A - D ' ' ' IF of NIH3T3 cells stably expressing Smo - mEos2 treated with mock ( control , A - A ' ' ' ) , the Smo agonist ( + SAG , B - B ' ' ' ) or the glycolytic inhibitors 3 - bromopyruvate ( + 3 - BP , C - C ' ' ' ) and 2 - deoxyglucose ( + 2 - DG , D - D ' ' ' ) . DAPI staining , shown in gray , was used to visualize cell nuclei . Acetylated tubulin ( AcTub ) , used as a ciliary marker , is shown in magenta , Smo - mEos2 is shown in green . Zoomed - in images of cilia marked by white arrows in A ' ' , B ' ' , C ' ' , and D ' ' are shown in A ' ' ' , B ' ' ' , C ' ' ' , and D ' ' ' . Scale bars = 5 μm , but 2 μm in A ' ' ' , B ' ' ' , C ' ' ' , and D ' ' ' . E , F Box plot showing quantification of Smo - mEos2 ciliary localization in NIH3T3 cells upon 3 - BP ( E ) and 2 - DG ( F ) treatment , respectively . Lower and upper hinges correspond to the first and third quartiles , vertical lines extend to the minimum and maximum values . Sample size is indicated for each perturbation . Treatment with glycolytic inhibitors induces Smo - mEos2 localization to the primary cilium . t - test , * * * p ≤ 0 . 001 .

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

A-D''' IF of NIH3T3 cells stably expressing Smo-mEos2 treated with mock (control, A-A'''), the Smo agonist (+ SAG, B-B''') or the glycolytic inhibitors 3-bromopyruvate (+3-BP, C-C''') and 2-deoxyglucose (+ 2-DG, D-D'''). DAPI staining, shown in gray, was used to visualize cell nuclei. Acetylated tubulin (AcTub), used as a ciliary marker, is shown in magenta, Smo-mEos2 is shown in green. Zoomed-in images of cilia marked by white arrows in A'', B'', C'', and D'' are shown in A''', B''', C''', and D'''. Scale bars= 5 μm, but 2 μm in A''', B''', C''', and D'''. E, F Box plot showing quantification of Smo-mEos2 ciliary localization in NIH3T3 cells upon 3-BP (E) and 2-DG (F) treatment, respectively. Lower and upper hinges correspond to the first and third quartiles, vertical lines extend to the minimum and maximum values. Sample size is indicated for each perturbation. Treatment with glycolytic inhibitors induces Smo-mEos2 localization to the primary cilium . t-test, ***p≤0.001.


A - B Time - controlled knock - down of ecd in the dorsal compartment of the wing discs ( see Fig 1C for the expression pattern of apGalts ) . IF of control ( A ) and apGalts > ecdRNAi ( B ) wing discs , stained for Hh . Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of control ( n = 4 ) and apGalts > ecdRNAi ( n = 5 ) wing discs . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Statistical analysis ( t - test ) revealed no significant differences in Hh expression in the dorsal compartment between control and apGalts > ecdRNAi wing discs . Thus , loss of Ecd does not affect Hh production and spreading . Wing discs were analyzed 48 h after RNAi induction . Scale bars = 50 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

A-B Time-controlled knock-down of ecd in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). IF of control (A) and apGalts>ecdRNAi (B) wing discs, stained for Hh. Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of control (n=4) and apGalts>ecdRNAi (n=5) wing discs. Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analysis (t-test) revealed no significant differences in Hh expression in the dorsal compartment between control and apGalts>ecdRNAi wing discs. Thus, loss of Ecd does not affect Hh production and spreading. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm.


C - F Time - controlled knock - down of ecd in the dorsal compartment of dispatched mutant wing discs ( dispS037707 , apGalts > ecdRNAi , see Fig 1C for the expression pattern of apGalts ) . IF of control ( C ) , apGalts > ecdRNAi ( D ) , dispS037707 ( E ) and dispS037707 , apGalts > ecdRNAi ( F ) wing discs expressing the decapentaplegic ( dpp ) reporter gene dpp - lacZ ( dppZ ) , stained for dppZ . Next to the images are quantifications of the respective stainings ( n = 4 ( C , F ) , n = 5 ( D ) , n = 3 ( E ) ) . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Statistical analyses ( t - test ) revealed a significant difference in dppZ expression in the dorsal compartment between control and apGalts > ecdRNAi ( p ≤ 0 . 01 ) ( C vs D ) , and dispS037707 and dispS037707 , apGalts > ecdRNAi ( p ≤ 0 . 01 ) wing discs ( E vs F ) . Loss of Ecd elevates the expression of dppZ in a broader than normal stripe . However , lack of Ecd function alone is not sufficient to trigger dpp expression , which instead depends on the presence of Hh . Wing discs were analyzed 48 h after RNAi induction . Scale bars = 50 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-F Time-controlled knock-down of ecd in the dorsal compartment of dispatched mutant wing discs (dispS037707, apGalts>ecdRNAi, see Fig 1C for the expression pattern of apGalts). IF of control (C), apGalts>ecdRNAi (D), dispS037707 (E) and dispS037707, apGalts>ecdRNAi (F) wing discs expressing the decapentaplegic (dpp) reporter gene dpp-lacZ (dppZ), stained for dppZ. Next to the images are quantifications of the respective stainings (n=4 (C, F), n=5 (D), n=3 (E)). Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analyses (t-test) revealed a significant difference in dppZ expression in the dorsal compartment between control and apGalts>ecdRNAi (p≤0.01) (C vs D), and dispS037707 and dispS037707, apGalts>ecdRNAi (p≤0.01) wing discs (E vs F). Loss of Ecd elevates the expression of dppZ in a broader than normal stripe. However, lack of Ecd function alone is not sufficient to trigger dpp expression, which instead depends on the presence of Hh. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm.


G - J IF of dispS037707 ( G , I ) and dispS037707 , apGalts > ecdRNAi ( H , J ) wing discs , stained for Smo ( G , H ) and Ci155 ( I , J ) . Next to the images are quantifications of the respective stainings ( n = 2 ( G , H ) , n = 3 ( I ) , n = 4 ( J ) ) . Graphs show mean ( thick line ) ± SD ( thin lines ) . Dashed yellow lines indicate the position of the A / P boundary . Although statistical analyses ( t - test ) revealed no significant differences in Smo and Ci155 expression in the dorsal compartment between control and dispS037707 , apGalts > ecdRNAi wing discs , loss of Ecd slightly induces an increased Smo accumulation and Ci155 stabilization independently of Hh . Wing discs were analyzed 48 h after RNAi induction . Scale bars = 50 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

G-J IF of dispS037707 (G, I) and dispS037707, apGalts>ecdRNAi (H, J) wing discs, stained for Smo (G, H) and Ci155 (I, J). Next to the images are quantifications of the respective stainings (n=2 (G, H), n=3 (I), n=4 (J)). Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Although statistical analyses (t-test) revealed no significant differences in Smo and Ci155 expression in the dorsal compartment between control and dispS037707, apGalts>ecdRNAi wing discs, loss of Ecd slightly induces an increased Smo accumulation and Ci155 stabilization independently of Hh. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm.


A - A ' ' ' Time - controlled knock - down of ecd or Gapdh in the dorsal compartment of the wing discs ( see Fig 1C for the expression pattern of apGalts ) . The plasma membrane potential was detected using DiBAC4 ( 3 ) . The fluorescence intensity of DiBAC4 ( 3 ) increases upon plasma membrane depolarization . ( A ) The change in plasma membrane potential upon genetic perturbation was quantified as the fold change in the ratio of DiBAC4 ( 3 ) signal intensity in the dorsal to ventral compartment ( control ( n = 19 ) , apGalts > ecdRNAi ( n = 36 ) and apGalts > GapdhRNAi ( n = 18 ) wing discs ) . Error bars indicate ± SD . t - test , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( A ' - A ' ' ' ) Representative images of DiBAC4 ( 3 ) assay in control ( A ' ) , apGalts > ecdRNAi ( A ' ' ) , and apGalts > GapdhRNAi ( A ' ' ' ) wing discs 48 h or 120 h after RNAi induction , respectively . V / D indicates the boundary between the ventral and dorsal compartments . Next to the maximal projections are shown the sum projections of cross - sections along the A / P axis . Loss of Ecd or Gapdh alters the plasma membrane potential . Scale bars = 50 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-A''' Time-controlled knock-down of ecd or Gapdh in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). The plasma membrane potential was detected using DiBAC4(3). The fluorescence intensity of DiBAC4(3) increases upon plasma membrane depolarization. (A) The change in plasma membrane potential upon genetic perturbation was quantified as the fold change in the ratio of DiBAC4(3) signal intensity in the dorsal to ventral compartment (control (n=19), apGalts>ecdRNAi (n=36) and apGalts>GapdhRNAi (n=18) wing discs). Error bars indicate ± SD. t-test, *p≤0.05, ***p≤0.001. (A'-A''') Representative images of DiBAC4(3) assay in control (A'), apGalts>ecdRNAi (A''), and apGalts>GapdhRNAi (A''') wing discs 48 h or 120 h after RNAi induction, respectively. V/D indicates the boundary between the ventral and dorsal compartments. Next to the maximal projections are shown the sum projections of cross-sections along the A/P axis. Loss of Ecd or Gapdh alters the plasma membrane potential. Scale bars= 50 μm.


A - A ' ' ' Time - controlled knock - down of ecd or Gapdh in the dorsal compartment of the wing discs ( see Fig 1C for the expression pattern of apGalts ) . The plasma membrane potential was detected using DiBAC4 ( 3 ) . The fluorescence intensity of DiBAC4 ( 3 ) increases upon plasma membrane depolarization . ( A ) The change in plasma membrane potential upon genetic perturbation was quantified as the fold change in the ratio of DiBAC4 ( 3 ) signal intensity in the dorsal to ventral compartment ( control ( n = 19 ) , apGalts > ecdRNAi ( n = 36 ) and apGalts > GapdhRNAi ( n = 18 ) wing discs ) . Error bars indicate ± SD . t - test , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . ( A ' - A ' ' ' ) Representative images of DiBAC4 ( 3 ) assay in control ( A ' ) , apGalts > ecdRNAi ( A ' ' ) , and apGalts > GapdhRNAi ( A ' ' ' ) wing

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

A-A''' Time-controlled knock-down of ecd or Gapdh in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). The plasma membrane potential was detected using DiBAC4(3). The fluorescence intensity of DiBAC4(3) increases upon plasma membrane depolarization. (A) The change in plasma membrane potential upon genetic perturbation was quantified as the fold change in the ratio of DiBAC4(3) signal intensity in the dorsal to ventral compartment (control (n=19), apGalts>ecdRNAi (n=36) and apGalts>GapdhRNAi (n=18) wing discs). Error bars indicate ± SD. t-test, *p≤0.05, ***p≤0.001. (A'-A''') Representative images of DiBAC4(3) assay in control (A'), apGalts>ecdRNAi (A''), and apGalts>GapdhRNAi (A''') wing discs


D - E ' ' Dissipation of the plasma membrane potential upon treatment of wing discs with gramicidin A ( gA ) . ( D ) Quantification of DiBAC4 ( 3 ) signal intensity in mock - treated ( - gA , n = 7 ) and gA - treated ( + gA , n = 5 ) wing discs . ( D ' , D ' ' ) Representative images of DiBAC4 ( 3 ) assay in mock - ( D ' ) and gA - treated ( D ' ' ) wing discs . Next to the maximal projections are shown sum projections of cross - sections along the A / P axis . ( E ) Quantification of PAC - NAE signal intensity in mock - ( n = 7 ) and gA - treated ( n = 9 ) wing discs , shown as fold change from control ( mock - treated ) . ( E ' - E ' ' ) Representative images of PAC - NAE uptake in mock - ( E ' ) and gA - treated ( E ' ' ) wing discs . GA treatment strongly reduces the uptake of PAC - NAE . Error bars indicate ± SD . t - test , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . Scale bars = 50 μm .

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D-E'' Dissipation of the plasma membrane potential upon treatment of wing discs with gramicidin A (gA). (D) Quantification of DiBAC4(3) signal intensity in mock-treated (- gA, n=7) and gA-treated (+ gA, n=5) wing discs. (D', D'') Representative images of DiBAC4(3) assay in mock- (D') and gA-treated (D'') wing discs. Next to the maximal projections are shown sum projections of cross-sections along the A/P axis. (E) Quantification of PAC-NAE signal intensity in mock- (n=7) and gA-treated (n=9) wing discs, shown as fold change from control (mock-treated). (E'-E'') Representative images of PAC-NAE uptake in mock- (E') and gA-treated (E'') wing discs. GA treatment strongly reduces the uptake of PAC-NAE. Error bars indicate ± SD. t-test, *p≤0.05, ***p≤0.001. Scale bars= 50 μm.


( a ) WT and Pgam5 KO MEFs evaluated by TEM . The black arrows indicate mitochondria ; ( b ) TEM analysis after 12 h CCCP treatment ; black arrowheads indicate DMS . Inset shows a DMS with an encapsulated mitochondrion . The black arrows indicate mitochondria . Scale bar = 0 . 5 micron ;

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(a) WT and Pgam5 KO MEFs evaluated by TEM. The black arrows indicate mitochondria; (b) TEM analysis after 12 h CCCP treatment; black arrowheads indicate DMS. Inset shows a DMS with an encapsulated mitochondrion. The black arrows indicate mitochondria. Scale bar=0.5 micron;


( c ) WT and KO MEFs were treated with CCCP for 0 , 3 or 6 h and cytosolic fractions were immunoblotted with anti - LC - 3 , anti - p62 or anti - actin as indicated .

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(c) WT and KO MEFs were treated with CCCP for 0, 3 or 6 h and cytosolic fractions were immunoblotted with anti-LC-3, anti-p62 or anti-actin as indicated.


( d ) Similar to c . WT and KO MEFs were treated with CCCP for 6 and 12 h and immunoblotted for anti - COXIV , anti - Tomm20 or anti - actin as indicated . For c and d , the numbers below each lane indicate the fractional band density compared with the 0 h time point , which has been set at 1 . 0 ;

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(d) Similar to c. WT and KO MEFs were treated with CCCP for 6 and 12 h and immunoblotted for anti-COXIV, anti-Tomm20 or anti-actin as indicated. For c and d, the numbers below each lane indicate the fractional band density compared with the 0 h time point, which has been set at 1.0;


( e ) Confocal photomicrographs of mt - Keima - transduced WT and KO MEFs treated with CCCP or DMSO vehicle for 12 h ( six individual cells from each group were scanned and representative cells are shown ) ; Intracellular mt - Keima excited at 450 nm was shown in green colour , while red colour indicated the excitation by 550 nm in the same cell . Scale bar = 20 μm .

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(e) Confocal photomicrographs of mt-Keima-transduced WT and KO MEFs treated with CCCP or DMSO vehicle for 12 h (six individual cells from each group were scanned and representative cells are shown); Intracellular mt-Keima excited at 450 nm was shown in green colour, while red colour indicated the excitation by 550 nm in the same cell. Scale bar=20 μm.


( f ) Mitochondrial extracts of WT and Pgam5 KO MEFs treated with DMSO ( − ) or CCCP ( + ) for 3 h and analysed by immunoblot as above . ( Note that mouse PINK1 antibody can only detect the full - length PINK1 ) . Arrow indicates the 63 kDa form of PINK1 .

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(f) Mitochondrial extracts of WT and Pgam5 KO MEFs treated with DMSO (−) or CCCP (+) for 3 h and analysed by immunoblot as above. (Note that mouse PINK1 antibody can only detect the full-length PINK1). Arrow indicates the 63 kDa form of PINK1.


( g ) Hela cells transduced with either nonspecific ( NS ) or PGAM5 shRNA viruses were treated with CCCP for 3 h , and mitochondrial fractions were immunoblotted for the proteins indicated . Full - length ( * 63 kDa ) and cleaved ( # 54 kDa ) PINK1 are indicated . For f and g , the numbers below indicate the full - length PINK1 band density compared with the first lane that has been set at 1 .

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(g) Hela cells transduced with either nonspecific (NS) or PGAM5 shRNA viruses were treated with CCCP for 3 h, and mitochondrial fractions were immunoblotted for the proteins indicated. Full-length (*63 kDa) and cleaved (#54 kDa) PINK1 are indicated. For f and g, the numbers below indicate the full-length PINK1 band density compared with the first lane that has been set at 1.0


( h ) Confocal micrographs of Hela cells as in g were transfected with PARKIN - YFP ( green ) plasmid before CCCP treatment , then ( i ) treated with CCCP for 12 h . Mitochondria were stained with anti - Tomm20 ( red ) 4 ' , 6 - diamidino - 2 - phenylindole stained nucleus was shown in blue . Co - localization was indicated by white colour . Scale bar = 10 μm . ( j ) Quantification of the co - localization percentage of Tomm20 ( red ) with parkin ( green ) per cell by Imaris software ( n = 7 for NS and PGAM5 shRNA group separately . Results are presented as mean and s . d . values and were analysed by the student t - test ) . All the blots and images are representative of three independent experiments . DMSO , dimethylsulphoxide .

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

(h) Confocal micrographs of Hela cells as in g were transfected with PARKIN-YFP (green) plasmid before CCCP treatment, then (i) treated with CCCP for 12 h. Mitochondria were stained with anti-Tomm20 (red) 4',6-diamidino-2-phenylindole stained nucleus was shown in blue. Co-localization was indicated by white colour. Scale bar=10 μm. (j) Quantification of the co-localization percentage of Tomm20 (red) with parkin (green) per cell by Imaris software (n=7 for NS and PGAM5 shRNA group separately. Results are presented as mean and s.d. values and were analysed by the student t-test). All the blots and images are representative of three independent experiments. DMSO, dimethylsulphoxide.


( a ) Mitochondria from MEFs from Parl WT ( + / + ) and KO ( − / − ) mice were treated with the indicated doses of proteinase K ( Prot . K ) for 30 min on ice and then immunoblotted for the indicated proteins . Full - length and cleaved PGAM5 bands are indicated with arrows .

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(a) Mitochondria from MEFs from Parl WT (+/+) and KO (−/−) mice were treated with the indicated doses of proteinase K (Prot.K) for 30 min on ice and then immunoblotted for the indicated proteins. Full-length and cleaved PGAM5 bands are indicated with arrows.


( b ) Pgam5 WT and KO MEFs were transduced with either nonspecific ( NS ) or Parl shRNA and either treated ( + ) or not ( − ) with CCCP for 3 h . Mitochondria were then purified and analysed by immunoblot for the indicated proteins .

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(b) Pgam5 WT and KO MEFs were transduced with either nonspecific (NS) or Parl shRNA and either treated (+) or not (−) with CCCP for 3 h. Mitochondria were then purified and analysed by immunoblot for the indicated proteins.


( c ) WT PINK1 as well as kinase dead ( KD ) or Parkinson ' s disease - associated PINK1 mutants ( C92F , A168P , H271Q , G309D , L347P , G386S , R464H ) were co - transfected into HEK293T cells with the same amount PGAM5 ( lane 1 - 9 ) or GFP ( lane 10 - 18 ) expression plasmids and total cell lysates were immunoblotting for PINK1 , PGAM5 and actin . The ratio of WT full - length ( 63 kDa ) versus cleaved ( 54 kDa ) PINK1 ( * / # ) is shown .

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(c) WT PINK1 as well as kinase dead (KD) or Parkinson's disease-associated PINK1 mutants (C92F, A168P, H271Q, G309D, L347P, G386S, R464H) were co-transfected into HEK293T cells with the same amount PGAM5 (lane 1-9) or GFP (lane 10-18) expression plasmids and total cell lysates were immunoblotting for PINK1, PGAM5 and actin. The ratio of WT full-length (63 kDa) versus cleaved (54 kDa) PINK1 (*/#) is shown.


( d ) Anti - myc immunoprecipitates ( IP ) of lysates from 293T cells transfected with combinations of myc - tagged PGAM5 and PINK1 - V5 with or without CCCP treatment and immunoblotting with anti - PINK1 or anti - myc .

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(d) Anti-myc immunoprecipitates (IP) of lysates from 293T cells transfected with combinations of myc-tagged PGAM5 and PINK1-V5 with or without CCCP treatment and immunoblotting with anti-PINK1 or anti-myc.


( e ) Four constructs expressing either : ( 1 ) no PGAM5 ( empty vector ) or ( 2 ) amino acids 1 - 98 , ( 3 ) 1 - 110 and ( 4 ) 98 - 289 truncated versions of PGAM5 ( left panel ) . Immunoblot of HEK293T cells expressing PINK1 - GFP and myc - tagged PGAM5 truncation mutants as indicated with anti - PINK1 or anti - myc for the input proteins as well as the anti - GFP IP ( right panel ) .

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(e) Four constructs expressing either: (1) no PGAM5 (empty vector) or (2) amino acids 1-98, (3) 1-110 and (4) 98-289 truncated versions of PGAM5 (left panel). Immunoblot of HEK293T cells expressing PINK1-GFP and myc-tagged PGAM5 truncation mutants as indicated with anti-PINK1 or anti-myc for the input proteins as well as the anti-GFP IP (right panel).


( f ) Co - transfection of GFP , WT PGAM5 , 2RA mutant ( R98A and R104A ) or the 2HA mutant ( H99A and H105A ) with a PINK1 expression plasmid into HEK293T cells and the input cell lysates as well as the anti - myc IP were immunoblotted with antibodies against PINK1 , PGAM5 or actin .

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(f) Co-transfection of GFP, WT PGAM5, 2RA mutant (R98A and R104A) or the 2HA mutant (H99A and H105A) with a PINK1 expression plasmid into HEK293T cells and the input cell lysates as well as the anti-myc IP were immunoblotted with antibodies against PINK1, PGAM5 or actin.


WT PINK1 was expressed in HEK293T cells with or without PGAM5 or a GFP control plasmid after ( g ) DMSO or ( h ) CCCP treatment . Cells were permeabilized with digitonin or Triton X - 100 separately , followed by treatment with the indicated amounts of trypsin on ice for 30 min . Cell lysates were immunoblotted for PINK1 , PGAM5 , Tomm20 or HSP60 as indicated . Full - length ( * 63 kDa ) and cleaved ( # 54 kDa ) PINK1 are shown .

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

WT PINK1 was expressed in HEK293T cells with or without PGAM5 or a GFP control plasmid after (g) DMSO or (h) CCCP treatment. Cells were permeabilized with digitonin or Triton X-100 separately, followed by treatment with the indicated amounts of trypsin on ice for 30 min. Cell lysates were immunoblotted for PINK1, PGAM5, Tomm20 or HSP60 as indicated. Full-length (*63 kDa) and cleaved (#54 kDa) PINK1 are shown.


( a ) Body weight was measured in 5 - month - old and 12 - month - old female Pgam5 WT and KO mice .

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(a) Body weight was measured in 5-month-old and 12-month-old female Pgam5 WT and KO mice.


Behavioral measurements on 12 - month - old female WT and KO mice ( WT n = 10 , KO n = 8 ) were as follows : ( b ) Open - field tests including time spent mobile , time spent immobile , whole area distance and centre area distance ;

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Behavioral measurements on 12-month-old female WT and KO mice (WT n=10, KO n=8) were as follows: (b) Open-field tests including time spent mobile, time spent immobile, whole area distance and centre area distance;


Behavioral measurements on 12 - month - old female WT and KO mice ( WT n = 10 , KO n = 8 ) were as follows : ( c ) Gait length , gait frequency in steps / ss and step angle ; ( d ) rearing test , ( e ) akinesia test and ( f ) ladder climb test and ( g ) vertical pole test . T - turn indicates the time mice spent to turn their bodies 180 degrees from the head up start position before descending the pole . T - down indicates the time mice spent to descend to the bottom of pole . Graphs show the mean and s . d . values and were analysed by the student ' s t - test , * P0 . 05 , * * P0 . 01 , * * * P0 . 001 , NS , not significant .

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Behavioral measurements on 12-month-old female WT and KO mice (WT n=10, KO n=8) were as follows: (c) Gait length, gait frequency in steps/ss and step angle; (d) rearing test, (e) akinesia test and (f) ladder climb test and (g) vertical pole test. T-turn indicates the time mice spent to turn their bodies 180 degrees from the head up start position before descending the pole. T-down indicates the time mice spent to descend to the bottom of pole. Graphs show the mean and s.d. values and were analysed by the student's t-test, *P0.05, **P0.01, ***P0.001, NS, not significant.


High - performance liquid chromatography - electrochemical detection quantitation of ( a ) striatal DA and its metabolites ( b ) dihydroxyphenylacetic acid ( DOPAC ) ( c ) Homovanillic acid ( HVA ) and ( d ) 3MT levels at three different time points ( 1 , 2 and 12 - month - old ) in Pgam5 WT or KO mice .

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High-performance liquid chromatography-electrochemical detection quantitation of (a) striatal DA and its metabolites (b) dihydroxyphenylacetic acid (DOPAC) (c) Homovanillic acid (HVA) and (d) 3MT levels at three different time points (1, 2 and 12-month-old) in Pgam5 WT or KO mice.


( e ) Representative images of TH - immunohistochemistry from WT and KO mice . The VTA region is indicated . Stereological quantitation of ( f ) TH and ( g ) Nissl - positive neurons in the SNpc ( n = 4 mice per group ) . Error bars represent the mean ± s . e . m . , n = 4 mice per group . ( two - way analysis of variance , * * * P0 . 01 , * P0 . 05 , NS , not significant ) . mo , month old .

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(e) Representative images of TH-immunohistochemistry from WT and KO mice. The VTA region is indicated. Stereological quantitation of (f) TH and (g) Nissl-positive neurons in the SNpc (n=4 mice per group). Error bars represent the mean±s.e.m., n=4 mice per group. (two-way analysis of variance, ***P0.01, *P0.05, NS, not significant). mo, month old.


The 18 - month - old Pgam5 WT and KO mice received either water as control ( n = 4 ) , or 5 mg kg − 1 L - DOPA and 12 . 5 mg kg − 1 Benserazide ( n = 5 ) by intraperitoneal injection . Twenty minutes later , mice were subjected to the open - field test and the centre area time in seconds ( s ) as well as the centre area distance in centimeters ( cm ) were measured as described in the Materials and Methods . Graphs show the mean and s . d . values and were analysed by the student t - test , NS , no significance ; * P0 . 05 ; * * P0 . 01 .

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

The 18-month-old Pgam5 WT and KO mice received either water as control (n=4), or 5 mg kg−1 L-DOPA and 12.5 mg kg−1 Benserazide (n=5) by intraperitoneal injection. Twenty minutes later, mice were subjected to the open-field test and the centre area time in seconds (s) as well as the centre area distance in centimeters (cm) were measured as described in the Materials and Methods. Graphs show the mean and s.d. values and were analysed by the student t-test, NS, no significance; *P0.05; **P0.01.


( a ) WT and dnm1Δ cells expressing Vph1 - RFP and Idp1 - GFP were grown in minimal synthetic dextrose medium and transferred to lactate medium at an initial density of OD600 0 . 08 . The cells were then sampled daily and analysed by fluorescence microscopy ( × 100 objective ) during stationary phase mitophagy in lactate medium . Scale bar , 5 μm .

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(a) WT and dnm1Δ cells expressing Vph1-RFP and Idp1-GFP were grown in minimal synthetic dextrose medium and transferred to lactate medium at an initial density of OD600 0.08. The cells were then sampled daily and analysed by fluorescence microscopy (× 100 objective) during stationary phase mitophagy in lactate medium. Scale bar, 5 μm.


( b ) WT , dnm1Δ and dnm1Δ mgm1Δ mutants were grown in SL medium as described in Methods , and samples ( 10 OD600 units ) were taken at each time point . Protein extracts were prepared and equal protein amounts ( 20 μg ) were subjected to SDS - polyacrylamide gel electrophoresis and immunoblotting with anti - GFP antibody .

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(b) WT, dnm1Δ and dnm1Δ mgm1Δ mutants were grown in SL medium as described in Methods, and samples (10 OD600 units) were taken at each time point. Protein extracts were prepared and equal protein amounts (20 μg) were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting with anti-GFP antibody.


( c ) Quantification of the percent free GFP at the day 4 time point in each of the three strains analysed in b , using four independent experiments for each strain . Error bars indicate s . d . analysis of variance , P0 . 001 ; N = 4 .

(c) Quantification of the percent free GFP at the day 4 time point in each of the three strains analysed in b, using four independent experiments for each strain. Error bars indicate s.d. analysis of variance, P0.001; N=4.


( c ) Density plot of the reproducibility of biological replicates .

(c) Density plot of the reproducibility of biological replicates.


( a ) Ratios from proteins with quantifications from all samples were log2 transformed and z - score normalized . Columns containing data from the different samples were clustered hierarchically and rows that contained protein entries were clustered by k - means . The proteins in each k - means cluster were tested for enrichment of GO CC ( indicated in red ) and GO BP ( indicated in blue ) terms .

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(a) Ratios from proteins with quantifications from all samples were log2 transformed and z-score normalized. Columns containing data from the different samples were clustered hierarchically and rows that contained protein entries were clustered by k-means. The proteins in each k-means cluster were tested for enrichment of GO CC (indicated in red) and GO BP (indicated in blue) terms.


( b ) Proteins associated with different cellular compartments were sequentially extracted and the average of their ratios were compared with averages of remaining proteins .

(b) Proteins associated with different cellular compartments were sequentially extracted and the average of their ratios were compared with averages of remaining proteins.


( d ) GO BP enrichment analysis of proteins in the different clusters compared with the remaining proteins .

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(d) GO BP enrichment analysis of proteins in the different clusters compared with the remaining proteins.


A comparison of the SILAC - derived abundance as a function of time , for several proteins that either show differential behaviour between WT and the mutants ( Idh2 , Idp1 and Aco2 ) and proteins that behave similarly between all the genetic backgrounds tested ( Hsp78 ) . Shown are mean protein ratios ( two replicates for mutants , three replicates for WT ) , error bars indicating peptide - based relative s . d .

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A comparison of the SILAC-derived abundance as a function of time, for several proteins that either show differential behaviour between WT and the mutants (Idh2, Idp1 and Aco2) and proteins that behave similarly between all the genetic backgrounds tested (Hsp78). Shown are mean protein ratios (two replicates for mutants, three replicates for WT), error bars indicating peptide-based relative s.d.


WT ( a ) , dnm1Δ ( b ) and atg1Δ cells ( c ) and atg32Δ cells ( d ) expressing chromosomally tagged GFP chimeras of Aco2 , Idh2 , Idp1 and Hsp78 were incubated in SL medium for 7 days , and 10 OD600 units were sampled at day 1 and day 7 and processed for immunoblotting with anti - GFP antibodies as described in the Methods section . Positive control used in c and d was from dnm1Δ cells expressing Idp1 - GFP from a plasmid at 4 - day incubation ( Fig . 1b , lane 4 ) . Release of free GFP in WT cells is reproducibly variable between these reporters , and varies between 0 ( Idh2 ) and 60 % ( Aco2 ) . ( e ) Densitometric quantification of the release of free GFP ( as % of total signal ) in four independent experiments , comparing the results between WT and dnm1Δ cells . Bars denote s . d . ( n = 4 ) .

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WT (a), dnm1Δ (b) and atg1Δ cells (c) and atg32Δ cells (d) expressing chromosomally tagged GFP chimeras of Aco2, Idh2, Idp1 and Hsp78 were incubated in SL medium for 7 days, and 10 OD600 units were sampled at day 1 and day 7 and processed for immunoblotting with anti-GFP antibodies as described in the Methods section. Positive control used in c and d was from dnm1Δ cells expressing Idp1-GFP from a plasmid at 4-day incubation (Fig. 1b, lane 4). Release of free GFP in WT cells is reproducibly variable between these reporters, and varies between 0 (Idh2) and 60% (Aco2). (e) Densitometric quantification of the release of free GFP (as % of total signal) in four independent experiments, comparing the results between WT and dnm1Δ cells. Bars denote s.d. (n=4).


( a ) Cells expressing chromosomally tagged GFP chimeras of Aco2 , Idh2 , Idp1 and Hsp78 as well as a plasmid - borne ( pDJB12 ) , mitochondrially targeted RFP ( mtRFP ) were incubated in SL - leucine medium for 3 days and imaged daily . Aco2 and Idp1 show clear mitophagic targeting ( white arrows point at specific examples ) by day 3 , whereas Idh2 and Hsp78 show weak or no mitophagy . In addition , Idh2 and Hsp78 show different degrees of segregation relative to the mtRFP marker , whereas Idp1 - GFP and Aco2 - GFP show a near - complete co - localization with mtRFP . Scale bar , 5 μm . ( b ) Quantification of the frequency of mitophagy from a ; the percentage of cells showing mitophagic profiles in the red ( magenta bars ; plasmid - borne mtRFP ) or green ( turquoise bars ; integrated C - terminal GFP chimerae ) was calculated for each GFP chimera . Idp - GFP , red N = 76 , green N = 82 ; Aco2 - GFP , red N = 79 , green N = 82 ; Idh2 - GFP , red N = 72 , green N = 72 ; Hsp78 - GFP , red N = 70 , green N = 70 .

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

(a) Cells expressing chromosomally tagged GFP chimeras of Aco2, Idh2, Idp1 and Hsp78 as well as a plasmid-borne (pDJB12), mitochondrially targeted RFP (mtRFP) were incubated in SL-leucine medium for 3 days and imaged daily. Aco2 and Idp1 show clear mitophagic targeting (white arrows point at specific examples) by day 3, whereas Idh2 and Hsp78 show weak or no mitophagy. In addition, Idh2 and Hsp78 show different degrees of segregation relative to the mtRFP marker, whereas Idp1-GFP and Aco2-GFP show a near-complete co-localization with mtRFP. Scale bar, 5 μm. (b) Quantification of the frequency of mitophagy from a; the percentage of cells showing mitophagic profiles in the red (magenta bars; plasmid-borne mtRFP) or green (turquoise bars; integrated C-terminal GFP chimerae) was calculated for each GFP chimera. Idp-GFP, red N=76, green N=82; Aco2-GFP, red N=79, green N=82; Idh2-GFP, red N=72, green N=72; Hsp78-GFP, red N=70, green N=70.


WT and dnm1Δ cells expressing integrated Hsp78 - GFP and plasmid - borne mtRFP were grown on lactate medium for 3 days . Cells were imaged daily and the images were analysed using ImageJ as described in Methods . Graphs illustrate the distribution of pixels associated with specific values of RFP and GFP channel intensities , respectively . Diagonal concentrations of dots indicate spatial correlation between the signals . The coloured photographic panels at the bottom illustrate the increased intensity correlation of the two channels at the 3 - day time point in the dnm1Δ mutant , relative to the WT cells . Scale bar , 5 μm .

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WT and dnm1Δ cells expressing integrated Hsp78-GFP and plasmid-borne mtRFP were grown on lactate medium for 3 days. Cells were imaged daily and the images were analysed using ImageJ as described in Methods. Graphs illustrate the distribution of pixels associated with specific values of RFP and GFP channel intensities, respectively. Diagonal concentrations of dots indicate spatial correlation between the signals. The coloured photographic panels at the bottom illustrate the increased intensity correlation of the two channels at the 3-day time point in the dnm1Δ mutant, relative to the WT cells. Scale bar, 5 μm.


B , C Serial transplantations of PR + / + and PR − / − mammary epithelia . ( B ) Fluorescence stereo microscopy of third ‐ generation mammary outgrowths derived from 8 ‐ week ‐ old PR + / + ; EGFP and PR − / − ; EGFP donor mice . ( C ) Table summarizing 3 independent serial transplant experiments with PR + / + ; EGFP and PR − / − ; EGFP . Each engrafted gland is represented by a micrograph ; black sectors represent area of fat pad filled by engrafted epithelium . Scale bar : 200 μm .

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B, C Serial transplantations of PR+/+ and PR−/− mammary epithelia. (B) Fluorescence stereo microscopy of third‐generation mammary outgrowths derived from 8‐week‐old PR+/+; EGFP and PR−/−; EGFP donor mice. (C) Table summarizing 3 independent serial transplant experiments with PR+/+; EGFP and PR−/−; EGFP. Each engrafted gland is represented by a micrograph; black sectors represent area of fat pad filled by engrafted epithelium. Scale bar: 200 μm.


D , E Serial transplantation of RANKL + / + and RANKL − / − mammary epithelia . ( D ) Fluorescence stereo microscopy of third ‐ generation mammary outgrowths derived from 5 ‐ week ‐ old RANKL + / + ; EGFP and RANKL − / − ; EGFP donor mice . Insets : higher magnification showing side branches present in the WT control ( arrowheads ) absent from RANKL − / − ; EGFP epithelium . ( E ) Table summarizing three independent serial transplant experiments with RANKL + / + ; EGFP and RANKL − / − ; EGFP donor mice . Scale bar : 200 μm .

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D, E Serial transplantation of RANKL+/+ and RANKL−/− mammary epithelia. (D) Fluorescence stereo microscopy of third‐generation mammary outgrowths derived from 5‐week‐old RANKL+/+; EGFP and RANKL−/−; EGFP donor mice. Insets: higher magnification showing side branches present in the WT control (arrowheads) absent from RANKL−/−; EGFP epithelium. (E) Table summarizing three independent serial transplant experiments with RANKL+/+; EGFP and RANKL−/−; EGFP donor mice. Scale bar: 200 μm.


F , G Serial transplantation of Wnt4 + / + and Wnt4 − / − mammary epithelia . ( F ) Fluorescence stereo microscopy of third ‐ generation mammary outgrowths derived from mammary buds of E12 . 5 and E13 . 5 Wnt4 + / + ; EGFP and Wnt4 − / − ; EGFP embryos . Scale bar : 200 μm . ( G ) Table summarizing three independent serial transplant experiments with Wnt4 + / + ; EGFP donor mice . Scale bar : 200 μm .

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F, G Serial transplantation of Wnt4+/+ and Wnt4−/− mammary epithelia. (F) Fluorescence stereo microscopy of third‐generation mammary outgrowths derived from mammary buds of E12.5 and E13.5 Wnt4+/+; EGFP and Wnt4−/−; EGFP embryos. Scale bar: 200 μm. (G) Table summarizing three independent serial transplant experiments with Wnt4+/+; EGFP donor mice. Scale bar: 200 μm.


H Box plot showing the difference between percentage of reconstitution between WT and MT contralateral grafts in each transplant generation . P values were determined by Mann - Whitney U ‐ test .

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H Box plot showing the difference between percentage of reconstitution between WT and MT contralateral grafts in each transplant generation. P values were determined by Mann-Whitney U‐test.


A Epifluorescence stereo microscopy of inguinal mammary gland from a 5 ‐ day ‐ old Wnt4 : : Cre ; mT / mG female ( n = 7 ) . Scale bars : 0 . 5 mm and 0 . 1 mm ( inset ) .

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A Epifluorescence stereo microscopy of inguinal mammary gland from a 5‐day‐old Wnt4::Cre; mT/mG female (n = 7). Scale bars: 0.5 mm and 0.1 mm (inset).


B , C Histology sections of mammary glands from a 5 ‐ day ‐ old ( B ; n = 7 ) and a 10 ‐ day ‐ old ( C ; n = 5 ) Wnt4 : : Cre ; mT / mG female stained by double immunofluorescence microscopy for EGFP ( green ) and PR ( magenta , not detected ) , counterstained with DAPI ( blue ) . Scale bar : 50 mm .

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B, C Histology sections of mammary glands from a 5‐day‐old (B; n = 7) and a 10‐day‐old (C; n = 5) Wnt4::Cre; mT/mG female stained by double immunofluorescence microscopy for EGFP (green) and PR (magenta, not detected), counterstained with DAPI (blue). Scale bar: 50 mm.


D - F EGFP ( green ) and PR ( magenta ) co ‐ immunofluorescence microscopy counterstained with DAPI ( blue ) on histology sections from mT / mG ; Wnt4 : : Cre mammary glands at different developmental stages . ( D ) TEB of a 4 ‐ week ‐ old female ( n = 4 ) ; scale bar : 30 μm . ( E ) Ducts of an 8 ‐ week ‐ old female ( n = 3 ) ; scale bar : 100 μm ; inset , scale bar : 20 μm . ( F ) Duct of a female at day 10 . 5 of pregnancy ( n = 3 ) ; scale bar : 150 μm ; inset , scale bar : 30 μm .

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D-F EGFP (green) and PR (magenta) co‐immunofluorescence microscopy counterstained with DAPI (blue) on histology sections from mT/mG; Wnt4::Cre mammary glands at different developmental stages. (D) TEB of a 4‐week‐old female (n = 4); scale bar: 30 μm. (E) Ducts of an 8‐week‐old female (n = 3); scale bar: 100 μm; inset, scale bar: 20 μm. (F) Duct of a female at day 10.5 of pregnancy (n = 3); scale bar: 150 μm; inset, scale bar: 30 μm.


G - I EGFP ( green ) , PR ( magenta ) , and p63 ( white ) triple co ‐ immunofluorescence microscopy counterstained with DAPI ( blue ) on histological sections from mT / mG ; Wnt4 : : Cre mammary glands at different developmental stages . ( G ) Ducts of 5 ‐ day ‐ old female ( n = 3 ) . ( H ) TEB of a 4 ‐ week ‐ old female ( n = 3 ) . ( I ) Duct of an 8 ‐ week ‐ old female ( n = 3 ) . Scale bars : 30 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

G-I EGFP (green), PR (magenta), and p63 (white) triple co‐immunofluorescence microscopy counterstained with DAPI (blue) on histological sections from mT/mG; Wnt4::Cre mammary glands at different developmental stages. (G) Ducts of 5‐day‐old female (n = 3). (H) TEB of a 4‐week‐old female (n = 3). (I) Duct of an 8‐week‐old female (n = 3). Scale bars: 30 μm.


J - O Epifluorescence stereo microscopy of mammary glands harvested from 15 ‐ day ‐ old Wnt4 : : GFP females either WT ( n = 18 ) ( J , M ) , ERα − / − ( n = 4 ) ( K , N ) , or PR − / − ( n = 3 ) ( L , O ) . dTomato expression ( J - L ) ; EGFP expression ( M - O ) is not abrogated in ERα − / − nor PR − / − epithelia . Arrowheads mark the main duct originating from the nipple . Scale bar : 50 μm .

J-O Epifluorescence stereo microscopy of mammary glands harvested from 15‐day‐old Wnt4::GFP females either WT (n = 18) (J, M), ERα−/− (n = 4) (K, N), or PR−/− (n = 3) (L, O). dTomato expression (J-L); EGFP expression (M-O) is not abrogated in ERα−/− nor PR−/− epithelia. Arrowheads mark the main duct originating from the nipple. Scale bar: 50 μm.


Q Bar plots showing relative PR and Wnt4 mRNA expression normalized to CK18 mRNA in mammary organoids from 5 pubertal ( 6 weeks old ) and 3 adult ( 11 weeks old ) mice exposed for 6 h to vehicle ( C ) , 17β ‐ estradiol ( 20 nmol ) ( E2 ) , or R5020 ( 20 nmol ) ( P ) . Bars represent the mean ± SD of 3 independent experiments .

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

Q Bar plots showing relative PR and Wnt4 mRNA expression normalized to CK18 mRNA in mammary organoids from 5 pubertal (6 weeks old) and 3 adult (11 weeks old) mice exposed for 6 h to vehicle (C), 17β‐estradiol (20 nmol) (E2), or R5020 (20 nmol) (P). Bars represent the mean ± SD of 3 independent experiments.


R - X Epifluorescence stereo microscopy of contralateral mammary glands that were engrafted with Wnt4 : : GFP epithelium from 8 ‐ week ‐ old females , either PRWT ( R , T , V , X ) or PR − / − ( S , U , W ) . dTomato expression ( R , S ) ; EGFP expression ( T , U ) double epifluorescence ( V , W , X ) on contralateral engrafted glands 3 weeks after surgery when recipients were 6 weeks old . Representative result from three independent experiments . Arrowheads point to TEBs ( V , X ) or to origin of growth ( W ) . Scale bar ( R - W ) : 5 mm , ( X ) : 1 mm .

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

R-X Epifluorescence stereo microscopy of contralateral mammary glands that were engrafted with Wnt4::GFP epithelium from 8‐week‐old females, either PRWT (R, T, V, X) or PR−/− (S, U, W). dTomato expression (R, S); EGFP expression (T, U) double epifluorescence (V, W, X) on contralateral engrafted glands 3 weeks after surgery when recipients were 6 weeks old. Representative result from three independent experiments. Arrowheads point to TEBs (V, X) or to origin of growth (W). Scale bar (R-W): 5 mm, (X): 1 mm.


A - D Representative epifluorescence stereo microscopy of inguinal mammary glands from a 10 ‐ day ‐ old mT / mG ( A , C ) and MMTV : : Cre ; mT / mG ( B , D ) female ( n = 7 ) . Scale bar : 1 mm .

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

A-D Representative epifluorescence stereo microscopy of inguinal mammary glands from a 10‐day‐old mT/mG (A, C) and MMTV::Cre; mT/mG (B, D) female (n = 7). Scale bar: 1 mm.


E Higher magnification of inguinal mammary gland from a 10 ‐ day ‐ old MMTV : : Cre ; mT / mG female . Scale bar : 0 . 2 mm . F Immunofluorescence microscopy for EGFP ( green ) on a mammary gland section from a 10 ‐ day ‐ old MMTV : : Cre ; mT / mG female counterstained with DAPI ( blue ) ( n = 7 ) . Scale bar : 30 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

E Higher magnification of inguinal mammary gland from a 10‐day‐old MMTV::Cre; mT/mG female. Scale bar: 0.2 mm.F Immunofluorescence microscopy for EGFP (green) on a mammary gland section from a 10‐day‐old MMTV::Cre; mT/mG female counterstained with DAPI (blue) (n = 7). Scale bar: 30 μm.


G Epifluorescence stereo microscopy of mammary glands from 10 ‐ day ‐ old littermates either MMTV : : Cre ; mT / mG Wnt4fl / + or MMTV : : Cre ; mT / mG ; Wnt4fl / fl . Scale bar : 1 mm . Arrowhead marks the main duct originating from the nipple . H Bar plot showing ratio of branching points in prepubertal Cre + ; Wnt4fl / fl ( n = 12 ) relative to Cre + ; Wnt4fl / + littermates ( n = 10 ) . Two ‐ tailed , paired Student ' s t ‐ test was used to calculate statistical significance .

G Epifluorescence stereo microscopy of mammary glands from 10‐day‐old littermates either MMTV::Cre; mT/mG Wnt4fl/+ or MMTV::Cre; mT/mG; Wnt4fl/fl. Scale bar: 1 mm. Arrowhead marks the main duct originating from the nipple.H Bar plot showing ratio of branching points in prepubertal Cre+;Wnt4fl/fl (n = 12) relative to Cre+;Wnt4fl/+ littermates (n = 10). Two‐tailed, paired Student's t‐test was used to calculate statistical significance.


I Fluorescence stereo microscopy of mammary glands from 35 ‐ day ‐ old littermates either MMTV : : Cre ; mT / m ; Wnt4fl / + or MMTV : : Cre ; mT / m ; Wnt4fl / fl . Note ductal elongation is delayed in Wnt4 ‐ deficient mammary epithelium compared to Wnt4fl / + control . LN : lymph node . Scale bar : 4 mm . J , K Bar plots showing number of terminal end buds ( TEBs ) ( J ) and area of mammary fat pad filled by the ductal system ( K ) . Ctrl ( Wnt4fl / wt or Wnt4fl / fl ; Cre − ) : n = 6 , fl / + ; Cre + : n = 4 , and fl / fl ; Cre + : n = 7 ) . Data are presented as the mean ± SD . Two ‐ tailed Student ' s t ‐ test was used to calculate statistical significance .

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

I Fluorescence stereo microscopy of mammary glands from 35‐day‐old littermates either MMTV::Cre; mT/m; Wnt4fl/+ or MMTV::Cre; mT/m;Wnt4fl/fl. Note ductal elongation is delayed in Wnt4‐deficient mammary epithelium compared to Wnt4fl/+ control. LN: lymph node. Scale bar: 4 mm.J, K Bar plots showing number of terminal end buds (TEBs) (J) and area of mammary fat pad filled by the ductal system (K). Ctrl (Wnt4fl/wt or Wnt4fl/fl; Cre−): n = 6, fl/+; Cre+: n = 4, and fl/fl; Cre+: n = 7). Data are presented as the mean ± SD. Two‐tailed Student's t‐test was used to calculate statistical significance.


L , M Bar plots showing BrdU incorporation index in inguinal mammary glands of 4 ‐ week ‐ old Wnt4fl / + ; Cre + ( n = 3 ) or Wnt4fl / fl ; Cre + ( n = 4 ) in the ducts ( L ) and in the TEBs ( M ) . Data are presented as the mean ± SD . Two ‐ tailed Student ' s t ‐ test was used to calculate statistical significance .

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

L, M Bar plots showing BrdU incorporation index in inguinal mammary glands of 4‐week‐old Wnt4fl/+; Cre+ (n = 3) or Wnt4fl/fl; Cre+ (n = 4) in the ducts (L) and in the TEBs (M). Data are presented as the mean ± SD. Two‐tailed Student's t‐test was used to calculate statistical significance.


A Whole ‐ mount microscopy of X ‐ gal ‐ stained Axin2 + / lacZ in E12 . 5 embryo showing β ‐ galactosidase expression in the mammary buds ( arrows ) ( n = 8 ) . Scale bar : 1 mm . Arrowheads mark mammary buds .

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

A Whole‐mount microscopy of X‐gal‐stained Axin2+/lacZ in E12.5 embryo showing β‐galactosidase expression in the mammary buds (arrows) (n = 8). Scale bar: 1 mm. Arrowheads mark mammary buds.


B - F Whole ‐ mount microscopy of X ‐ gal ( blue ) ‐ and carmine alum ( red ) ‐ stained mammary glands harvested from Axin2 + / lacZ mice at distinct developmental stages . ( B ) At postnatal day 1 , β ‐ galactosidase activity detected in the nipple area ( n = 6 ) . Scale bar : 1 mm . ( C , D ) In 5 ‐ week ‐ old mammary glands , reporter activity was detected around the ducts ( small arrows ) and in the neck region of the terminal end buds ( TEBs ) ( large arrows ) ( C ) ( n = 8 ) . Scale bars : 400 μm ( C ) and 100 μm ( D ) . ( E ) At 8 . 5 day of pregnancy , reporter expression was detected in the ducts . Higher magnification ( inset ) suggests myoepithelial expression ( n = 10 ) . Scale bar : 1 mm . ( F ) Whole ‐ mount at day 14 . 5 of pregnancy : reporter activity is limited to ducts ( n = 5 ) . Scale bar : 200 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

B-F Whole‐mount microscopy of X‐gal (blue)‐ and carmine alum (red)‐stained mammary glands harvested from Axin2+/lacZ mice at distinct developmental stages. (B) At postnatal day 1, β‐galactosidase activity detected in the nipple area (n = 6). Scale bar: 1 mm. (C, D) In 5‐week‐old mammary glands, reporter activity was detected around the ducts (small arrows) and in the neck region of the terminal end buds (TEBs) (large arrows) (C) (n = 8). Scale bars: 400 μm (C) and 100 μm (D). (E) At 8.5 day of pregnancy, reporter expression was detected in the ducts. Higher magnification (inset) suggests myoepithelial expression (n = 10). Scale bar: 1 mm. (F) Whole‐mount at day 14.5 of pregnancy: reporter activity is limited to ducts (n = 5). Scale bar: 200 μm.


G Histology section of Axin2 : : LacZ mammary gland at day 8 . 5 of pregnancy counterstained with nuclear red ; luminal epithelial cells show no detectable β ‐ galactosidase activity but myoepithelial cells do . Scale bar : 200 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

G Histology section of Axin2::LacZ mammary gland at day 8.5 of pregnancy counterstained with nuclear red; luminal epithelial cells show no detectable β‐galactosidase activity but myoepithelial cells do. Scale bar: 200 μm.


H β ‐ galactosidase activity ( blue ) colocalizes with the myoepithelial marker p63 ( green ) detected by immunofluorescence microscopy . Arrows point to myoepithelial cells . L , lumen . Scale bar : 100 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

H β‐galactosidase activity (blue) colocalizes with the myoepithelial marker p63 (green) detected by immunofluorescence microscopy. Arrows point to myoepithelial cells. L, lumen. Scale bar: 100 μm.


I Representative whole ‐ mount stereo microscopy of X ‐ gal ( blue ) ‐ and carmine alum ( magenta ) ‐ stained mammary gland biopsies taken from 14 ‐ week ‐ old Axin2 : : LacZ females collected at diestrus and estrus , respectively ( n = 3 ) . Scale bar : 200 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

I Representative whole‐mount stereo microscopy of X‐gal (blue)‐ and carmine alum (magenta)‐stained mammary gland biopsies taken from 14‐week‐old Axin2::LacZ females collected at diestrus and estrus, respectively (n = 3). Scale bar: 200 μm.


J Relative Wnt4 and Axin2 mRNA expression in mammary glands from three mice in estrus versus diestrus assessed by semiquantitative qRT - PCR normalized to 18S rRNA . Two ‐ tailed , paired Student ' s t ‐ test was used to calculate statistical significance .

J Relative Wnt4 and Axin2 mRNA expression in mammary glands from three mice in estrus versus diestrus assessed by semiquantitative qRT-PCR normalized to 18S rRNA. Two‐tailed, paired Student's t‐test was used to calculate statistical significance.


K Stereo microscopy of X ‐ gal ‐ and carmine alum ‐ stained mammary glands from ovariectomized Axin2 : : LacZ females treated for 72 h with vehicle ( n = 4 ) ( left ) , 17 ‐ β ‐ estradiol ( E2 ) ( n = 6 ) ( center ) , 17 ‐ β ‐ estradiol and progesterone ( E2 and P ) ( n = 8 ) ( right ) . Scale bar : 200 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

K Stereo microscopy of X‐gal‐ and carmine alum‐stained mammary glands from ovariectomized Axin2::LacZ females treated for 72 h with vehicle (n = 4) (left), 17‐β‐estradiol (E2) (n = 6) (center), 17‐β‐estradiol and progesterone (E2 and P) (n = 8) (right). Scale bar: 200 μm.


L , M Stereo microscopy of contralateral glands whole ‐ mounted and X ‐ gal stained after engraftment with mammary buds from Axin2 : : LacZ transgenic and Wnt4 + / + or Wnt4 − / − female E12 . 5 and E13 . 5 embryos ( L ) or Axin2 : : LacZ transgenic and either PR + / − or PR − / − 8 ‐ week ‐ old females ( M ) , at day 8 . 5 of pregnancy . β ‐ galactosidase expression reflecting Axin2 transcription is readily detected in PR + / − as well as Wnt4 + / + mammary epithelia but not in the PR − / − ( n = 6 ) and Wnt4 − / − counterparts ( n = 6 ) . Blue : X ‐ gal staining ; magenta : carmine alum counterstain . Scale bars : 200 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

L, M Stereo microscopy of contralateral glands whole‐mounted and X‐gal stained after engraftment with mammary buds from Axin2::LacZ transgenic and Wnt4+/+ or Wnt4−/− female E12.5 and E13.5 embryos (L) or Axin2::LacZ transgenic and either PR+/− or PR−/− 8‐week‐old females (M), at day 8.5 of pregnancy. β‐galactosidase expression reflecting Axin2 transcription is readily detected in PR+/− as well as Wnt4+/+ mammary epithelia but not in the PR−/− (n = 6) and Wnt4−/− counterparts (n = 6). Blue: X‐gal staining; magenta: carmine alum counterstain. Scale bars: 200 μm.


A X ‐ gal ‐ stained whole ‐ mounts of Axin2 : : LacZ embryos at E14 . 5 either Wnt4 + / + or Wnt4 − / − ( n = 5 or 4 , respectively ) . Arrows mark mammary buds . Scale bar : 5 mm .

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

A X‐gal‐stained whole‐mounts of Axin2::LacZ embryos at E14.5 either Wnt4+/+or Wnt4−/− (n = 5 or 4, respectively). Arrows mark mammary buds. Scale bar: 5 mm.


B Whole ‐ mount analysis of X ‐ gal ‐ and carmine alum ‐ stained mammary glands harvested from 6 ‐ week ‐ old littermates either Wnt4fl / wt ; MMTV : : Cre + ; Axin2 + / lacZ ( n = 5 ) or Wnt4fl / flMMTV : : Cre + ; Axin2 + / lacZ ( n = 4 ) . Left insets : higher magnification ; right insets : histology section of the same gland . Note expression of reporter detected around the neck of TEBs in Wnt4 ‐ deficient and littermate control is comparable . LN , lymph node . Scale bars : 1 mm , and inset scale bar : 100 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

B Whole‐mount analysis of X‐gal‐ and carmine alum‐stained mammary glands harvested from 6‐week‐old littermates either Wnt4fl/wt;MMTV::Cre+;Axin2+/lacZ (n = 5) or Wnt4fl/flMMTV::Cre+;Axin2+/lacZ (n = 4). Left insets: higher magnification; right insets: histology section of the same gland. Note expression of reporter detected around the neck of TEBs in Wnt4‐deficient and littermate control is comparable. LN, lymph node. Scale bars: 1 mm, and inset scale bar: 100 μm.


( A - D ) Serum NfL concentrations of preataxic ( green ) and ataxic ( red ) SCA3 subjects and controls ( blue ) were measured in two independent cohorts , each with a different Simoa approach : cohort # 1 , recruited by the ESMI consortium ( A , C ) , and cohort # 2 , recruited by the EuroSCA / RiSCA consortium ( B , D ) . Boxes show the ranges between lower and upper quartiles , the central bands show the medians , and the whiskers show data within 1 . 5 ∙ IQR of the median , with dots representing outliers . Groups were compared with Mann Whitney U tests ( * * * p < . 001 , * * p < . 01 , ns p ≥ . 05 , two - tailed , Bonferroni - corrected ; In the scatter plots , the individual NfL values were plotted as a function of subjects ' age . The dashed grey lines visualise the optimal cut - offs for differentiating ataxic SCA3 subjects from controls in each cohort ( cohort # 1 : 20 . 0 pg / ml , 98 . 7 % sensitivity , 92 . 2 % specificity ; cohort # 2 : 50 . 9 pg / ml , 92 . 6 % sensitivity , 100 % specificity ; cut - offs were derived by maximising Youden ' s index irrespective of age ) . Note the logarithmic scale of the y - axes .

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

(A-D) Serum NfL concentrations of preataxic (green) and ataxic (red) SCA3 subjects and controls (blue) were measured in two independent cohorts, each with a different Simoa approach: cohort #1, recruited by the ESMI consortium (A, C), and cohort #2, recruited by the EuroSCA/RiSCA consortium (B, D). Boxes show the ranges between lower and upper quartiles, the central bands show the medians, and the whiskers show data within 1.5∙IQR of the median, with dots representing outliers. Groups were compared with Mann Whitney U tests (*** p<.001, ** p<.01, ns p≥.05, two-tailed, Bonferroni-corrected; In the scatter plots, the individual NfL values were plotted as a function of subjects' age. The dashed grey lines visualise the optimal cut-offs for differentiating ataxic SCA3 subjects from controls in each cohort (cohort #1: 20.0 pg/ml, 98.7% sensitivity, 92.2% specificity; cohort #2: 50.9 pg/ml, 92.6% sensitivity, 100% specificity; cut-offs were derived by maximising Youden's index irrespective of age). Note the logarithmic scale of the y-axes.


( E , F ) Serum pNfH levels of preataxic and ataxic SCA3 subjects and controls were also measured in both cohorts , each with a different Simoa approach ( two - tailed Mann Whitney U tests , Bonferroni - corrected ;

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

(E, F) Serum pNfH levels of preataxic and ataxic SCA3 subjects and controls were also measured in both cohorts, each with a different Simoa approach (two-tailed Mann Whitney U tests, Bonferroni-corrected;


( A ) Serum NfL levels of ataxic SCA3 subjects significantly correlated with disease severity , as quantified by the Scale for the Rating and Assessment of Ataxia ( SARA ) score ( r = 0 . 43 , p < . 001 , Pearson ' s correlation , two - tailed test ) .

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

(A) Serum NfL levels of ataxic SCA3 subjects significantly correlated with disease severity, as quantified by the Scale for the Rating and Assessment of Ataxia (SARA) score (r=0.43, p<.001, Pearson's correlation, two-tailed test).


( B ) The cross - sectional NfL levels also reflected longitudinal disease progression , as quantified by the annual SARA score change ( available for 35 subjects ) . Boxes show the ranges between lower and upper quartiles , the central bands show the medians , and the whiskers show data within 1 . 5 ∙ IQR of the median , with dots representing outliers . Subjects with high disease progression ( annual SARA score increase ≥ 0 . 71 points / year , n = 18 , median split ) had significantly higher serum levels of NfL than subjects with low disease progression

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The cross-sectional NfL levels also reflected longitudinal disease progression, as quantified by the annual SARA score change (available for 35 subjects). Boxes show the ranges between lower and upper quartiles, the central bands show the medians, and the whiskers show data within 1.5∙IQR of the median, with dots representing outliers. Subjects with high disease progression (annual SARA score increase ≥0.71 points/year, n=18, median split) had significantly higher serum levels of NfL than subjects with low disease progression


( B ) The cross - sectional NfL levels also reflected longitudinal disease progression , as quantified by the annual SARA score change ( available for 35 subjects ) . Boxes show the ranges between lower and upper quartiles , the central bands show the medians , and the whiskers show data within 1 . 5 ∙ IQR of the median , with dots representing outliers . Subjects with high disease progression ( annual SARA score increase ≥ 0 . 71 points / year , n = 18 , median split ) had significantly higher serum levels of NfL than subjects with low disease

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


( C ) We modelled serum NfL levels ( log - transformed ) in SCA3 carriers ( n = 123 ) with the predictors age and ATXN3 CAG repeat length , their squares and all possible interactions ( for details , see Results and Methods ) . The highly significant predictors ( age , its square , and repeat length , and the intercept , all p < . 001 , explained variance : R2 = 0 . 37 ) were used to generate the diagram . For a given age , each increase in CAG repeat count was associated with higher NfL concentrations . The steepness of the slopes declined with increasing age . In controls ( black , n = 125 ) , the relation between NfL level ( log - transformed ) and age was linear .

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(C) We modelled serum NfL levels (log-transformed) in SCA3 carriers (n=123) with the predictors age and ATXN3 CAG repeat length, their squares and all possible interactions (for details, see Results and Methods). The highly significant predictors (age, its square, and repeat length, and the intercept, all p<.001, explained variance: R2=0.37) were used to generate the diagram. For a given age, each increase in CAG repeat count was associated with higher NfL concentrations. The steepness of the slopes declined with increasing age. In controls (black, n=125), the relation between NfL level (log-transformed) and age was linear.


( A ) Serum NfL levels in preataxic SCA3 subjects ( cross - sectional data ) were plotted over the time from the individually estimated ataxia onset . NfL levels increased significantly with proximity to the estimated onset ( F ( 1 , 21 ) = 39 . 68 , p < . 001 , R2 = 0 . 64 ) . For benchmarking the levels of preataxic subjects , the blue dashed line visualises the optimal cut - off for differentiating ataxic SCA3 subjects from controls in the pooled cohort ( 20 . 3 pg / ml , 99 . 0 % sensitivity , 95 . 2 % specificity ) .

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(A) Serum NfL levels in preataxic SCA3 subjects (cross-sectional data) were plotted over the time from the individually estimated ataxia onset. NfL levels increased significantly with proximity to the estimated onset (F(1,21)=39.68, p<.001, R2=0.64). For benchmarking the levels of preataxic subjects, the blue dashed line visualises the optimal cut-off for differentiating ataxic SCA3 subjects from controls in the pooled cohort (20.3 pg/ml, 99.0% sensitivity, 95.2% specificity).


( B ) To determine the time at which NfL levels become significantly increased in the preataxic stage of SCA3 , NfL levels of preataxic carriers need to be related to NfL levels of controls at the same age , as NfL levels physiologically increase with age . For each preataxic subject , the difference between the measured NfL value ( green dot ) and the NfL value predicted for control subjects of the same age ( solid blue line ) was visualised by the length of the vertical green line . Standardisation of this difference relative to the NfL distribution in controls

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(B) To determine the time at which NfL levels become significantly increased in the preataxic stage of SCA3, NfL levels of preataxic carriers need to be related to NfL levels of controls at the same age, as NfL levels physiologically increase with age. For each preataxic subject, the difference between the measured NfL value (green dot) and the NfL value predicted for control subjects of the same age (solid blue line) was visualised by the length of the vertical green line. Standardisation of this difference relative to the NfL distribution in controls


( B ) To determine the time at which NfL levels become significantly increased in the preataxic stage of SCA3 , NfL levels of preataxic carriers need to be related to NfL levels of controls at the same age , as NfL levels physiologically increase with age . For each preataxic subject , the difference between the measured NfL value ( green dot ) and the NfL value predicted for control subjects of the same age ( solid blue line ) was visualised by the length of the vertical green line . Standardisation of this difference relative to the NfL distribution in

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( C ) In preataxic SCA3 subjects , NfL z - scores increased significantly ( F ( 1 , 21 ) = 30 . 78 , p < . 001 , R2 = 0 . 58 ) with subjects approaching the expected age of onset . NfL levels of preataxic subjects were significantly increased compared to controls ( i . e . z - score > 1 . 96 ) 7 . 5 years before the expected onset , indicated by the non - overlapping 95 % confidence intervals of SCA3 subjects ( black solid line ) and controls ( blue dashed line ) .

(C) In preataxic SCA3 subjects, NfL z-scores increased significantly (F(1,21)=30.78, p<.001, R2=0.58) with subjects approaching the expected age of onset. NfL levels of preataxic subjects were significantly increased compared to controls (i.e. z-score >1.96) 7.5 years before the expected onset, indicated by the non-overlapping 95% confidence intervals of SCA3 subjects (black solid line) and controls (blue dashed line).


( A , B ) Longitudinal intraindividual stability of NfL ( A ) and pNfH ( B ) serum levels over 6 weeks , assessed in both ataxic ( red ) and preataxic ( green ) SCA3 and control ( blue ) subjects ( n = 21 ) . Data of the same individuals are linked by lines . ICC , intraclass correlation coefficient ( estimate and 95 % CI ) .

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(A, B) Longitudinal intraindividual stability of NfL (A) and pNfH (B) serum levels over 6 weeks, assessed in both ataxic (red) and preataxic (green) SCA3 and control (blue) subjects (n=21). Data of the same individuals are linked by lines. ICC, intraclass correlation coefficient (estimate and 95% CI).


( C ) Sample size estimations were performed for future intervention trials using the reduction of Nf levels towards control levels as outcome measure . The estimated number of subjects per study arm is plotted over the assumed therapeutic effect for lowering the Nf level in SCA3 subjects to levels observed in healthy controls ( NfL : dashed line , pNfH : dotted line ) .

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(C) Sample size estimations were performed for future intervention trials using the reduction of Nf levels towards control levels as outcome measure. The estimated number of subjects per study arm is plotted over the assumed therapeutic effect for lowering the Nf level in SCA3 subjects to levels observed in healthy controls (NfL: dashed line, pNfH: dotted line).


( A - B ) Plasma concentrations of NfL ( A ) and pNfH ( B ) were measured in the 304Q knock - in SCA3 mouse model . Heterozygous animals become symptomatic at ≈ 8 months of age .

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(A-B) Plasma concentrations of NfL (A) and pNfH (B) were measured in the 304Q knock-in SCA3 mouse model. Heterozygous animals become symptomatic at ≈8 months of age.


( C - F ) Tissue levels of soluble and aggregated mutant ataxin - 3 were measured in cerebellum ( C , E ) and frontal lobe ( D , F ) .

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(C-F) Tissue levels of soluble and aggregated mutant ataxin-3 were measured in cerebellum (C, E) and frontal lobe (D, F).


The figure shows the degree to which each variable differs between heterozygous SCA3 mice and wildtype controls for a given age . For each variable , we quantified the deviation of hetero zygous mice from controls of the same age by calculating the effect size r of the group comparison ( independent t - tests ) . An effect size of 0 indicates that the value in heterozygous mice is not different from controls , while an effect size of 1 would indicate strong abnormality . Note that the drop in the effect size of Nf levels from 12 months to 18 months thus does not imply a putative reduction of Nf levels , but rather the loss of the effect in mutants relative to controls , as controls show strong age - related Nf increases at 18 months of age . Negative effect size values were set to 0 .

The figure shows the degree to which each variable differs between heterozygous SCA3 mice and wildtype controls for a given age. For each variable, we quantified the deviation of heterozygous mice from controls of the same age by calculating the effect size r of the group comparison (independent t-tests). An effect size of 0 indicates that the value in heterozygous mice is not different from controls, while an effect size of 1 would indicate strong abnormality. Note that the drop in the effect size of Nf levels from 12 months to 18 months thus does not imply a putative reduction of Nf levels, but rather the loss of the effect in mutants relative to controls, as controls show strong age-related Nf increases at 18 months of age. Negative effect size values were set to 0.


( A ) We assessed samples of cerebellar cortex , deep cerebellar nuclei , frontal cortex , pons and pyramidal tract by immunohistochemistry with ataxin - 3 staining , comparing heterozygous 304Q SCA3 mice at 2 , 6 , 12 and 18 months of age with wildtype animals at 18 months . ML : molecular layer , PC : Purkinje cells , GL : granular layer , scale bars : 20 µm .

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(A) We assessed samples of cerebellar cortex, deep cerebellar nuclei, frontal cortex, pons and pyramidal tract by immunohistochemistry with ataxin-3 staining, comparing heterozygous 304Q SCA3 mice at 2, 6, 12 and 18 months of age with wildtype animals at 18 months. ML: molecular layer, PC: Purkinje cells, GL: granular layer, scale bars: 20 µm.


( B ) We histologically assessed the number and integrity of Purkinje cells in the cerebellar cortex by NfL , pNfH , Nissl and calbindin staining , comparing heterozygous 304Q SCA3 mice at 2 , 6 , 12 and 18 months of age with wildtype animals . ML : molecular layer , PC : Purkinje cells , GL : granular layer , scale bars : 20 µm .

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(B) We histologically assessed the number and integrity of Purkinje cells in the cerebellar cortex by NfL, pNfH, Nissl and calbindin staining, comparing heterozygous 304Q SCA3 mice at 2, 6, 12 and 18 months of age with wildtype animals. ML: molecular layer, PC: Purkinje cells, GL: granular layer, scale bars: 20 µm.


( B ) We histologically assessed the number and integrity of Purkinje cells in the cerebellar cortex by NfL , pNfH , Nissl and calbindin staining , comparing heterozygous 304Q SCA3 mice at 2 , 6 , 12 and 18 months of age with wildtype animals .

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(B) We histologically assessed the number and integrity of Purkinje cells in the cerebellar cortex by NfL, pNfH, Nissl and calbindin staining, comparing heterozygous 304Q SCA3 mice at 2, 6, 12 and 18 months of age with wildtype animals.


Select 2D class averages of the particles that were used to calculate the 2019 - nCoV S reconstruction ( left ) . Side and top views of the prefusion structure of the 2019 - nCoV S protein with a single RBD in the " up " conformation ( right ) . The two RBD " down " protomers are shown as cryo - EM density in either white or gray and the RBD " up " protomer is shown in ribbons , colored corresponding to the schematic in Fig 1A .

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Select 2D class averages of the particles that were used to calculate the 2019-nCoV S reconstruction (left). Side and top views of the prefusion structure of the 2019-nCoV S protein with a single RBD in the "up" conformation (right). The two RBD "down" protomers are shown as cryo-EM density in either white or gray and the RBD "up" protomer is shown in ribbons, colored corresponding to the schematic in Fig 1A.


SPR sensorgram showing the binding kinetics for human ACE2 and immobilized 2019 - nCoV S . Data are shown as black lines and the best fit of the data to a 1 : 1 binding model is shown in red .

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SPR sensorgram showing the binding kinetics for human ACE2 and immobilized 2019-nCoV S. Data are shown as black lines and the best fit of the data to a 1:1 binding model is shown in red.


Negative - stain EM 2D class averages of 2019 - nCoV S bound by ACE2 . Averages have been rotated so that ACE2 is positioned above the 2019 - nCoV S protein with respect to the viral membrane . A cartoon depicting the ACE2 - bound 2019 - nCoV S protein is shown ( right ) with ACE2 in blue and S protein monomers colored tan , pink and green .

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Negative-stain EM 2D class averages of 2019-nCoV S bound by ACE2. Averages have been rotated so that ACE2 is positioned above the 2019-nCoV S protein with respect to the viral membrane. A cartoon depicting the ACE2-bound 2019-nCoV S protein is shown (right) with ACE2 in blue and S protein monomers colored tan, pink and green.


A biolayer interferometry sensorgram that shows binding to ACE2 by the 2019 - nCoV RBD - SD1 . Binding data are shown as a black line and the best fit of the data to a 1 : 1 binding model is shown in red .

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A biolayer interferometry sensorgram that shows binding to ACE2 by the 2019-nCoV RBD-SD1. Binding data are shown as a black line and the best fit of the data to a 1:1 binding model is shown in red.


Biolayer interferometry to measure cross - reactivity of the SARS - CoV RBD - directed antibodies S230 , m396 and 80R . Sensortips with immobilized antibodies were dipped into wells containing 2019 - nCoV RBD - SD1 and the resulting data are shown as a black line .

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Biolayer interferometry to measure cross-reactivity of the SARS-CoV RBD-directed antibodies S230, m396 and 80R. Sensortips with immobilized antibodies were dipped into wells containing 2019-nCoV RBD-SD1 and the resulting data are shown as a black line.


( E ) . Relative luciferase activity of the sensor ( + ) reporter or sensor ( - ) reporter induced by expression vectors of the top 5 genes identified in the screen or the GFP vector in HEK293 - FT cells . Error bars show SD ; n = 3 . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * .

(E). Relative luciferase activity of the sensor (+) reporter or sensor (-) reporter induced by expression vectors of the top 5 genes identified in the screen or the GFP vector in HEK293-FT cells. Error bars show SD; n=3. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.


( F ) . Heat map of relative miRNA expression of various miRNAs in HEK - 293FT cells transfected with expression vectors of the top 5 genes from the screen , or the ctrl ( GFP ) vector . Red color represents suppressed expression compared with ctrl ( GFP ) . n = 3 .

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(F). Heat map of relative miRNA expression of various miRNAs in HEK-293FT cells transfected with expression vectors of the top 5 genes from the screen, or the ctrl (GFP) vector. Red color represents suppressed expression compared with ctrl (GFP). n=3.


( A ) . Volcano plot of TaqMan array showing the mean average expression and p - value of miRNA expression profiles between TruB1 KD and ctrl ( scramble ) . N = 3 . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test . MiRNAs suppressed ( Relative expression TruB1 KD / ctrl < 0 . 8 and p - value < 0 . 1 ) are enclosed by the red square .

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(A). Volcano plot of TaqMan array showing the mean average expression and p-value of miRNA expression profiles between TruB1 KD and ctrl (scramble). N=3. Significance was assessed using 2‐tailed Student's t‐test. MiRNAs suppressed (Relative expression TruB1 KD / ctrl <0.8 and p-value < 0.1) are enclosed by the red square.


( D ) . Relative expression of let - 7 family miRNAs and other miRNAs in HEK293FT cells with TruB1 KD or ctrl ( siRNA ) determined by qRT - PCR . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD .

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(D). Relative expression of let-7 family miRNAs and other miRNAs in HEK293FT cells with TruB1 KD or ctrl (siRNA) determined by qRT-PCR. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.


( E ) . Relative expression of primary - let - 7 family miRNAs as in ( D ) determined by qRT - PCR . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD .

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(E). Relative expression of primary-let-7 family miRNAs as in (D) determined by qRT-PCR. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.


( C ) . Relative miRNA expression of let - 7a in HEK - 293 cells infected with tetracycline - inducible expressing lentiviruses for TruB1 , mt1 , mt2 or GFP 5 days after doxycycline treatment , as determined by qRT - PCR . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD .

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(C). Relative miRNA expression of let-7a in HEK-293 cells infected with tetracycline-inducible expressing lentiviruses for TruB1, mt1, mt2 or GFP 5 days after doxycycline treatment, as determined by qRT-PCR. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.


( D ) . Northern blotting for let - 7a and RNU6B in HeLa cells infected with lentiviruses encoding tetracycline - inducible expression of TruB1 , mt1 , mt2 , or GFP , 5 days after doxycycline treatment .

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(D). Northern blotting for let-7a and RNU6B in HeLa cells infected with lentiviruses encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP, 5 days after doxycycline treatment.


( G ) . Location of pseudouridine sites detected by the CMC primer extension method . Total RNA purified from HEK - 293FT cells were treated with CMC . CMC treated RNA were reverse - transcribed with RI - labelled specific primers for tRNAphe or pri - let - 7b . ddATP was used for sequence control . Pseudouridines are indicated by black arrows .

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(G). Location of pseudouridine sites detected by the CMC primer extension method. Total RNA purified from HEK-293FT cells were treated with CMC. CMC treated RNA were reverse-transcribed with RI-labelled specific primers for tRNAphe or pri-let-7b. ddATP was used for sequence control. Pseudouridines are indicated by black arrows.


( A ) . RIP analysis of pri - let - 7a1 and Flag - TruB1 from HEK - 293FT cells . RNA was extracted from IP material and analyzed by qRT - PCR . HEK - 293FT cells were infected with lentiviruses encoding tetracycline - inducible expression of TruB1 , mt1 , mt2 , or GFP and treated with doxycycline . Error bars show SD ; n = 3 . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * .

(A). RIP analysis of pri-let-7a1 and Flag-TruB1 from HEK-293FT cells. RNA was extracted from IP material and analyzed by qRT-PCR. HEK-293FT cells were infected with lentiviruses encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP and treated with doxycycline. Error bars show SD; n=3. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.


( B ) . EMSA of 32p - ATP - labelled pri - let - 7a1 or pri - let - 7a1 loop mt mixed with recombinant TruB1 , mt1 or mt2 at several doses . RNP : Ribonucleoprotein complexes .

(B). EMSA of 32p-ATP-labelled pri-let-7a1 or pri-let-7a1 loop mt mixed with recombinant TruB1, mt1 or mt2 at several doses. RNP: Ribonucleoprotein complexes.


( E ) . Tag densities represented as dots . Density of miRNA clusters in HITS - CLIP were normalized by miRNA expression as determined by TaqMan array .

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(E). Tag densities represented as dots. Density of miRNA clusters in HITS-CLIP were normalized by miRNA expression as determined by TaqMan array.


( A ) . In vitro processing assay for RI - labelled pri - let - 7a1 . Autoradiographs of gels showing pri - let - 7a1 treated with whole cell lysate ( WCL ) from HEK - 293FT cells transfected with GFP , TruB1 , mt1 , or mt2 ( overexpression , left ) , and TruB1 KD or ctrl ( siRNA , right ) . Dicer KD was used to show the height of pre - let - 7a1 . ( B ) RI intensities of ( A ) were quantified and normalized to ctrl . Relative processing rate of pri - let - 7a1 into pre - and mature - are shown . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD .

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(A). In vitro processing assay for RI-labelled pri-let-7a1. Autoradiographs of gels showing pri-let-7a1 treated with whole cell lysate (WCL) from HEK-293FT cells transfected with GFP, TruB1, mt1, or mt2 (overexpression, left), and TruB1 KD or ctrl (siRNA, right). Dicer KD was used to show the height of pre-let-7a1. (B) RI intensities of (A) were quantified and normalized to ctrl. Relative processing rate of pri-let-7a1 into pre- and mature- are shown. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.


( C ) . RIP assay of pri - let - 7a1 and DGCR8 from HEK - 293FT cells . Western blotting for input or immunoprecipitate ( IP ) using anti - DGCR8 antibody and anti - actin antibody are shown on top . RNA was extracted from IP material and analyzed by qRT - PCR ( bottom ) . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . ( D ) . RIP assay of pri - let - 7a1 and Flag - tagged TruB1 from HEK - 293FT cells with Lin28B KD or ctrl ( siRNA ) . Western blotting for input or IP material using anti - Flag antibody , anti - Lin28B antibody , and anti - actin antibody are shown on top . RNA was extracted from IP material and analyzed by qRT - PCR ( bottom ) . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars

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(C). RIP assay of pri-let-7a1 and DGCR8 from HEK-293FT cells. Western blotting for input or immunoprecipitate (IP) using anti-DGCR8 antibody and anti-actin antibody are shown on top. RNA was extracted from IP material and analyzed by qRT-PCR (bottom). Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. (D). RIP assay of pri-let-7a1 and Flag-tagged TruB1 from HEK-293FT cells with Lin28B KD or ctrl (siRNA). Western blotting for input or IP material using anti-Flag antibody, anti-Lin28B antibody, and anti-actin antibody are shown on top. RNA was extracted from IP material and analyzed by qRT-PCR (bottom). Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show