured as described in panel a.(c) Vero cells were infected with the parental MERS-CoV/EMC strain (Re-EMC/MERS) or the recombinant mutant strain (Re-Nsp15-A25V) with an amino acid substitution at A25V in NSP15. Viral titer was measured as described in panel a.


Figure 2 : ( a ) Intracellular SARS - CoV - 2 RNA ( 6 hpi ) . VeroE6 / TMPRSS2 cells were infected with SARS - CoV - 2 at MOI = 1 in the presence of 10 □ μM compounds for 6 h . Cellular viral RNA was quantified by real - time PCR using the E gene primer / probe set . ( b ) Culture Medium SARS - CoV - 2 RNA ( 27 hpi ) . VeroE6 / TMPRSS2 cells were infected with SARS - CoV - 2 at MOI = 0 . 01 in the presence of ciclesonide for 27 h . Viral RNA in culture medium was quantified by real - time PCR using the E gene primer / probe set . Cell viability in the presence of ciclesonide was quantified at 27 hpi by WST assay .

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Figure 2:(a) Intracellular SARS-CoV-2 RNA (6 hpi). VeroE6/TMPRSS2 cells were infected with SARS-CoV-2 at MOI = 1 in the presence of 10□μM compounds for 6 h. Cellular viral RNA was quantified by real-time PCR using the E gene primer/probe set.(b) Culture Medium SARS-CoV-2 RNA (27 hpi). VeroE6/TMPRSS2 cells were infected with SARS-CoV-2 at MOI = 0.01 in the presence of ciclesonide for 27 h. Viral RNA in culture medium was quantified by real-time PCR using the E gene primer/probe set. Cell viability in the presence of ciclesonide was quantified at 27 hpi by WST assay.


Figure 1Cell height was measured using AFM for HeLa - FUCCI in the indicated phase of mitosis . Below : representative images of synchronised HeLa - FUCCIRepresentative images of synchronised HeLa stained with an anti - geminin to confirm the indicated phase of mitosis ( scale bar 15 µm ) . Nucleus height was measured by immunofluorescence using Hoechst stainingRepresentative images of synchronised MEFs stained with an anti - geminin to confirm the indicated phase of mitosisNucleus height of synchronised MEFs ( black dot ) and synchronised MRC - 5 ( blue dot ) were measured by immunofluorescence using Hoechst staining . Data are presented as mean ± s . e . m . Nucleus height was measured by immunofluorescence using lamin A / C staining . Representative HeLa - FUCCI cells in S - G2 - M ( green ) and G1 phase ( red ) in Ctrl , Blebb and Blebb + AP conditions . Scale bar = 31 µm . Corresponding percentage of HeLa - FUCCI in mitosis . Representative flow cytometry histograms for EdU positive cells in Ctrl , Blebb and Blebb + AP conditions . Corresponding percentage of EdU positive cells .

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Figure 1Cell height was measured using AFM for HeLa-FUCCI in the indicated phase of mitosis. Below: representative images of synchronised HeLa-FUCCIRepresentative images of synchronised HeLa stained with an anti-geminin to confirm the indicated phase of mitosis (scale bar 15 µm).Nucleus height was measured by immunofluorescence using Hoechst stainingRepresentative images of synchronised MEFs stained with an anti-geminin to confirm the indicated phase of mitosisNucleus height of synchronised MEFs (black dot) and synchronised MRC-5 (blue dot) were measured by immunofluorescence using Hoechst staining. Data are presented as mean ± s.e.m.Nucleus height was measured by immunofluorescence using lamin A/C staining.Representative HeLa-FUCCI cells in S-G2-M (green) and G1 phase (red) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 31 µm.Corresponding percentage of HeLa-FUCCI in mitosis.Representative flow cytometry histograms for EdU positive cells in Ctrl, Blebb and Blebb + AP conditions. Corresponding percentage of EdU positive cells.


Figure 2Analysis of transcription factor activity ( Affymetrix combo protein - DNA array ) for Ctrl , Blebb and Blebb + AP conditions . Each square corresponds to a specific transcription factor ( TF ) activity and is color coded to show the relative activity . ① To identify TFs regulated by nuclear shape , we selected TFs that were more active in Ctrl and Blebb + AP compared to blebbistation - treated cells ( Blebb ) . TF ' s target genes and GAPDH mRNA levels were analyzed by real - time qPCR . Results are expressed as relative mRNA expression levels . TEAD activity was analyzed in HeLa cells co - transfected with a renilla plasmid as a luciferase reporter plasmid controlled by the TEAD - responsive promoter , and with a renilla plasmid as a gene reporter ; HeLa cells were cultured on 1 and 50 kPa ( n = 3 , t - test * * * p < 0 . 01 ) or treated with blebbistatin and blebbistatin + AP ( n = 6 ; t - test * p < 0 . 5 ) . AP1 activity was assessed in the indicated conditions using AP1 reporter assay . Heatmap of mRNA expression profiles of genes regulated by TEAD and AP1 .

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Figure 2Analysis of transcription factor activity (Affymetrix combo protein-DNA array) for Ctrl, Blebb and Blebb + AP conditions. Each square corresponds to a specific transcription factor (TF) activity and is color coded to show the relative activity. ① To identify TFs regulated by nuclear shape, we selected TFs that were more active in Ctrl and Blebb + AP compared to blebbistation-treated cells (Blebb).TF's target genes and GAPDH mRNA levels were analyzed by real-time qPCR. Results are expressed as relative mRNA expression levels.TEAD activity was analyzed in HeLa cells co-transfected with a renilla plasmid as a luciferase reporter plasmid controlled by the TEAD-responsive promoter, and with a renilla plasmid as a gene reporter; HeLa cells were cultured on 1 and 50 kPa (n = 3, t-test ***p<0.01) or treated with blebbistatin and blebbistatin + AP (n = 6; t-test *p<0.5).AP1 activity was assessed in the indicated conditions using AP1 reporter assay.Heatmap of mRNA expression profiles of genes regulated by TEAD and AP1.


Figure 3Representative cells stained for p - Jun Ser63 ( magenta ) and for DNA ( cyan ) in Ctrl , Blebb and Blebb + AP conditions . Scale bar = 10 µm . Nuclear heights were measured using Hoechst staining . ( b ) Corresponding quantifications of p - Jun Ser63 nuclear intensity . Representative cells stained for YAP ( magenta ) and for DNA ( cyan ) in Ctrl , Blebb and Blebb + AP conditions . Scale bar = 10 µm . Nuclear heights were measured using Hoechst staining . Corresponding quantifications of YAP activity ( ratio nucleus / cytosol ) . Quantifications of YAP activity ( nucleo - cytoplasmic ratio ) and p - Jun Ser63 nuclear intensity in Ctrl , Blebb , Blebb + AP condition and in Ctrl , Y27632 and Y27632 + AP . Quantifications of YAP activity ( nucleo - cytoplasmic ratio ) and p - Jun Ser63 nuclear intensity in Ctrl , Cytochalasin D ( CytoD ) , CytoD + AP condition . Quantifications of YAP activity ( nucleo - cytoplasmic ratio ) and p - Jun Ser63 nuclear intensity in cells depleted or not for SUN1 and SUN2 . Representative cells cultured on circular micropatterns with surfaces of 1500 and 500 µm2 and stained for p - Jun Ser63 ( magenta ) , YAP ( cyan ) and for DNA ( green ) . Additionally , cells were cultured on the micropatterns of 500 µm2 and an agarose pad was used to flatten their nuclei . Scale bar = 10 µm . Nuclear heights were measured using Hoechst staining . Corresponding quantifications of YAP activity ( nucleo - cytoplasmic ratio ) and p - Jun Ser63 nuclear intensity .

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Figure 3Representative cells stained for p-Jun Ser63 (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. (b) Corresponding quantifications of p-Jun Ser63 nuclear intensity.Representative cells stained for YAP (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. Corresponding quantifications of YAP activity (ratio nucleus/cytosol).Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in Ctrl, Blebb, Blebb + AP condition and in Ctrl, Y27632 and Y27632 + AP.Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in Ctrl, Cytochalasin D (CytoD), CytoD + AP condition.Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in cells depleted or not for SUN1 and SUN2.Representative cells cultured on circular micropatterns with surfaces of 1500 and 500 µm2 and stained for p-Jun Ser63 (magenta), YAP (cyan) and for DNA (green). Additionally, cells were cultured on the micropatterns of 500 µm2 and an agarose pad was used to flatten their nuclei. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. Corresponding quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity.


Figure 4Quantifications of YAP activity in Ctrl , Blebb and Blebb + AP conditions , treated or not with importazol ( nucleo - cytoplasmic ratio ) . Immunoblots of p - YAP ( ser127 ) , YAP , p - LATS ( Thr1079 ) , LATS , p - MST ( Thr1083 ) , MST , Kibra , PTPN14 and GADPH for Ctrl , Blebb and Blebb + AP conditions . Corresponding quantification of p - YAP ( Ser125 ) relative to Ctrl and normalized to GAPDH . Corresponding quantification of p - LATS ( thr1079 ) relative to Ctrl and normalized to GAPDH . Quantifications of p - Jun Ser63 nuclear intensity in Ctrl , Blebb , and Blebb + AP conditions for cells treated with DMSO , SB203580 , a MAP Kinase inhibitor and SP600125 , a JNK inhibitor . Representative cells stained for p - Jun Ser63 ( magenta ) and for DNA ( cyan ) in Ctrl , Blebb and Blebb + AP conditions for cells depleted or not for lamin - A / C . Scale bar = 10 µm . Corresponding quantification of p - Jun Ser63 nuclear intensity .

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Figure 4Quantifications of YAP activity in Ctrl, Blebb and Blebb + AP conditions, treated or not with importazol (nucleo-cytoplasmic ratio).Immunoblots of p-YAP (ser127), YAP, p-LATS (Thr1079), LATS, p-MST (Thr1083), MST, Kibra, PTPN14 and GADPH for Ctrl, Blebb and Blebb + AP conditions.Corresponding quantification of p-YAP (Ser125) relative to Ctrl and normalized to GAPDH. Corresponding quantification of p-LATS (thr1079) relative to Ctrl and normalized to GAPDH.Quantifications of p-Jun Ser63 nuclear intensity in Ctrl, Blebb, and Blebb + AP conditions for cells treated with DMSO, SB203580, a MAP Kinase inhibitor and SP600125, a JNK inhibitor.Representative cells stained for p-Jun Ser63 (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions for cells depleted or not for lamin-A/C. Scale bar = 10 µm. Corresponding quantification of p-Jun Ser63 nuclear intensity.


Figure 4Results from multiplex ACE2 competition assay are shown for the three spike - derived antigens : RBD , S1 - domain ( S1 ) and homotrimeric spike ( Spike ) . Color - coded beads coated with the respective antigens were co - incubated with biotinylated ACE2 and dilution series of NM1267 ( 8 pM to 126 nM ) followed by measuring residual binding of ACE2 . MFI signals were normalized to the maximum detectable signal per antigen given by the ACE2 - only control . IC50 values were calculated from a four - parametric sigmoidal model . Data are presented as mean ± s . d . of three technical replicates . E Neutralization potency of NM1267 was analyzed in Caco ‑ 2 cells using the SARS - CoV - 2 - mNG infectious clones . Infection rate normalized to virus - only infection control is illustrated as percent of infection ( % Infection ) . IC50 value was calculated from a four - parametric sigmoidal model and data are presented as mean ± s . e . m . of three biological replicates ( n = 3 ) .

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Figure 4Results from multiplex ACE2 competition assay are shown for the three spike-derived antigens: RBD, S1-domain (S1) and homotrimeric spike (Spike). Color-coded beads coated with the respective antigens were co-incubated with biotinylated ACE2 and dilution series of NM1267 (8 pM to 126 nM) followed by measuring residual binding of ACE2. MFI signals were normalized to the maximum detectable signal per antigen given by the ACE2-only control. IC50 values were calculated from a four-parametric sigmoidal model. Data are presented as mean ± s.d. of three technical replicates.E Neutralization potency of NM1267 was analyzed in Caco‑2 cells using the SARS-CoV-2-mNG infectious clones. Infection rate normalized to virus-only infection control is illustrated as percent of infection (% Infection). IC50 value was calculated from a four-parametric sigmoidal model and data are presented as mean ± s.e.m. of three biological replicates (n = 3).


Figure 5B For the NeutrobodyPlex , antigen - coated beads comprising RBD , S1 or spike were co - incubated with serum samples from 5 patients and a dilution series of NM1267 ( 1 µM to 6 pM ) ( n = 1 ) . Mean fluorescent intensities ( MFI ) derived from antigen - bound IgGs in the presence of bipNb normalized to the MFI values of IgGs in the serum - only samples , illustrated as MFI ( % control ) , are shown .

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Figure 5B For the NeutrobodyPlex, antigen-coated beads comprising RBD, S1 or spike were co-incubated with serum samples from 5 patients and a dilution series of NM1267 (1 µM to 6 pM) (n = 1). Mean fluorescent intensities (MFI) derived from antigen-bound IgGs in the presence of bipNb normalized to the MFI values of IgGs in the serum-only samples, illustrated as MFI (% control), are shown.


Figure 7A , B Total IgGs derived from spike - binding IgGs were plotted against normalized MFI values from IgGs binding to RBD ( MFI RBD ( % control ) ) in the presence of both concentrations of NM1267 ( 1 µM ; 1 nM , ) . C , D Total IgGs derived from N - binding IgGs were plotted against normalized MFI values from IgGs binding to RBD ( MFI RBD ( % control ) ) in the presence of both concentrations of NM1267 ( 1 µM ; 1 nM , ) . F

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Figure 7A ,B Total IgGs derived from spike-binding IgGs were plotted against normalized MFI values from IgGs binding to RBD (MFI RBD (%control)) in the presence of both concentrations of NM1267 (1 µM; 1 nM,). C, D Total IgGs derived from N-binding IgGs were plotted against normalized MFI values from IgGs binding to RBD (MFI RBD (%control)) in the presence of both concentrations of NM1267 (1 µM; 1 nM,). F


Figure 1A PhosTag - based Western blot probed for YAP reveals increased levels of unphosphorylated ( downshift ) YAP in HEK293A cells in response to cyclic 0 . 1Hz 200mbar hydrostatic pressure ( + ) compared to control ( - ) . B PhosTag - based Western blot probed for TAZ reveals increased levels of unphosphorylated ( downshift ) TAZ in HEK293A cells in response to cyclic 0 . 1Hz 200mbar hydrostatic pressure ( + ) compared to control ( - ) . C Western blots from cell lysates as in ( A ) and ( B ) show total protein levels in control ( - ) and in cell lysates obtained from cells exposed to hydrostatic pressure ( + ) stimulation in HEK293A cells . Note the decrease in the inhibitory p127 YAP signal as well as increase of YAP / TAZ targets CTGF and CYR61 levels upon hydrostatic pressure . D PhosTag - based Western blot probed for YAP reveals increased levels of unphosphorylated ( downshift ) YAP in 143B cells in response to hydrostatic pressure ( + ) compared to control ( - ) . E Western blot shows total protein levels in control ( - ) and with hydrostatic pressure ( + ) stimulation in 143B cells from cell lysates as in D . F HEK293A cells at steady state ( upper ) or upon cyclic 0 . 1Hz 200mbar hydrostatic pressure ( lower ) were analysed by immunofluorescence . Cells are labelled for Hoechst ( blue ) and YAP ( green ) . Scale bar = 20µm . Graph on the right depict quantification of nuclear - to - cytoplasmic ( Nucl / Cyt ) ratio of YAP in cells as shown on the left . Each dot represents a single cell and data are pooled from three independent experiments . Error bars represent mean ± 95 % CI . Mann - Whitney U test . * * * p < 0 . 001 . G 143B cells at steady state ( upper ) or upon cyclic 0 . 1Hz 200mbar hydrostatic pressure ( lower ) . Graph on the right show quantification of nuclear - to - cytoplasmic ( Nucl / Cyt ) ratio of YAP in 143B from images as those shown on the left . Each dot represents a single cell and data are pooled from three independent experiments . Error bars represent mean ± 95 % CI . Mann - Whitney U test . * * * p < 0 . 001 . H Western blot confirming no YAP and TAZ expression in two independent ( # 1 and # 2 ) YAP / TAZ double knockout ( Y / T DKO ) clones . I Relative expression levels of YAP / TAZ target genes CYR61 and CTGF in HEK293A WT and Y / T DKO clones in response to cyclic 0 . 1Hz 200mbar hydrostatic pressure . Data from six independent experiments Error bars represent mean ± SD . Kruskal - Wallis test with Dunn ' s post hoc . * * p = 0 . 0661 ( WT CYR61 ) , * * * p = 0 . 0001 ( WT CTGF ) , p = 0 . 3924 ( Y / T DKO # 1 CYR61 ) , p = 0 . 4616 ( Y / T DKO # 1 CTGF ) , p > 0 . 999 ( Y / T DKO # 2 CYR61 ) , p = 0 . 0782 ( Y / T DKO # 2 CTGF ) . J CYR61 and CTGF gene expression levels induced by hydrostatic pressure in ( I ) of Y / T DKO # 1 and # 2 normalised to WT . Kruskal - Wallis test with Dunn ' s post hoc . * p = 0 . 0259 ( Y / T DKO # 1 CYR61 ) , * * p = 0 . 0007 ( Y / T DKO # 1 CTGF ) , * p = 0 . 0155 ( Y / T DKO # 2 CYR61 ) , * * * p < 0 . 001 ( Y / T DKO # 2 CTGF ) .

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Figure 1A PhosTag-based Western blot probed for YAP reveals increased levels of unphosphorylated (downshift) YAP in HEK293A cells in response to cyclic 0.1Hz 200mbar hydrostatic pressure (+) compared to control (-). B PhosTag-based Western blot probed for TAZ reveals increased levels of unphosphorylated (downshift) TAZ in HEK293A cells in response to cyclic 0.1Hz 200mbar hydrostatic pressure (+) compared to control (-).C Western blots from cell lysates as in (A) and (B) show total protein levels in control (-) and in cell lysates obtained from cells exposed to hydrostatic pressure (+) stimulation in HEK293A cells. Note the decrease in the inhibitory p127 YAP signal as well as increase of YAP/TAZ targets CTGF and CYR61 levels upon hydrostatic pressure.D PhosTag-based Western blot probed for YAP reveals increased levels of unphosphorylated (downshift) YAP in 143B cells in response to hydrostatic pressure (+) compared to control (-).E Western blot shows total protein levels in control (-) and with hydrostatic pressure (+) stimulation in 143B cells from cell lysates as in D.F HEK293A cells at steady state (upper) or upon cyclic 0.1Hz 200mbar hydrostatic pressure (lower) were analysed by immunofluorescence. Cells are labelled for Hoechst (blue) and YAP (green). Scale bar = 20µm. Graph on the right depict quantification of nuclear-to-cytoplasmic (Nucl/Cyt) ratio of YAP in cells as shown on the left. Each dot represents a single cell and data are pooled from three independent experiments. Error bars represent mean ± 95% CI. Mann-Whitney U test. ***p < 0.001. G 143B cells at steady state (upper) or upon cyclic 0.1Hz 200mbar hydrostatic pressure (lower). Graph on the right show quantification of nuclear-to-cytoplasmic (Nucl/Cyt) ratio of YAP in 143B from images as those shown on the left. Each dot represents a single cell and data are pooled from three independent experiments. Error bars represent mean ± 95% CI. Mann-Whitney U test. ***p < 0.001.H Western blot confirming no YAP and TAZ expression in two independent (#1 and #2) YAP/TAZ double knockout (Y/T DKO) clones.I Relative expression levels of YAP/TAZ target genes CYR61 and CTGF in HEK293A WT and Y/T DKO clones in response to cyclic 0.1Hz 200mbar hydrostatic pressure. Data from six independent experiments Error bars represent mean ± SD. Kruskal-Wallis test with Dunn's post hoc. **p=0.0661 (WT CYR61), ***p= 0.0001 (WT CTGF), p=0.3924 (Y/T DKO#1 CYR61), p=0.4616 (Y/T DKO#1 CTGF), p>0.999 (Y/T DKO#2 CYR61), p=0.0782 (Y/T DKO#2 CTGF).J CYR61 and CTGF gene expression levels induced by hydrostatic pressure in (I) of Y/T DKO #1 and #2 normalised to WT. Kruskal-Wallis test with Dunn's post hoc. *p=0.0259 (Y/T DKO#1 CYR61), **p=0.0007 (Y/T DKO#1 CTGF), *p=0.0155 (Y/T DKO#2 CYR61), ***p<0.001 (Y/T DKO#2 CTGF).