( c ) Atg7 − / − brown adipose tissues ( BAT ) display decreased ERK phosphorylation . Immunoblots for indicated proteins in Hom from Con and Atg7 − / − BAT . The bars represent mean ± s . e . m . * * P0 . 01 compared with Con ; Student ' s t - test , n = 4 - 6 .

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(c) Atg7−/− brown adipose tissues (BAT) display decreased ERK phosphorylation. Immunoblots for indicated proteins in Hom from Con and Atg7−/−BAT. The bars represent mean±s.e.m. **P0.01 compared with Con; Student's t-test, n=4-6.


( d ) Atg7 − / − livers exhibit decreased nuclear ERK phosphorylation . Immunoblots show the indicated proteins in homogenate ( Hom , lane 1 ) and nuclear fractions ( lanes 2 - 5 ) from Con and Atg7 − / − livers . The bars represent mean ± s . e . m . * * * P0 . 001 compared with Con ; Student ' s t - test , n = 4 - 5 .

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(d) Atg7−/− livers exhibit decreased nuclear ERK phosphorylation. Immunoblots show the indicated proteins in homogenate (Hom, lane 1) and nuclear fractions (lanes 2-5) from Con and Atg7−/− livers. The bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test, n=4-5.


( e ) LC3 C terminus glycine - deleted ( ΔG ) mutants exhibit decreased ERK phosphorylation . Immunoblots for indicated proteins in total lysates from CFP - LC3 - and CFP - LC3ΔG - transfected NIH / 3T3 cells exposed or not to EGF ( 10 min ) . The bars represent mean ± s . e . m . * * * P0 . 001 compared with Con ; Student ' s t - test , n = 8 .

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(e) LC3 C terminus glycine-deleted (ΔG) mutants exhibit decreased ERK phosphorylation. Immunoblots for indicated proteins in total lysates from CFP-LC3- and CFP-LC3ΔG-transfected NIH/3T3 cells exposed or not to EGF (10 min). The bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test, n=8.


( a ) Atg5 − / − MEFs display reduced ERK phosphorylation during nutrient deprivation . Quantification of P - ERK levels normalized to total ERK ( detected by immunoblotting ) in WT and Atg5 − / − MEFs cultured in the absence of serum for indicated times . The bars represent mean ± s . e . m . * P0 . 05 compared with corresponding WT value ; * * * P0 . 001 , * * * * P0 . 0001 compared with 10 min serum - starved Atg5 − / − MEFs ; ANOVA - Bonferroni post hoc test , n = 3 .

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(a) Atg5−/− MEFs display reduced ERK phosphorylation during nutrient deprivation. Quantification of P-ERK levels normalized to total ERK (detected by immunoblotting) in WT and Atg5−/− MEFs cultured in the absence of serum for indicated times. The bars represent mean±s.e.m. *P0.05 compared with corresponding WT value; ***P0.001, ****P0.0001 compared with 10 min serum-starved Atg5−/− MEFs; ANOVA-Bonferroni post hoc test, n=3.


( b ) Serum - deprived Atg5 − / − MEFs display decreased ERK and p90RSK phosphorylation . Immunoblots for the indicated proteins in total lysates from 2 h serum - deprived WT and Atg5 − / − MEFs . The bars represent mean ± s . e . m . * P0 . 05 , * * * P0 . 001 compared with Con ; Student ' s t - test , n = 3 .

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(b) Serum-deprived Atg5−/− MEFs display decreased ERK and p90RSK phosphorylation. Immunoblots for the indicated proteins in total lysates from 2 h serum-deprived WT and Atg5−/− MEFs. The bars represent mean±s.e.m. *P0.05, ***P0.001 compared with Con; Student's t-test, n=3.


( c ) Atg5 − / − MEFs display decreased nuclear ERK activity . Immunoblots for phosphorylated HA ( haemagglutinin ) - tagged Elk1 in total lysates from 2 h serum - deprived WT and Atg5 − / − MEFs cells exposed or not to EGF ( 10 min ) .

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(c) Atg5−/− MEFs display decreased nuclear ERK activity. Immunoblots for phosphorylated HA (haemagglutinin)-tagged Elk1 in total lysates from 2 h serum-deprived WT and Atg5−/− MEFs cells exposed or not to EGF (10 min).


( d ) Atg5 − / − MEFs display decreased Elk1 - driven gene expression . Elk1 - driven ZFP36 mRNA levels from 2 hserum - deprived WT and Atg5 − / − MEFs cells exposed or not to EGF ( 10 min ) . The bars represent mean ± s . e . m . * * * P0 . 001 compared with Con ; Student ' s t - test , n = 3 . (

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(d) Atg5−/− MEFs display decreased Elk1-driven gene expression. Elk1-driven ZFP36 mRNA levels from 2 hserum-deprived WT and Atg5−/− MEFs cells exposed or not to EGF (10 min). The bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test, n=3. (


( e ) Decreased ERK phosphorylation in Atg5 − / − MEFs occurs independently of changes in ERK phosphatases . Immunoblots for the indicated proteins in 2 h serum - deprived WT MEFs transfected with scrambled siRNA ( scr ) , and Atg5 − / − MEFs transfected with scr or siRNAs against MKP3 or PP2A in the presence or absence of EGF . The bars represent mean ± s . e . m . , n = 3 .

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(e) Decreased ERK phosphorylation in Atg5−/− MEFs occurs independently of changes in ERK phosphatases. Immunoblots for the indicated proteins in 2 h serum-deprived WT MEFs transfected with scrambled siRNA (scr), and Atg5−/− MEFs transfected with scr or siRNAs against MKP3 or PP2A in the presence or absence of EGF. The bars represent mean±s.e.m., n=3.


( a ) Mutations in FRS on ERK2 decrease colocalization of ERK2 with ATG5 - ATG12 , LC3 and WIPI1 . Immunofluorescence ( IF ) showing colocalization ( depicted as white pixels by ' colocalization finder application ' ) of WT - ERK2 - HA , FRS ERK2 mutants ( L198A - , L232A - , L198A / L232A - , Y261A - ERK2 - HA ) or common docking ( CD ) mutant ( D319N - ERK2 - HA ) with ATG5 - ATG12 ( panels 1 - 6 ) , LC3 ( panels 7 - 12 ) or WIPI1 ( panels 13 - 18 ) in EGF - treated NIH / 3T3 cells . ERK2 is stained in red , and autophagy proteins are stained in green . Scale bar , 10 μm . The bars represent mean ± s . e . m . * * P0 . 01 , * * * * P0 . 0001 compared with WT - ERK2 - transfected cells ; Student ' s t - test , 50 cells analysed from n = 2 . (

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(a) Mutations in FRS on ERK2 decrease colocalization of ERK2 with ATG5-ATG12, LC3 and WIPI1. Immunofluorescence (IF) showing colocalization (depicted as white pixels by 'colocalization finder application') of WT-ERK2-HA, FRS ERK2 mutants (L198A-, L232A-, L198A/L232A-, Y261A-ERK2-HA) or common docking (CD) mutant (D319N-ERK2-HA) with ATG5-ATG12 (panels 1-6), LC3 (panels 7-12) or WIPI1 (panels 13-18) in EGF-treated NIH/3T3 cells. ERK2 is stained in red, and autophagy proteins are stained in green. Scale bar, 10 μm. The bars represent mean±s.e.m. **P0.01, ****P0.0001 compared with WT-ERK2-transfected cells; Student's t-test, 50 cells analysed from n=2. (


( b ) Mutations in FRS on ERK2 decrease colocalization of ERK2 with nuclearATG5 - ATG12 or LC3 . IF showing colocalization ( white pixels ) of WT - ERK2 - HA ( red ) , FRS ERK2 mutants ( L198A - , L232A - , L198A / L232A - , Y261A - ERK2 - HA ) or common docking ( CD ) mutant ( D319N - ERK2 - HA ) with ATG5 - ATG12 ( panels 1 - 6 ) or LC3 - II ( panels 7 - 12 ) in EGF - treated NIH / 3T3 cells . Scale bar , 5 μm . The bars represent mean ± s . e . m . * * P0 . 01 , * * * P0 . 001 , * * * * P0 . 0001 compared with WT - ERK2 - transfected cells ; Student ' s t - test , 50 cells analysed from n = 2 . Arrows depict colocalization .

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(b) Mutations in FRS on ERK2 decrease colocalization of ERK2 with nuclearATG5-ATG12 or LC3. IF showing colocalization (white pixels) of WT-ERK2-HA (red), FRS ERK2 mutants (L198A-, L232A-, L198A/L232A-, Y261A-ERK2-HA) or common docking (CD) mutant (D319N-ERK2-HA) with ATG5-ATG12 (panels 1-6) or LC3-II (panels 7-12) in EGF-treated NIH/3T3 cells. Scale bar, 5 μm. The bars represent mean±s.e.m. **P0.01, ***P0.001, ****P0.0001 compared with WT-ERK2-transfected cells; Student's t-test, 50 cells analysed from n=2. Arrows depict colocalization.


( c ) ATG5 - ATG12 conjugation is required for ERK phosphorylation . Immunoblots for P - ERK , total ERK and β - actin in NIH / 3T3 cells transfected with WT ATG5 or the conjugation - defective ATG5 K130R mutant and treated with EGF ( 10 min ) . The bars represent mean ± s . e . m . * P0 . 05 compared with WT ATG5 - transfected cells ; Student ' s t - test , n = 3 .

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(c) ATG5-ATG12 conjugation is required for ERK phosphorylation. Immunoblots for P-ERK, total ERK and β-actin in NIH/3T3 cells transfected with WT ATG5 or the conjugation-defective ATG5 K130R mutant and treated with EGF (10 min). The bars represent mean±s.e.m. *P0.05 compared with WT ATG5-transfected cells; Student's t-test, n=3.


a ) ATG4B depletion increases steady - state LC3 - II levels . Immunoblots for indicated proteins in NIH / 3T3 cells transfected with scrambled siRNAs ( scr ) or siRNAs against ATG4B ( siATG4B ) in presence / absence of EGF . Bars represent mean ± s . e . m . * P0 . 05 , * * * P0 . 001 compared with scr ; Student ' s t - test , n = 4 .

a) ATG4B depletion increases steady-state LC3-II levels. Immunoblots for indicated proteins in NIH/3T3 cells transfected with scrambled siRNAs (scr) or siRNAs against ATG4B (siATG4B) in presence/absence of EGF. Bars represent mean±s.e.m. *P0.05, ***P0.001 compared with scr; Student's t-test, n=4.


( b ) siATG4B cells exhibit increased autophagic vesicles . Representative electron micrographs depicting autophagic vesicles ( indicated by black arrows ) in scr and siATG4B NIH / 3T3 cells .

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(b) siATG4B cells exhibit increased autophagic vesicles. Representative electron micrographs depicting autophagic vesicles (indicated by black arrows) in scr and siATG4B NIH/3T3 cells.


( c ) ATG4B depletion does not increase autophagic flux in serum - fed cells . Immunoblots for indicated proteins in lysates from scr or siATG4B NIH / 3T3 cells in presence / absence of lysosomal inhibitors , ammonium chloride and leupeptin ( Inh ) for 2 h . Scale bars , 1 μm . Bars represent mean ± s . e . m . , n = 3 .

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(c) ATG4B depletion does not increase autophagic flux in serum-fed cells. Immunoblots for indicated proteins in lysates from scr or siATG4B NIH/3T3 cells in presence/absence of lysosomal inhibitors, ammonium chloride and leupeptin (Inh) for 2 h. Scale bars, 1 μm. Bars represent mean±s.e.m., n=3.


( d ) siATG4B cells display increased nuclear LC3 - II . Images for LC3 - II ( red ) in scr or siATG4B NIH / 3T3 cells in presence / absence of EGF ( 10 min ) . Scale bar , 5 μm . Bars represent mean ± s . e . m . * * * P0 . 001 compared with scr ; Student ' s t - test , 60 cells from n = 2 .

(d) siATG4B cells display increased nuclear LC3-II. Images for LC3-II (red) in scr or siATG4B NIH/3T3 cells in presence/absence of EGF (10 min). Scale bar, 5 μm. Bars represent mean±s.e.m. ***P0.001 compared with scr; Student's t-test, 60 cells from n=2.


( e ) ATG4B deficiency increases P - ERK / LC3 - II colocalization . Immunofluorescence ( IF ) for P - ERK ( green ) / LC3 ( red ) colocalization in scr or siATG4B NIH / 3T3 cells in presence / absence of EGF ( 10 min ) . Scale bars , 10 μm . Bars represent mean ± s . e . m . * * P0 . 01 , * * * P0 . 001 compared with scr ; Student ' s t - test , 60 cells from n = 2 .

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(e) ATG4B deficiency increases P-ERK/LC3-II colocalization. Immunofluorescence (IF) for P-ERK (green)/LC3 (red) colocalization in scr or siATG4B NIH/3T3 cells in presence/absence of EGF (10 min). Scale bars, 10 μm. Bars represent mean±s.e.m. **P0.01, ***P0.001 compared with scr; Student's t-test, 60 cells from n=2.


( f ) ATG4B - deficient cells display increased nuclear P - ERK / LC3 colocalization . IF depicting P - ERK ( green ) / LC3 ( red ) colocalization in nuclei of scr or siATG4B NIH / 3T3 cells in presence / absence of EGF . Scale bars , 5 μm . Bars represent mean ± s . e . m . * * P0 . 01 , * * * P0 . 001 compared with scr ; Student ' s t - test , 60 cells from n = 2 .

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(f) ATG4B-deficient cells display increased nuclear P-ERK/LC3 colocalization. IF depicting P-ERK (green)/LC3 (red) colocalization in nuclei of scr or siATG4B NIH/3T3 cells in presence/absence of EGF. Scale bars, 5 μm. Bars represent mean±s.e.m. **P0.01, ***P0.001 compared with scr; Student's t-test, 60 cells from n=2.


( g ) ATG4B deficiency augments EGF - induced MEK and ERK phosphorylation . Immunoblots for P - and total MEK and ERK , LC3 and GAPDH in total lysates from scr or siATG4B NIH / 3T3 cells in presence / absence of EGF . Bars represent mean ± s . e . m . , n = 3 .

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(g) ATG4B deficiency augments EGF-induced MEK and ERK phosphorylation. Immunoblots for P- and total MEK and ERK, LC3 and GAPDH in total lysates from scr or siATG4B NIH/3T3 cells in presence/absence of EGF. Bars represent mean±s.e.m., n=3.


h ) ATG4B deficiency increases nuclear P - ERK levels . Native ( top ) / inverted images ( bottom ) showing nuclear P - ERK content . P - ERK fluorescence in untreated ( Con ) / EGF - treated scr or siATG4B NIH / 3T3 cells is shown . Scale bars , 5 μm . Bars represent mean ± s . e . m . * * P0 . 01 , * * * P0 . 001 compared with scr ; Student ' s t - test , 60 cells from n = 2 . Nuclei are blue ( DAPI ) .

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h) ATG4B deficiency increases nuclear P-ERK levels. Native (top)/inverted images (bottom) showing nuclear P-ERK content. P-ERK fluorescence in untreated (Con)/EGF-treated scr or siATG4B NIH/3T3 cells is shown. Scale bars, 5 μm. Bars represent mean±s.e.m. **P0.01, ***P0.001 compared with scr; Student's t-test, 60 cells from n=2. Nuclei are blue (DAPI).


A HEK 293 cells were transfected with mCherry - tagged Parkin and FLAG - tagged UBQLN2 for 24 h , and then were treated with 5 µg / mL Antimycin A and Oligomycin A ( A / O ) for 4 h and 24 h . The cells were stained with anti - FLAG ( green ) antibody and anti - TOM20 ( gray ) , and then were visualized using confocal microscopy . Scale bars , 10 µm . Insets were higher magnifications of the dashed box area . Scale bars , 2 . 5 µm . B , C The dashed square is magnified as shown in the boxed region in A with corresponding line scan indicating Parkin and UBQLN2 co - localization on the depolarized mitochondria ( the line scan indicates pixel intensity of Parkin and UBQLN2 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HEK 293 cells were transfected with mCherry-tagged Parkin and FLAG-tagged UBQLN2 for 24 h, and then were treated with 5 µg/mL Antimycin A and Oligomycin A (A/O) for 4 h and 24 h. The cells were stained with anti-FLAG (green) antibody and anti-TOM20 (gray), and then were visualized using confocal microscopy. Scale bars, 10 µm. Insets were higher magnifications of the dashed box area. Scale bars, 2.5 µm. B, C The dashed square is magnified as shown in the boxed region in A with corresponding line scan indicating Parkin and UBQLN2 co-localization on the depolarized mitochondria (the line scan indicates pixel intensity of Parkin and UBQLN2).


D HEK 293 cells were transfected with mCherry - tagged Parkin and EGFP - tagged UBQLN2 . 24 h after transfection , cells were treated with 1 µg / mL A / O for 4 h , and then were harvested for the isolation of mitochondria . Whole cell ( Total ) , cytosol ( Cyto ) and mitochondria ( Mito ) were lysed and analyzed by western blotting with the indicated antibodies .

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D HEK 293 cells were transfected with mCherry-tagged Parkin and EGFP-tagged UBQLN2. 24 h after transfection, cells were treated with 1 µg/mL A/O for 4 h, and then were harvested for the isolation of mitochondria. Whole cell (Total), cytosol (Cyto) and mitochondria (Mito) were lysed and analyzed by western blotting with the indicated antibodies.


A Domain structure of Parkin in upper region . HEK 293 cells were transfected with HA - tagged UBQLN2 and FLAG - tagged Parkin WT or mutants ( K161N , T240R , C418R , C431F and P437L ) for 24 h , and then were treated with 5 µg / mL A / O for 5 h . The cells were stained with anti - HA ( green ) and FLAG ( red ) antibodies . Then the cells were visualized using confocal microscopy . Scale bars , 10 µm . Lower panel were higher magnifications of the dashed box area . Scale bars , 2 . 5 µm . B , C The quantification data of the Parkin or UBQLN2 with mitochondrial localization in A . At least 60 anti - HA - UBQLN2 or anti - FLAG - Parkin positive cells were counted for each sample . Data from three independent experiments were represented as means ± SD . , ns , not significantly different ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , one - way ANOVA .

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A Domain structure of Parkin in upper region. HEK 293 cells were transfected with HA-tagged UBQLN2 and FLAG-tagged Parkin WT or mutants (K161N, T240R, C418R, C431F and P437L) for 24 h, and then were treated with 5 µg/mL A/O for 5 h. The cells were stained with anti-HA (green) and FLAG (red) antibodies. Then the cells were visualized using confocal microscopy. Scale bars, 10 µm. Lower panel were higher magnifications of the dashed box area. Scale bars, 2.5 µm. B, C The quantification data of the Parkin or UBQLN2 with mitochondrial localization in A. At least 60 anti-HA-UBQLN2 or anti-FLAG-Parkin positive cells were counted for each sample. Data from three independent experiments were represented as means ± SD., ns, not significantly different; **, p<0.01; ***, p<0.001, one-way ANOVA.


B HEK 293 cells were transfected with indicated siRNA . After 48 h , the cells were re - transfected with FLAG - Parkin and mt - mKeima for 17 h . Then the cells were treated with 5 µg / mL A / O for 5 h , and visualized using confocal microscopy . Intracellular mt - mKeima signal excited at 458 nm ( measuring mitochondria with a neutral pH ) was shown in green color , while red color indicated the mt - mKeima fluorescence excited at 561 nm ( measuring mitochondria with an acidic pH ) in the same cell . Scale bars , 10 µm .

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B HEK 293 cells were transfected with indicated siRNA. After 48 h, the cells were re-transfected with FLAG-Parkin and mt-mKeima for 17 h. Then the cells were treated with 5 µg/mL A/O for 5 h, and visualized using confocal microscopy. Intracellular mt-mKeima signal excited at 458 nm (measuring mitochondria with a neutral pH) was shown in green color, while red color indicated the mt-mKeima fluorescence excited at 561 nm (measuring mitochondria with an acidic pH) in the same cell. Scale bars, 10 µm.


D The cells were transfected and treated as B . Cells were analyzed by FACS . The percentage of cells with lysosomal positive mt - mKeima are shown on the representative FACS data . Data from three independent experiments were represented as means ± SD . , * * , p < 0 . 01 ; * * * , p < 0 . 001 , one - way ANOVA .

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D The cells were transfected and treated as B. Cells were analyzed by FACS. The percentage of cells with lysosomal positive mt-mKeima are shown on the representative FACS data. Data from three independent experiments were represented as means ± SD., **, p<0.01; ***, p<0.001, one-way ANOVA.


G HEK 293 cells were transfected with indicated siRNAs . After 24 h , the cells were re - transfected with FLAG tag or FLAG - tagged UBQLN2WT or ∆ UBA , along with mCherry - Parkin for 24 h . The cells then were treated with or without 1 µg / mL A / O for 24 h . The cell lysates were subjected to immunoblot with indicated antibodies .

G HEK 293 cells were transfected with indicated siRNAs. After 24 h, the cells were re-transfected with FLAG tag or FLAG-tagged UBQLN2WT or ∆UBA, along with mCherry-Parkin for 24 h. The cells then were treated with or without 1 µg/mL A/O for 24 h. The cell lysates were subjected to immunoblot with indicated antibodies.


A Domain structure of UBQLN2 in upper region . HEK 293 cells were transfected with FLAG - tagged UBQLN2 - WT or ALS - linked mutants ( ∆ UBA , ∆ UBL , P497H or P509S ) , along with mCherry - Parkin . After 24 h , the cells were treated with 5 µg / mL A / O for 5 h . The cells were stained with anti - FLAG ( green ) and anti - TOM20 ( gray ) antibodies , and then were visualized using confocal microscopy . Scale bars , 10 µm . Insets were higher magnifications of the dashed box area . Scale bars , 2 . 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Domain structure of UBQLN2 in upper region. HEK 293 cells were transfected with FLAG-tagged UBQLN2-WT or ALS-linked mutants (∆UBA, ∆UBL, P497H or P509S), along with mCherry-Parkin. After 24 h, the cells were treated with 5 µg/mL A/O for 5 h. The cells were stained with anti-FLAG (green) and anti-TOM20 (gray) antibodies, and then were visualized using confocal microscopy. Scale bars, 10 µm. Insets were higher magnifications of the dashed box area. Scale bars, 2.5 µm.


C The cells were transfected with mCherry - Parkin and HA - tagged UBQLN2 - WT or ALS - mutants as A and subjected to immunofluorescent assay as in A , but were treated with 1 µg / mL A / O for 24 h . Scale bar , 10 µm . Yellow arrow indicates uncleared mitochondria .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The cells were transfected with mCherry-Parkin and HA-tagged UBQLN2-WT or ALS-mutants as A and subjected to immunofluorescent assay as in A, but were treated with 1 µg/mL A/O for 24 h. Scale bar, 10 µm. Yellow arrow indicates uncleared mitochondria.


E HEK 293T cells were transfected with indicated siRNAs . After 24 h , the cells were re - transfected with FLAG - tagged UBQLN2 - WT or mutants , along with mCherry - Parkin for 24h . Then the cells were treated with or without 1 µg / mL A / O for 18 h . The cell lysates were subjected to immunoblot with indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HEK 293T cells were transfected with indicated siRNAs. After 24 h, the cells were re-transfected with FLAG-tagged UBQLN2-WT or mutants, along with mCherry-Parkin for 24h. Then the cells were treated with or without 1 µg/mL A/O for 18 h. The cell lysates were subjected to immunoblot with indicated antibodies.


A HEK 293 cells were transfected with mCherry - tagged Parkin , HA - tagged UBQLN2 , FLAG - tagged HSP70 and BFP - mito for 24 h , and were then treated with 5 µg / mL A / O for 4 h . The cells were stained with anti - HA ( green ) and FLAG ( gray ) antibodies , and were visualized using confocal microscopy . Scale bar , 10 µm . Right panel were higher magnifications of the dashed box area . Scale bars , 2 . 5 µm .

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

A HEK 293 cells were transfected with mCherry-tagged Parkin, HA-tagged UBQLN2, FLAG-tagged HSP70 and BFP-mito for 24 h, and were then treated with 5 µg/mL A/O for 4 h. The cells were stained with anti-HA (green) and FLAG (gray) antibodies, and were visualized using confocal microscopy. Scale bar, 10 µm. Right panel were higher magnifications of the dashed box area. Scale bars, 2.5 µm.


D HEK 293T cells expressing HA - tagged UBQLN2 and FLAG - tagged HSP70 ( WT or ΔC ) were incubated with 5 µg / mL A / O for 5 h . The cells were then subjected to immunoprecipitation assay using anti - FLAG antibody .

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

D HEK 293T cells expressing HA-tagged UBQLN2 and FLAG-tagged HSP70 (WT or ΔC) were incubated with 5 µg/mL A/O for 5 h. The cells were then subjected to immunoprecipitation assay using anti-FLAG antibody.


E HEK 293T cells expressing HA tagged UBQLN2 - WT , ΔUBA or P497H ) and FLAG - tagged HSP70 were incubated with 5 µg / mL A / O for 5 h . Immunoprecipitation assay were performed using UBQLN2 antibody .

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

E HEK 293T cells expressing HA tagged UBQLN2-WT, ΔUBA or P497H) and FLAG-tagged HSP70 were incubated with 5 µg/mL A/O for 5 h. Immunoprecipitation assay were performed using UBQLN2 antibody.


G The quantification data of UBQLN2 recruitment to mitochondria after 5 µg / mL A / O treatment . HEK 293T cells were transfected with indicated siRNAs . The cells were re - transfected with HA - tagged UBQLN2 - WT , mCherry - Parkin , along with BFP - mito , and treated with 5 µg / mL A / O for 5 h . The cells were subjected to immunofluorescent assay using anti - HA antibody . Data from three independent experiments were represented as means ± SD . , Sixty cells were counted for each sample . * * , p < 0 . 01 , one - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The quantification data of UBQLN2 recruitment to mitochondria after 5 µg/mL A/O treatment. HEK 293T cells were transfected with indicated siRNAs. The cells were re-transfected with HA-tagged UBQLN2-WT, mCherry-Parkin, along with BFP-mito, and treated with 5 µg/mL A/O for 5 h. The cells were subjected to immunofluorescent assay using anti-HA antibody. Data from three independent experiments were represented as means ± SD., Sixty cells were counted for each sample. **, p<0.01, one-way ANOVA.


A HEK 293T cells expressing mCherry - Parkin were pre - treated with proteasome inhibitor ( 20 µM MG132 ) for 12 h , and treated with 1 µg / mL A / O for 2 h . Then the cells were fractionated and proteins were immunoblotted for relative antibodies . B The level of Mfn2 ( OMM protein ) , TOM20 , COXIV and HSP60 was quantified and normalized relative to GAPDH , and was shown in A . The data from three independent experiments were presented as mean ± SD . , ns , not significantly different ; * * , p < 0 . 01 ; * * * , p < 0 . 001 , one - way ANOVA .

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

A HEK 293T cells expressing mCherry-Parkin were pre-treated with proteasome inhibitor (20 µM MG132) for 12 h, and treated with 1 µg/mL A/O for 2 h. Then the cells were fractionated and proteins were immunoblotted for relative antibodies. B The level of Mfn2 (OMM protein), TOM20, COXIV and HSP60 was quantified and normalized relative to GAPDH, and was shown in A. The data from three independent experiments were presented as mean± SD., ns, not significantly different; **, p<0.01; ***, p<0.001, one-way ANOVA.


HEK 293T cells were transfected with indicated siRNAs for 48 h . C The cells were re - transfected with mCherry - Parkin for 12 h and were pre - treated with or without 20 µM MG132 for 12 h and treated with1 µg / mL A / O for 2 h . The cell lysates were subjected to immunoblot with indicated antibodies . The data from three independent experiments were presented as mean ± SD . , ns , not significantly different ; * , p < 0 . 05 ; * * , p < 0 . 01 , one - way ANOVA .

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

HEK 293T cells were transfected with indicated siRNAs for 48 h. C The cells were re-transfected with mCherry-Parkin for 12 h and were pre-treated with or without 20 µM MG132 for 12 h and treated with1 µg/mL A/O for 2 h. The cell lysates were subjected to immunoblot with indicated antibodies. The data from three independent experiments were presented as mean± SD., ns, not significantly different; *, p<0.05; **, p<0.01, one-way ANOVA.


HEK 293T cells were transfected with indicated siRNAs for 48 h . The cells were transfected with HA - tagged UBQLN2 - WT or ∆ 379 - 462 , along with mCherry - Parkin for 12 h . Then the cells were treated with 1 µg / mL A / O for 2 h . The cell lysates were subjected to immunoblot using indicated antibodies . G Relative densities from F were shown in G . The data from three independent experiments were presented as means ± SD . , ns , not significantly different ; * , p < 0 . 05 ; * * , p < 0 . 01 , one - way ANOVA .

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

HEK 293T cells were transfected with indicated siRNAs for 48 h. The cells were transfected with HA-tagged UBQLN2-WT or ∆379-462, along with mCherry-Parkin for 12 h. Then the cells were treated with 1 µg/mL A/O for 2 h. The cell lysates were subjected to immunoblot using indicated antibodies. G Relative densities from F were shown in G. The data from three independent experiments were presented as means ± SD., ns, not significantly different; *, p<0.05; **, p<0.01, one-way ANOVA.


HEK 293T cells were transfected with indicated siRNAs for 48 h . H The cells were re - transfected with HA - tagged UBQLN2 - WT , along with mCherry - Parkin for 12 h . Then the cells were pretreated with 20 µM MG132 for 12 h and treated with 5 µg / mL A / O for 4 h . Mitochondria isolated from cell lysates were treated with or without 10 µg / mL proteinase K ( Pro . K ) and then were subjected to immunoblot with indicated antibodies . Mito : the isolated mitochondria ; Mito ( Pro . K ) : mitochondria after Pro . K treatment . COXIV level in " Mito ( Pro . K ) " group was quantified and normalized relative to HSP60 in " Mito group " . The data from three independent experiments were presented as mean ± SD . , * * p < 0 . 01 , * * * , p < 0 . 001 , one - way ANOVA .

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

HEK 293T cells were transfected with indicated siRNAs for 48 h. H The cells were re-transfected with HA-tagged UBQLN2-WT, along with mCherry-Parkin for 12 h. Then the cells were pretreated with 20 µM MG132 for 12 h and treated with 5 µg/mL A/O for 4 h. Mitochondria isolated from cell lysates were treated with or without 10 µg/mL proteinase K (Pro.K) and then were subjected to immunoblot with indicated antibodies. Mito: the isolated mitochondria; Mito (Pro.K): mitochondria after Pro.K treatment. COXIV level in "Mito (Pro.K)" group was quantified and normalized relative to HSP60 in "Mito group". The data from three independent experiments were presented as mean± SD., ** p<0.01, ***, p<0.001, one-way ANOVA.


HEK 293T cells were transfected with indicated siRNAs for 48 h . I The cells were re - transfected with HA , HA - UBQLN2 - WT or P497H , along with GFP - Parkin and FLAG - PHB2 for 20 h , and then were treated with or without 5 µg / mL A / O for 4 h . Images of situ PLA using rabbit anti - LC3B and mouse anti - FLAG antibodies . Blue : nuclei ( DAPI ) ; white dots : PLA positive puncta . Scale bar , 10 µm . J Over 500 cells were counted for each sample in I . The data from three independent experiments were presented as mean ± SD . , * , p < 0 . 05 ; * * p < 0 . 01 , * * * , p < 0 . 001 , one - way ANOVA .

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

HEK 293T cells were transfected with indicated siRNAs for 48 h. I The cells were re-transfected with HA, HA-UBQLN2-WT or P497H, along with GFP-Parkin and FLAG-PHB2 for 20 h, and then were treated with or without 5 µg/mL A/O for 4 h. Images of situ PLA using rabbit anti-LC3B and mouse anti-FLAG antibodies. Blue: nuclei (DAPI); white dots: PLA positive puncta. Scale bar, 10 µm. J Over 500 cells were counted for each sample in I. The data from three independent experiments were presented as mean± SD., *, p<0.05; ** p<0.01, ***, p<0.001, one-way ANOVA.


A Mouse cortical neurons ( DIV 7 ) were infected with lentivirus carrying mCherry - Parkin for 45 h , and then the neurons were treated with 5 µg / mL A / O for 5 h and subjected to immunofluorescent assay using anti - UBQLN2 ( green ) and anti - HSP60 ( gray ) antibodies . Scale bars , 10 µm . Lower panel were higher magnifications of the dashed box area . Scale bar , 2 . 5 µm .

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

A Mouse cortical neurons (DIV 7) were infected with lentivirus carrying mCherry-Parkin for 45 h, and then the neurons were treated with 5 µg/mL A/O for 5 h and subjected to immunofluorescent assay using anti-UBQLN2 (green) and anti-HSP60 (gray) antibodies. Scale bars, 10 µm. Lower panel were higher magnifications of the dashed box area. Scale bar, 2.5 µm.


Mouse cortical neurons ( DIV 7 ) were transfected with indicated siRNAs for 24 h . B The neurons were infected with lentivirus carrying mCherry - Parkin for 24 h and then were treated with 1 µg / mL A / O for 48 h . The neurons were stained with DAPI , anti - UBQLN2 ( green ) and anti - HSP60 ( gray ) antibodies and visualized . Scale bars , 10 µm . Yellow arrows indicate uncleared mitochondria .

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

Mouse cortical neurons (DIV 7) were transfected with indicated siRNAs for 24 h. B The neurons were infected with lentivirus carrying mCherry-Parkin for 24 h and then were treated with 1 µg/mL A/O for 48 h. The neurons were stained with DAPI, anti-UBQLN2 (green) and anti-HSP60 (gray) antibodies and visualized. Scale bars, 10 µm. Yellow arrows indicate uncleared mitochondria.


Mouse cortical neurons ( DIV 7 ) were transfected with indicated siRNAs for 24 h . F The neurons were infected with lentivirus carrying mCherry - Parkin for 36 h and were treated with 100 ng / mL Antimycin A ( Antimycin A alone , AA ) and 1 µM DFP for 10 h . The neurons were subjected to immunofluorescent assay using anti - TOM20 ( magenta ) and anti - LAMP 2 ( gray ) antibodies . Scale bars , 10 µm . Right panel were higher magnifications of the dashed box area . Scale bars , 2 . 5 µm . Yellow arrows indicate the co - localization between mitochondria and lysosomes .

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

Mouse cortical neurons (DIV 7) were transfected with indicated siRNAs for 24 h. F The neurons were infected with lentivirus carrying mCherry-Parkin for 36 h and were treated with 100 ng/mL Antimycin A (Antimycin A alone, AA) and 1 µM DFP for 10 h. The neurons were subjected to immunofluorescent assay using anti-TOM20 (magenta) and anti-LAMP 2 (gray) antibodies. Scale bars, 10 µm. Right panel were higher magnifications of the dashed box area. Scale bars, 2.5 µm. Yellow arrows indicate the co-localization between mitochondria and lysosomes.


Mouse cortical neurons ( DIV 7 ) were transfected with indicated siRNAs for 24 h . H The neurons were infected with lentivirus carrying mCherry - Parkin for 24 h . Then , the neurons were treated with 100 ng / mL Antimycin A and 1 µM DFP for another 10 h and subjected to immunofluorescent assay using anti - MAP2 antibody . Scale bars , 10 µm . I , J The neurite length ( I ) and cell death ( J ) in more than 30 neurons were quantified from each group in H . The data from three independent experiments were presented as the means ± SD . , * , p < 0 . 05 ; * * * , p < 0 . 001 , one - way ANOVA .

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

Mouse cortical neurons (DIV 7) were transfected with indicated siRNAs for 24 h. H The neurons were infected with lentivirus carrying mCherry-Parkin for 24 h. Then, the neurons were treated with 100 ng/mL Antimycin A and 1 µM DFP for another 10 h and subjected to immunofluorescent assay using anti-MAP2 antibody. Scale bars, 10 µm. I, J The neurite length (I) and cell death (J) in more than 30 neurons were quantified from each group in H. The data from three independent experiments were presented as the means ± SD., *, p<0.05; ***, p<0.001, one-way ANOVA.


a , b , LDH release , IL - 1β secretion ( a ) and immunoblots for caspase - 1 , caspase - 11 , IL - 1β , IL - 18 and IL - 1α ( b )

O O O O O O O O O O O O O O O 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, LDH release, IL-1β secretion (a) and immunoblots for caspase-1, caspase-11, IL-1β, IL-18 and IL-1α(b)


and immunoblots for caspase - 1 , caspase - 11 , IL - 1β , IL - 18 and IL - 1α ( b ) from unprimed BMDMs infected for 16h with the indicated bacteria ( grown to stationary phase ) .

and immunoblots for caspase-1, caspase-11, IL-1β, IL-18 and IL-1α (b) from unprimed BMDMs infected for 16h with the indicated bacteria (grown to stationary phase).


c , Time course measuring LDH release from unprimed or IFN - γ - primed BMDMs infected with S .

c, Time course measuring LDH release from unprimed or IFN-γ-primed BMDMs infected with S. typhimurium


d , e , LDH release and IL - 1β secretion from primed BMDMs infected with SPI - 1 - expressing logarithmic phase S . typhimurium , treated with monosodium urate , alum and nigericin or transfected with poly ( dA : dT ) and LPS .

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

d, e, LDH release and IL-1β secretion from primed BMDMs infected with SPI-1-expressing logarithmic phase S. typhimurium, treated with monosodium urate, alum and nigericin or transfected with poly(dA:dT) and LPS.


LDH release and IL - 1β secretion from unprimed wild - type and Gbp2 − / − BMDMs infected for 16h with the indicated bacteria ( grown to stationary phase ) . Graphs show mean and s . d . of quadruplicate wells and data are representative of two ( b ) and three ( a , c - f ) independent experiments . * Crossreactive band ; * * P < 0 . 01 ; NS , not significant ( two - tailed t - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LDH release and IL-1β secretion from unprimed wild-type and Gbp2−/− BMDMs infected for 16h with the indicated bacteria (grown to stationary phase). Graphs show mean and s.d. of quadruplicate wells and data are representative of two (b) and three (a, c-f) independent experiments. *Crossreactive band; **P<0.01; NS, not significant (two-tailed t-test).


a , b , Quantification of live ( mCherry - positive ) and dead ( mCherry - negative ) S . typhimurium per cell by immunofluorescence ( a ) or as percent of total by flow -

O O O O O O O O O O O O O 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, Quantification of live (mCherry-positive) and dead (mCherry-negative) S. typhimurium per cell by immunofluorescence (a) or as percent of total by flow-cytometry


a , b , Quantification of live ( mCherry - positive ) and dead ( mCherry - negative ) S . typhimurium per cell by immunofluorescence ( a ) or as percent of total by flow - cytometry ( b ) in unprimed BMDMs at 16 h post - infection .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Quantification of live (mCherry-positive) and dead (mCherry-negative) S. typhimurium per cell by immunofluorescence (a) or as percent of total by flow-cytometry (b) in unprimed BMDMs at 16 h post-infection.


c , Immunostaining for GBP2 and quantification of live and dead Salmonella at 4 h post - infection . Arrowheads , bacteria shown in insets .

O O 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, Immunostaining for GBP2 and quantification of live and dead Salmonella at 4 h post-infection. Arrowheads, bacteria shown in insets.


d , Quantification of GBP - positive bacteria , LDH release and IL - 1β secretion at indicated time points from BMDMs infected with Salmonella , live or killed by different means .

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

d, Quantification of GBP-positive bacteria, LDH release and IL-1β secretion at indicated time points from BMDMs infected with Salmonella, live or killed by different means.


e , Immunohistochemistry for GBP2 and Salmonella on spleen tissue from Salmonella ( mCherry - positive ) - infected mice ( representative of n = 3 per group ) . S . tm . , S . typhimurium .

e, Immunohistochemistry for GBP2 and Salmonella on spleen tissue from Salmonella (mCherry-positive)-infected mice (representative of n = 3 per group). S. tm., S. typhimurium.


f , g , Quantification of GBP - positive Salmonella in anti - IFN - γ - treated or control animals ( f ) and live and dead bacteria among GBP2 - negative / - positive Salmonella ( g ) ( n = 3 per group ) . Scale bars , 10 µm ( c ) , 1 µm ( e ) . Graphs show mean and 5 - 95 percentile ( box plots ) or s . d . of technical triplicates , and data are representative of three independent experiments . * P 0 . 05 , * * P 0 . 01 ( two - tailed t - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f, g, Quantification of GBP-positive Salmonella in anti-IFN-γ-treated or control animals (f) and live and dead bacteria among GBP2-negative/-positive Salmonella (g) (n = 3 per group). Scale bars, 10 µm (c), 1 µm (e). Graphs show mean and 5-95 percentile (box plots) or s.d. of technical triplicates, and data are representative of three independent experiments. *P  0.05, **P  0.01 (two-tailed t-test).


a , b , Unprimed BMDMs infected with S . typhimurium for 4 h and immunostained for LC3 and GBP2 . Arrowheads , bacteria shown in insets . Scale bars , 10 & amp ; amp ; mgr ; m .

O O O O O O O O O O O O O O O O O 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, Unprimed BMDMs infected with S. typhimurium for 4 h and immunostained for LC3 and GBP2. Arrowheads, bacteria shown in insets. Scale bars, 10 &amp;mgr;m.


. c , Quantification of results from b .

. c, Quantification of results from b.


b . d - g , LDH release and immunoblots for caspase - 1 and caspase - 11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3 - methyladenine ( 3 - MA ) .

O O O O O O O O O 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

b. d-g, LDH release and immunoblots for caspase-1 and caspase-11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3-methyladenine (3-MA).


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

b. d-g, LDH release and immunoblots for caspase-1 and caspase-11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3-methyladenine (3-MA).


h , i , LDH release and IL - 1b secretion from BMDMs infected for 16 h or transfected with LPS . Graphs show mean and s . d . of quadruplicate wells and data are representative of two ( e , i ) and three ( a - d , f - h ) independent experiments . * P 0 . 05 , * * P 0 . 01 ; NS , not significant ( two - tailed t - test ) .

h, i, LDH release and IL-1b secretion from BMDMs infected for 16 h or transfected with LPS. Graphs show mean and s.d. of quadruplicate wells and data are representative of two (e, i) and three (a-d, f-h) independent experiments. *P  0.05, **P  0.01; NS, not significant (two-tailed t-test).


a , Quantification of galectin - 8 - positive Salmonella in unprimed BMDMs at 4 h post - infection .

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

a, Quantification of galectin-8-positive Salmonella in unprimed BMDMs at 4 h post-infection. b


c , Unprimed BMDMs infected with S . typhimurium for 4 h and immunostained for galectin - 8 , GBP2 and LC3 . Arrowheads , bacteria shown in insets . Scale bars , 10 µm . d , Quantification of galectin - 8 / LC3 - double - positive Salmonella at indicated time points post - infection .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Unprimed BMDMs infected with S. typhimurium for 4 h and immunostained for galectin-8, GBP2 and LC3. Arrowheads, bacteria shown in insets. Scale bars, 10 µm. d, Quantification of galectin-8/LC3-double-positive Salmonella at indicated time points post-infection.


e - h , Quantification of cytosolic and vacuolar bacteria by flow cytometry in BMDMs infected with mCherry - positive S . typhimurium ( e - g ) or S . flexneri ( h , wild - type or ΔT3SS ) for 4 h . Graphs show mean and s . d . or 5 - 95 percentile ( Box plots ) of technical triplicates . Data are representative of 2 ( g , h ) , 3 ( a - d ) and 4 ( e , f ) independent experiments . * P 0 . 05 , * * P 0 . 01 ( two - tailed t - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-h, Quantification of cytosolic and vacuolar bacteria by flow cytometry in BMDMs infected with mCherry-positive S. typhimurium (e-g) or S. flexneri (h, wild-type or ΔT3SS) for 4 h. Graphs show mean and s.d. or 5-95 percentile (Box plots) of technical triplicates. Data are representative of 2 (g, h), 3 (a-d) and 4 (e, f) independent experiments. *P  0.05, **P  0.01 (two-tailed t-test).


Determination of serum testosterone ( B ) and 17β - estradiol levels , in post - pubescent male , castrated male ( CtM ) , and female Hanwoo ( n = 5 in each group ) .

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

Determination of serum testosterone (B) and 17β-estradiol levels, in post-pubescent male, castrated male (CtM), and female Hanwoo (n = 5 in each group).


Determination of the body weight , in post - pubescent male , castrated male ( CtM ) , and female Hanwoo ( n = 5 in each group ) .

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

Determination of the body weight , in post-pubescent male, castrated male (CtM), and female Hanwoo (n = 5 in each group).


D . PCoA of the rectal bacterial 16S rRNA gene sequences based on the weighted UniFrac distance matrix .

D. PCoA of the rectal bacterial 16S rRNA gene sequences based on the weighted UniFrac distance matrix.


E . Discriminant taxa of the male and CtM rectal microbiota , as determined by the linear discriminant analysis effect size ( LEfSe ) . The different abundances are represented by LDA scores .

O O O O O O O O O O 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. Discriminant taxa of the male and CtM rectal microbiota, as determined by the linear discriminant analysis effect size (LEfSe). The different abundances are represented by LDA scores.


F . The serum metabolome profiles were analyzed using GC - TOF - MS and clustered by PCoA based on the Bray - Curtis dissimilarity matrix .

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

F. The serum metabolome profiles were analyzed using GC-TOF-MS and clustered by PCoA based on the Bray-Curtis dissimilarity matrix.


G . The relative abundances of serum metabolites displayed as bar graphs ( n = 5 in each group ) .

G. The relative abundances of serum metabolites displayed as bar graphs (n = 5 in each group).


H . The BCAA serum levels quantified enzymatically , as described in the materials and methods section .

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

H. The BCAA serum levels quantified enzymatically, as described in the materials and methods section.


A . Serum testosterone levels in the adult male and CtM Hanwoo ( n = 10 in each group ) . The scattered dots in the bar graph represent the individual replicates .

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

A. Serum testosterone levels in the adult male and CtM Hanwoo (n = 10 in each group). The scattered dots in the bar graph represent the individual replicates.


PCoA , based on the weighted UniFrac distance matrix , of the bacterial 16S rRNA gene sequence data for the rumen , ileum , and colon , shown for the different segments of the gastrointestinal tract ( B )

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

PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the rumen, ileum, and colon, shown for the different segments of the gastrointestinal tract (B)


C . PCoA , based on the weighted UniFrac distance matrix , of the bacterial 16S rRNA gene sequence data or the rumen , ileum , and colon , combined data ( C ) . D . Microbial dissimilarity ( calculated based on the weighted UniFrac distance matrix , 100 values in each group ) between the male and CtM groups in the different intestinal compartments . The lines , boxes , and whiskers in the box plot diagrams represent the median , first and third quartiles , and min - to - max distribution of replicate values , respectively .

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

C. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data or the rumen, ileum, and colon, combined data (C). D. Microbial dissimilarity (calculated based on the weighted UniFrac distance matrix, 100 values in each group) between the male and CtM groups in the different intestinal compartments. The lines, boxes, and whiskers in the box plot diagrams represent the median, first and third quartiles, and min-to-max distribution of replicate values, respectively.


E . The relative abundances of abundant bacterial taxa ( > 0 . 5 % of the mean abundance ) .

E. The relative abundances of abundant bacterial taxa (> 0.5% of the mean abundance).


B . The intestinal branched - chain amino acid ( BCAA ) levels in the adult male and CtM Hanwoo ( n = 10 in each group ) . BCAA levels in the luminal contents of the ileum , cecum , and colon were quantified by an enzymatic method using a BCAA assay kit . The lines , boxes , and whiskers in the box plot diagrams represent the median , first and third quartiles , and min - to - max distribution of replicate values , respectively . The scattered dots in the box plot diagrams represent the individual replicates .

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

B. The intestinal branched-chain amino acid (BCAA) levels in the adult male and CtM Hanwoo (n = 10 in each group). BCAA levels in the luminal contents of the ileum, cecum, and colon were quantified by an enzymatic method using a BCAA assay kit. The lines, boxes, and whiskers in the box plot diagrams represent the median, first and third quartiles, and min-to-max distribution of replicate values, respectively. The scattered dots in the box plot diagrams represent the individual replicates.


A . Representative images of dressed bodies of the adult male and CtM Hanwoo ( n = 10 in each group ) .

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

A. Representative images of dressed bodies of the adult male and CtM Hanwoo (n = 10 in each group).


B . Dressed body weight , thickness of the dorsal subcutaneous fat , total weight of the extramuscular fat ( posterior subcutaneous fat , mesenteric fat , and retroperitoneal fat ) , and serum levels of the branched - chain amino acids ( BCAAs ) .

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

B. Dressed body weight, thickness of the dorsal subcutaneous fat, total weight of the extramuscular fat (posterior subcutaneous fat, mesenteric fat, and retroperitoneal fat), and serum levels of the branched-chain amino acids (BCAAs).


C . Representative images of fresh striploin muscle from the adult male and CtM carcasses ( n = 5 in each group ) .

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. Representative images of fresh striploin muscle from the adult male and CtM carcasses (n = 5 in each group).


D . The intramuscular fat area in size - normalized muscle .

D. The intramuscular fat area in size-normalized muscle.


E . The intramuscular metabolome profiles analyzed using GC - TOF - MS and clustered by PCoA based on the Bray - Curtis dissimilarity

E. The intramuscular metabolome profiles analyzed using GC-TOF-MS and clustered by PCoA based on the Bray-Curtis dissimilarity matrix


F , G . The relative abundances of intramuscular metabolites ( F ) and β - hydroxybutyrate ( 3 - HB ) ( G ) in the male and CtM samples .

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

F, G. The relative abundances of intramuscular metabolites (F) and β-hydroxybutyrate (3-HB) (G) in the male and CtM samples.


H . The serum and mucle ketone body levels , quantified enzymatically , as described in the Materials and Methods section ( n = 10 in each group ) .

H. The serum and mucle ketone body levels, quantified enzymatically, as described in the Materials and Methods section (n = 10 in each group).


C . PCoA , based on the weighted UniFrac distance matrix , of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon , shown according to the different diets and antibiotic treatment .

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

C. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon, shown according to the different diets and antibiotic treatment.


D . The relative abundances of the family Peptostreptococcaceae are displayed as a box and dot plot .

D. The relative abundances of the family Peptostreptococcaceae are displayed as a box and dot plot.


E . The serum branched - chain amino acid ( BCAA ) levels quantified enzymatically , as described in the Materials and Methods section .

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

E. The serum branched-chain amino acid (BCAA) levels quantified enzymatically, as described in the Materials and Methods section.


F . The total weight of the extramuscular fat , including posterior subcutaneous fat , mesenteric fat , and retroperitoneal fat .

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

F. The total weight of the extramuscular fat, including posterior subcutaneous fat, mesenteric fat, and retroperitoneal fat.


G , H . Representative images ( G ) and weight ( H ) of the hindlimb intermuscular fat . The images are from a longitudinal section of the hind leg . The fat weight data are presented as a percentage of the body weight .

O O O O O O O O O O O O O O O O O O O O O O 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, H. Representative images (G) and weight (H) of the hindlimb intermuscular fat. The images are from a longitudinal section of the hind leg. The fat weight data are presented as a percentage of the body weight.


I . Serum and hindlimb muscle ketone body levels quantified enzymatically .

I. Serum and hindlimb muscle ketone body levels quantified enzymatically.


B . The serum testosterone levels in the Sham - R and castrated male ( CtM ) - R mice .

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

B. The serum testosterone levels in the Sham-R and castrated male (CtM)-R mice.


C . PCoA , based on the weighted UniFrac distance matrix , of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon .

O O O O O O 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. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon.


E . The relative abundances of the family Peptostreptococcaceae are displayed as a box and dot plot .

E. The relative abundances of the family Peptostreptococcaceae are displayed as a box and dot plot.


F . Weights of the posterior subcutaneous fat , epididymal fat , mesenteric fat , and retroperitoneal fat in the Sham - R and CtM - R mice .

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

F. Weights of the posterior subcutaneous fat, epididymal fat, mesenteric fat, and retroperitoneal fat in the Sham-R and CtM-R mice.


G , H . Representative images ( G ) and weight ( H ) of the hindlimb intermuscular fat in the Sham - R and CtM - R mice . Images were obtained from a longitudinal section of the hind leg . The fat weight data are presented as a percentage of the body weight .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, H. Representative images (G) and weight (H) of the hindlimb intermuscular fat in the Sham-R and CtM-R mice. Images were obtained from a longitudinal section of the hind leg. The fat weight data are presented as a percentage of the body weight.


I . Serum and hindlimb muscle ketone body levels , quantified enzymatically .

I. Serum and hindlimb muscle ketone body levels, quantified enzymatically.


B . Body weight gain in response to the different levels of dietary BCAAs .

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

B. Body weight gain in response to the different levels of dietary BCAAs.


C . Representative images of gross anatomy .

C. Representative images of gross anatomy.


D . The total weight of the extramuscular fat ( posterior subcutaneous fat , epididymal fat , mesenteric fat , and retroperitoneal fat ) in the mice fed high - fat diet ( HFD ) with or without BCAAs .

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

D. The total weight of the extramuscular fat (posterior subcutaneous fat, epididymal fat, mesenteric fat, and retroperitoneal fat) in the mice fed high-fat diet (HFD) with or without BCAAs.


E . The serum BCAA levels in the mice fed HFD with or without BCAAs .

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

E. The serum BCAA levels in the mice fed HFD with or without BCAAs.


F . PCoA , based on the weighted UniFrac distance matrix , of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon are shown . The body weight gain data are presented as a percentage of the initial body weight . The fat weight data are presented as a percentage of the body weight .

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

F. PCoA, based on the weighted UniFrac distance matrix, of the bacterial 16S rRNA gene sequence data for the luminal contents of the ileum and colon are shown. The body weight gain data are presented as a percentage of the initial body weight. The fat weight data are presented as a percentage of the body weight.


G . Serum and hindlimb muscle ketone body levels , quantified enzymatically .

G. Serum and hindlimb muscle ketone body levels, quantified enzymatically.


A - E Double - immunolabeling of Sororin ( green ) and SYCP3 ( red ) on spread spermatocytes at ( A ) zygotene ( Zyg . ) , ( B ) pachytene ( Pac . ) , ( C ) diplotene ( Dip . ) , ( D ) late diplotene ( L . dip . ) , and ( E ) prometaphase I ( ProM I ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread spermatocytes at (A) zygotene (Zyg.), (B) pachytene (Pac.), (C) diplotene (Dip.), (D) late diplotene (L. dip.), and (E) prometaphase I (ProM I).


F - H Double - immunolabeling of Sororin ( green ) and SYCP1 ( pseudocoloured in pink ) on spread spermatocytes at ( F ) zygotene , ( G ) pachytene , and ( H ) diplotene .

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

F-H Double-immunolabeling of Sororin (green) and SYCP1 (pseudocoloured in pink) on spread spermatocytes at (F) zygotene, (G) pachytene, and (H) diplotene.


I - K Double - immunolabeling of Sororin ( green ) and HORMAD1 ( pseudocoloured in blue ) on spread spermatocytes at ( I ) zygotene , ( J ) pachytene , and ( K ) diplotene . The sex bivalent ( XY ) and their pseudoautosomal regions ( arrows ) , as well as the round body ( arrowheads ) are indicated . Scale bar : 10 µm .

I-K Double-immunolabeling of Sororin (green) and HORMAD1 (pseudocoloured in blue) on spread spermatocytes at (I) zygotene, (J) pachytene, and (K) diplotene. The sex bivalent (XY) and their pseudoautosomal regions (arrows), as well as the round body (arrowheads) are indicated. Scale bar: 10 µm.


A , C Double - immunolabeling of Sororin ( green ) and SYCP3 ( red ) on spread spermatocytes at ( A ) early zygotene ( E . zyg . ) and ( C ) late zygotene ( L . zyg . ) . B , D Double - immunolabeling of SMC3 ( pseudocoloured in cyan ) and SYCP3 ( red ) on spread spermatocytes at ( B ) early zygotene and ( D ) late zygotene .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,C Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread spermatocytes at (A) early zygotene (E. zyg.) and (C) late zygotene (L. zyg.).B,D Double-immunolabeling of SMC3 (pseudocoloured in cyan) and SYCP3 (red) on spread spermatocytes at (B) early zygotene and (D) late zygotene.


E Double - immunolabeling of Sororin ( green ) and REC8 - myc ( pseudocoloured in purple ) on a spread mid zygotene spermatocyte . The sex univalents ( X , Y ) are indicated . Scale bar : 10 µm .

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

E Double-immunolabeling of Sororin (green) and REC8-myc (pseudocoloured in purple) on a spread mid zygotene spermatocyte. The sex univalents (X,Y) are indicated. Scale bar: 10 µm.


Double - immunolabeling of Sororin ( green ) and SYCP3 ( red ) on spread pachytene - like ( Pac . - like ) and zygotene - like ( Zyg . - like ) spermatocytes of different genotypes . Unsynapsed axial elements of sex chromosomes ( X , Y ) are indicated . Arrowheads indicate nucleolar - associated round bodies . Scale bar : 10 µm .

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

Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread pachytene-like (Pac.-like) and zygotene-like (Zyg.-like) spermatocytes of different genotypes. Unsynapsed axial elements of sex chromosomes (X,Y) are indicated. Arrowheads indicate nucleolar-associated round bodies. Scale bar: 10 µm.


Double - immunolabeling of Sororin ( green ) and SYCP3 ( red ) , and counterstaining of the chromatin with DAPI ( blue ) on spread spermatocytes at ( A ) metaphase I ( M I ) , ( G ) anaphase I ( A I ) , ( H ) interkinesis ( Int . ) , ( I ) prophase II ( Pro . II ) , and ( J ) metaphase II ( M II ) . The sex bivalent ( XY ) is indicated . Arrows indicate the centriolar area at cell poles . Arrowheads indicate the Sororin labeling at the centromere of Y chromosome . B - F Selected metaphase I autosomal ( B and C ) and sex ( E and F ) bivalents . The sex chromosomes ( X , Y ) are indicated . K , L Enlarged metaphase II centromeres . Scale bar : 10 µm .

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

Double-immunolabeling of Sororin (green) and SYCP3 (red), and counterstaining of the chromatin with DAPI (blue) on spread spermatocytes at (A) metaphase I (M I), (G) anaphase I (A I), (H) interkinesis (Int.), (I) prophase II (Pro. II), and (J) metaphase II (M II). The sex bivalent (XY) is indicated. Arrows indicate the centriolar area at cell poles. Arrowheads indicate the Sororin labeling at the centromere of Y chromosome.B-F Selected metaphase I autosomal (B and C) and sex (E and F) bivalents. The sex chromosomes (X,Y) are indicated.K,L Enlarged metaphase II centromeres. Scale bar: 10 µm.


Double - immunolabeling of Sororin ( green ) and kinetochores ( ACA serum , red ) , and counterstaining of the chromatin with DAPI ( blue ) on spread spermatocytes at ( A ) metaphase I ( M I ) , ( B ) metaphase II ( M II ) , and ( C ) anaphase II ( A II ) . Arrowheads indicate the cell poles . The centromere of the Y chromosome and chromatids ( Y ) is indicated . Scale bar : 10 µm .

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

Double-immunolabeling of Sororin (green) and kinetochores (ACA serum, red), and counterstaining of the chromatin with DAPI (blue) on spread spermatocytes at (A) metaphase I (M I), (B) metaphase II (M II), and (C) anaphase II (A II). Arrowheads indicate the cell poles. The centromere of the Y chromosome and chromatids (Y) is indicated. Scale bar: 10 µm.


Double - immunolabeling of Sororin ( green ) and SYCP3 ( red ) , and counterstaining of the chromatin with DAPI ( blue ) on spread Sgo2 - / - spermatocytes at ( A ) pachytene ( Pac . ) , ( B ) diplotene ( Dip . ) , ( C ) metaphase I ( M I ) , and ( D ) metaphase II - like ( M II - like ) . The sex bivalent ( XY ) is indicated . Arrowheads indicate round bodies . Arrows in ( A ) indicate the pseudoautomal region in the sex bivalent , and the centriolar area at cell poles in ( C , D ) . Scale bar : 10 µm .

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

Double-immunolabeling of Sororin (green) and SYCP3 (red), and counterstaining of the chromatin with DAPI (blue) on spread Sgo2-/- spermatocytes at (A) pachytene (Pac.), (B) diplotene (Dip.), (C) metaphase I (M I), and (D) metaphase II-like (M II-like). The sex bivalent (XY) is indicated. Arrowheads indicate round bodies. Arrows in (A) indicate the pseudoautomal region in the sex bivalent, and the centriolar area at cell poles in (C,D). Scale bar: 10 µm.


Double - immunolabeling of Sororin ( green ) and SYCP3 ( red ) on spread cultured wild - type control ( WT Control ) , and okadaic acid treated wild - type ( WT + OA ) and Smc1β - / - ( Smc1β - / - + OA ) spermatocytes . A - B WT control cultured spermatocytes in ( A ) late zygotene ( L . zyg . ) and ( B ) metaphase I ( M I ) . Arrows indicate the cell poles . C - D WT cultured spermatocytes treated with okadaic acid in ( C ) late zygotene ( L . zyg . ) and ( D ) metaphase I - like ( M I - like ) . E - F Smc1β - / - cultured spermatocytes treated with okadaic acid in ( E ) pachytene - like ( Pac . - like ) and ( F ) metaphase I - like ( M I - like ) . Arrowheads indicate nucleolar - associated round bodies . The unsynapsed axial elements of the sex chromosomes ( X , Y ) in prophase I , as well as the sex bivalent ( XY ) in metaphases I are indicated . Scale bar : 10 µm .

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

Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread cultured wild-type control (WT Control), and okadaic acid treated wild-type (WT + OA) and Smc1β-/- (Smc1β-/- + OA) spermatocytes.A-B WT control cultured spermatocytes in (A) late zygotene (L. zyg.) and (B) metaphase I (M I). Arrows indicate the cell poles.C-D WT cultured spermatocytes treated with okadaic acid in (C) late zygotene (L. zyg.) and (D) metaphase I-like (M I-like).E-F Smc1β-/- cultured spermatocytes treated with okadaic acid in (E) pachytene-like (Pac.-like) and (F) metaphase I-like (M I-like). Arrowheads indicate nucleolar-associated round bodies. The unsynapsed axial elements of the sex chromosomes (X,Y) in prophase I, as well as the sex bivalent (XY) in metaphases I are indicated. Scale bar: 10 µm.


( A ) Western blot analysis of whole head homogenates from transgenic flies expressing a Myc - tagged PINK1 genomic construct and a UAS - RNAi construct driven by elav - GAL4 . The RNAi constructs used were as follows : the non - specific control mCherry - RNAi ( Control - R ) , Lon - RNAi1 ( Lon - R1 ) , and Lon - RNAi2 ( Lon - R2 ) . An anti - Myc antibody was used to detect PINK1 and an anti - Actin antibody was used as a protein loading control . ( B ) Densitometry of the PINK1 - Myc bands from the indicated genotypes was performed using Fiji software . The PINK1 - Myc band intensities were then normalized to their respective Actin loading controls , and these ratios were in turn normalized to the Control - R PINK1 / Actin ratio .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of whole head homogenates from transgenic flies expressing a Myc-tagged PINK1 genomic construct and a UAS-RNAi construct driven by elav-GAL4. The RNAi constructs used were as follows: the non-specific control mCherry-RNAi (Control-R), Lon-RNAi1 (Lon-R1), and Lon-RNAi2 (Lon-R2). An anti-Myc antibody was used to detect PINK1 and an anti-Actin antibody was used as a protein loading control. (B) Densitometry of the PINK1-Myc bands from the indicated genotypes was performed using Fiji software. The PINK1-Myc band intensities were then normalized to their respective Actin loading controls, and these ratios were in turn normalized to the Control-R PINK1/Actin ratio.


( C ) Western blot analysis of Lon protease ( Lon ) , Mitochondrial transcription factor A ( TFAM ) , Heat shock protein 60 ( Hsp60 ) and Actin in whole head homogenate from control and Lon deficient animals . ( D ) Quantification of the Lon band intensities from the indicated genotypes performed as described in B . ( E ) Quantification of the TFAM bands from the indicated genotypes performed as described in B . ( F ) Quantification of the Hsp60 bands from the indicated genotypes performed as described in B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of Lon protease (Lon), Mitochondrial transcription factor A (TFAM), Heat shock protein 60 (Hsp60) and Actin in whole head homogenate from control and Lon deficient animals. (D) Quantification of the Lon band intensities from the indicated genotypes performed as described in B. (E) Quantification of the TFAM bands from the indicated genotypes performed as described in B. (F) Quantification of the Hsp60 bands from the indicated genotypes performed as described in B.


( G ) Flow cytometry analysis of dissociated cells from the brains of flies expressing the indicated RNAi constructs . The graph shows the normalized fluorescence intensity of TMRE , an indicator of mitochondrial membrane potential , relative to age - matched control animals . Fluorescence intensity was normalized to control samples prepared and analyzed on the same day as experimental samples . The mitochondrial membrane potential uncoupling agent CCCP was added to samples of the indicated genotypes to illustrate the effect of mitochondrial depolarization on TMRE signal intensity . All experiments described in this figure were repeated at least three times per genotype . Error bars represent the standard error of the mean ( s . e . m . ) . * p < 0 . 05 , * * p < 0 . 005 by Student t 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 O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Flow cytometry analysis of dissociated cells from the brains of flies expressing the indicated RNAi constructs. The graph shows the normalized fluorescence intensity of TMRE, an indicator of mitochondrial membrane potential, relative to age-matched control animals. Fluorescence intensity was normalized to control samples prepared and analyzed on the same day as experimental samples. The mitochondrial membrane potential uncoupling agent CCCP was added to samples of the indicated genotypes to illustrate the effect of mitochondrial depolarization on TMRE signal intensity. All experiments described in this figure were repeated at least three times per genotype. Error bars represent the standard error of the mean (s.e.m.). *p<0.05, **p<0.005 by Student t test.


PINK1 accumulates in the mitochondrial and postmitochondrial supernatant fractions upon knockdown of Lon . ( A ) Mitochondrial ( Mito ) and postmitochondrial supernatant ( Sup ) protein fractions isolated from the heads of flies of the indicated genotypes were analyzed by western blot with antibodies to Myc , Complex V β ( Comp V β ) , NADH dehydrogenase ( ubiquinone ) Fe - S protein 3 ( NDUFS3 ) , voltage - dependent anion channel ( VDAC ) and Actin . elav - GAL4 was used to drive expression of the RNAi constructs used in these experiments . ( B ) Densitometry of the MPP - PINK1 bands in the mitochondrial fractions from the indicated genotypes was performed using Fiji software . PINK1 band intensities were normalized to VDAC intensity , and this ratio was in turn normalized to the MPP - PINK1 / VDAC ratio in Control - R animals . ( C ) Quantification of the Rho - PINK1 band intensities in mitochondrial fractions from the indicated genotypes was performed as described in B . ( D ) Quantification of the AFG - PINK1 band intensities in mitochondrial fractions from the indicated genotypes was performed as described in B . ( E ) Quantification of the MPP - PINK1 band intensities in the supernatant fractions from the indicated genotypes was performed as described in B except that Actin rather than VDAC was used as the loading control . ( F ) Quantification of the Rho - PINK1 band intensities in supernatant fractions from the indicated genotypes was performed as described in E . ( G ) Quantification of the AFG - PINK1 band intensities in supernatant fractions from the indicated genotypes was performed as described in E . Results shown are representative of at least three independent subcellular fractionation experiments per genotype . Error bars represent s . e . m . * p < 0 . 05 by Student t 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

PINK1 accumulates in the mitochondrial and postmitochondrial supernatant fractions upon knockdown of Lon.(A) Mitochondrial (Mito) and postmitochondrial supernatant (Sup) protein fractions isolated from the heads of flies of the indicated genotypes were analyzed by western blot with antibodies to Myc, Complex V β (Comp V β), NADH dehydrogenase (ubiquinone) Fe-S protein 3 (NDUFS3), voltage-dependent anion channel (VDAC) and Actin. elav-GAL4 was used to drive expression of the RNAi constructs used in these experiments. (B) Densitometry of the MPP-PINK1 bands in the mitochondrial fractions from the indicated genotypes was performed using Fiji software. PINK1 band intensities were normalized to VDAC intensity, and this ratio was in turn normalized to the MPP-PINK1/VDAC ratio in Control-R animals. (C) Quantification of the Rho-PINK1 band intensities in mitochondrial fractions from the indicated genotypes was performed as described in B. (D) Quantification of the AFG-PINK1 band intensities in mitochondrial fractions from the indicated genotypes was performed as described in B. (E) Quantification of the MPP-PINK1 band intensities in the supernatant fractions from the indicated genotypes was performed as described in B except that Actin rather than VDAC was used as the loading control. (F) Quantification of the Rho-PINK1 band intensities in supernatant fractions from the indicated genotypes was performed as described in E. (G) Quantification of the AFG-PINK1 band intensities in supernatant fractions from the indicated genotypes was performed as described in E. Results shown are representative of at least three independent subcellular fractionation experiments per genotype. Error bars represent s.e.m. *p<0.05 by Student t test.


A ) Mitochondrial fractions isolated from the heads of flies expressing Control - R were divided in half . One of the two samples was treated with 0 . 5 µg / ml Proteinase K and both samples were incubated at 4 ° C for 20 minutes . Samples were then subjected to western blot analysis using antibodies to the outer membrane protein Mitofusin ( Mfn ) , the inner membrane - associated matrix protein Complex V β ( Comp V β ) , and the matrix luminal protein pyruvate dehydrogenase ( PDH ) . elav - GAL4 was used to drive expression of all the RNAi constructs used in this and the following experiments . ( B ) Densitometry of Mfn , Comp V β , and PDH bands from experiments represented by panel A were performed using Fiji software , and the ratios of the band intensities in the sample containing ProK to the sample lacking ProK 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mitochondrial fractions isolated from the heads of flies expressing Control-R were divided in half. One of the two samples was treated with 0.5 µg/ml Proteinase K and both samples were incubated at 4°C for 20 minutes. Samples were then subjected to western blot analysis using antibodies to the outer membrane protein Mitofusin (Mfn), the inner membrane-associated matrix protein Complex V β (Comp V β), and the matrix luminal protein pyruvate dehydrogenase (PDH). elav-GAL4 was used to drive expression of all the RNAi constructs used in this and the following experiments. (B) Densitometry of Mfn, Comp V β, and PDH bands from experiments represented by panel A were performed using Fiji software, and the ratios of the band intensities in the sample containing ProK to the sample lacking ProK are shown.


( C ) Mitochondrial fractions isolated from the heads of flies expressing Control - R and PINK1 - Myc were divided in half . Each sample was treated with the indicated concentrations of ProK . Following incubation , samples were subjected to western blot analysis using antibodies to the indicated proteins . ( D ) Densitometry of the PINK1 - Myc , Comp V β , and PDH bands from experiments represented by panel C were performed as described in B . The Mfn quantification data from panel B is also included for comparison .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mitochondrial fractions isolated from the heads of flies expressing Control-R and PINK1-Myc were divided in half. Each sample was treated with the indicated concentrations of ProK. Following incubation, samples were subjected to western blot analysis using antibodies to the indicated proteins. (D) Densitometry of the PINK1-Myc, Comp V β, and PDH bands from experiments represented by panel C were performed as described in B. The Mfn quantification data from panel B is also included for comparison.


( E ) Mitochondrial fractions isolated from the heads of Lon - R2 and PINK1 - Myc expressing flies were divided in half . Each sample was treated with the indicated concentrations of ProK . Following incubation , samples were subjected to western blot analysis using antibodies to the indicated proteins . ( F ) Densitometry of the PINK1 - Myc , Comp V β , and PDH bands from experiments represented by panel E were performed as described in B . All experiments described were repeated at least three times . Error bars represent s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mitochondrial fractions isolated from the heads of Lon-R2 and PINK1-Myc expressing flies were divided in half. Each sample was treated with the indicated concentrations of ProK. Following incubation, samples were subjected to western blot analysis using antibodies to the indicated proteins. (F) Densitometry of the PINK1-Myc, Comp V β, and PDH bands from experiments represented by panel E were performed as described in B. All experiments described were repeated at least three times. Error bars represent s.e.m.


PINK1 accumulates in the mitochondrialmatrix upon knockdown of Lon . Immunofluorescent staining of thoracic muscles expressing matrix - targeted YFP ( Mito - YFP ) under control of the sqh genomic promoter , together with either ( A ) Myc - tagged MitochondrialRho ( Miro - Myc ) , ( B ) FLAG - tagged Optic atrophy 1 ( Opa1 - FLAG ) , ( C ) endogenous Cytochrome C ( Cyto C ) , ( D ) Myc - tagged PINK1 ( PINK1 - Myc ) , or ( E ) Lon - RNAi1 ( Lon - R1 ) and PINK1 - Myc . The muscle - specific Mhc - GAL4 driver was used to drive expression of Miro - Myc in panel A and Opa1 - FLAG in panel B , and the muscle - specific Dmef2 - GAL4 driver was used to drive expression of Lon - R1 in panel E . ( F ) We determined the percent volume shared between green objects ( representing the matrix marker Mito - YFP ) and red objects ( representing the mitochondrial proteins analyzed in A - E ) . Red objects that shared at least 60 % of their volume with Mito - YFP were considered to co - localize . Bar represents 2 µm .

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

PINK1 accumulates in the mitochondrialmatrix upon knockdown of Lon.Immunofluorescent staining of thoracic muscles expressing matrix-targeted YFP (Mito-YFP) under control of the sqh genomic promoter, together with either (A) Myc-tagged MitochondrialRho (Miro-Myc), (B) FLAG-tagged Optic atrophy 1 (Opa1-FLAG), (C) endogenous Cytochrome C (Cyto C), (D) Myc-tagged PINK1 (PINK1-Myc), or (E) Lon-RNAi1 (Lon-R1) and PINK1-Myc. The muscle-specific Mhc-GAL4 driver was used to drive expression of Miro-Myc in panel A and Opa1-FLAG in panel B, and the muscle-specific Dmef2-GAL4 driver was used to drive expression of Lon-R1 in panel E. (F) We determined the percent volume shared between green objects (representing the matrix marker Mito-YFP) and red objects (representing the mitochondrial proteins analyzed in A-E). Red objects that shared at least 60% of their volume with Mito-YFP were considered to co-localize. Bar represents 2 µm.


A ) The eye severity score of flies co - overexpressing PINK1 with Control - R ( n = 74 ) , Lon - R1 ( n = 163 ) , or Lon - R2 ( n = 90 ) using the ey - GAL4 driver .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The eye severity score of flies co-overexpressing PINK1 with Control-R (n = 74), Lon-R1 (n = 163), or Lon-R2 (n = 90) using the ey-GAL4 driver.


( B ) Comparison of the frequency of thoracic indentations in flies expressing the PINK1 - RNAi construct ( PINK1 - R ) in an otherwise wild - type background to the frequency in animals that also bear heterozygous mutations in Lon : PINK1 - RNAi + / + ( n = 306 ) , PINK1 - RNAi LonEP / + ( n = 74 ) , and PINK1 - RNAi LonDf / + ( n = 83 ) . LonEP refers to the LonG3998 P - element allele of Lon , and LonDf refers to the Df ( 3L ) Exel9011 deletion that removes the Lon gene along with several other genes . The Dmef2 - GAL4 driver was used to drive expression of the PINK1 - R construct in experiments described in panel B . Error bars represent s . e . m . * p < 0 . 05 , * * p < 0 . 005 by Student t 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Comparison of the frequency of thoracic indentations in flies expressing the PINK1-RNAi construct (PINK1-R) in an otherwise wild-type background to the frequency in animals that also bear heterozygous mutations in Lon: PINK1-RNAi +/+ (n = 306), PINK1-RNAi LonEP/+ (n = 74), and PINK1-RNAi LonDf/+ (n = 83). LonEP refers to the LonG3998 P-element allele of Lon, and LonDf refers to the Df(3L)Exel9011 deletion that removes the Lon gene along with several other genes. The Dmef2-GAL4 driver was used to drive expression of the PINK1-R construct in experiments described in panel B. Error bars represent s.e.m. *p<0.05, **p<0.005 by Student t test.


B Representative images showing co - expression of the stage - specific markers Nestin , Tbr2 , Doublecortin ( DCX ) , and Calbindin ( all in green ) , with the β - Galactosidase ( β - Gal , red ) reporter in 8 - week old BATGAL and Axin2LacZ / + mice . Nuclei are counterstained with DAPI ( in blue ) . Scale bar = 10µm . Insets show a 1 . 5x magnification of selected cells ( position indicated by dashed box ) . Scale bar = 5µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative images showing co-expression of the stage-specific markers Nestin, Tbr2, Doublecortin (DCX), and Calbindin (all in green), with the β-Galactosidase (β-Gal, red) reporter in 8-week old BATGAL and Axin2LacZ/+ mice. Nuclei are counterstained with DAPI (in blue). Scale bar= 10µm. Insets show a 1.5x magnification of selected cells (position indicated by dashed box). Scale bar= 5µm.


C Fraction of Nestin - , Tbr2 - , DCX - and Calbindin - positive cells expressing β - Gal in BATGAL and Axin2LacZ / + animals show stage specific canonical Wnt signaling during adult hippocampal neurogenesis [ BATGAL : Tbr2 vs . DCX p = 0 . 0056 , DCX vs . Calbindin p < 0 . 0001 ; Axin2LacZ : Nestin vs . Tbr2 p = 0 . 0003 , Tbr2 vs . DCX p = 0 . 0115 , DCX vs . Calbindin p < 0 . 0001 ] ( n = 3 animals per mouse model and marker ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fraction of Nestin-, Tbr2-, DCX- and Calbindin-positive cells expressing β-Gal in BATGAL and Axin2LacZ/+ animals show stage specific canonical Wnt signaling during adult hippocampal neurogenesis [BATGAL: Tbr2 vs. DCX p=0.0056, DCX vs. Calbindin p<0.0001; Axin2LacZ: Nestin vs. Tbr2 p=0.0003, Tbr2 vs. DCX p=0.0115, DCX vs. Calbindin p<0.0001] (n= 3 animals per mouse model and marker).


D Detection of the corrected total cell fluorescence ( CTCF ) of the β - Gal reporter in Nestin - , Tbr2 - , DCX - and Calbindin - positive cells in BATGAL mice corroborate stage specific activity pattern of canonical Wnt signaling during lineage progression [ Nestin vs . Tbr2 p < 0 . 0001 , Tbr2 vs . DCX p = 0 . 0029 , DCX vs . Calbindin p < 0 . 0001 ] ( Nestin : n = 236 cells , Tbr2 : n = 134 cells , DCX : n = 200 cells , Calbindin : n = 300 cells ) .

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

D Detection of the corrected total cell fluorescence (CTCF) of the β-Gal reporter in Nestin-, Tbr2-, DCX- and Calbindin-positive cells in BATGAL mice corroborate stage specific activity pattern of canonical Wnt signaling during lineage progression [Nestin vs. Tbr2 p <0.0001, Tbr2 vs. DCX p=0.0029, DCX vs. Calbindin p<0.0001] (Nestin: n= 236 cells, Tbr2: n= 134 cells, DCX: n= 200 cells, Calbindin: n= 300 cells).


F Quantification of BrdU + cells expressing β - Gal reveals biphasic activity of canonical Wnt signaling during adult hippocampal neurogenesis [ 0 vs . 7dpi p = 0 . 0036 , 7 vs . 28dpi p = 0 . 0115 , 7 vs . 42dpi p = 0 . 0007 ] ( 0dpi time - point ; n = 4 animals ) , 3dpi ( n = 3 animals ) , 7dpi ( n = 3 animals ) , 14dpi ( n = 4 animals ) , 28dpi ( n = 6 animals ) and 42dpi ( n = 6 animals ) .

F Quantification of BrdU+ cells expressing β-Gal reveals biphasic activity of canonical Wnt signaling during adult hippocampal neurogenesis [0 vs. 7dpi p=0.0036, 7 vs. 28dpi p=0.0115, 7 vs. 42dpi p=0.0007] (0dpi time-point; n= 4 animals), 3dpi (n= 3 animals), 7dpi (n= 3 animals), 14dpi (n= 4 animals), 28dpi (n= 6 animals) and 42dpi (n= 6 animals).


G Total cell fluorescence of β - Gal in BrdU + cells indicate a biphasic pattern in canonical Wnt signaling strength during lineage progression [ 0 vs . 3dpi p < 0 . 0001 , 7 vs . 14dpi p < 0 . 0001 , 14 vs . 28dpi p < 0 . 0001 , 28 vs . 42dpi p = 0 . 0333 ] ( 0dpi n = 194 cells , 3dpi n = 245 cells , 7dpi n = 224 cells , 14dpi n = 72 cells , 28dpi n = 103 cells and 42dpi n = 114 cells )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Total cell fluorescence of β-Gal in BrdU+ cells indicate a biphasic pattern in canonical Wnt signaling strength during lineage progression [0 vs. 3dpi p<0.0001, 7 vs. 14dpi p<0.0001, 14 vs. 28dpi p<0.0001, 28 vs. 42dpi p=0.0333] (0dpi n= 194 cells, 3dpi n= 245 cells, 7dpi n= 224 cells, 14dpi n= 72 cells, 28dpi n= 103 cells and 42dpi n= 114 cells)


B Representative images of transduced adult - born neurons at 17dpi . CAG - dnLEF - IRES - GFP ( dnLEF , green ) and CAG - RFP ( control , red ) double transduced cells ( arrows ) and CAG - RFP single transduced cells ( arrowheads ) . Scale bar = 20

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative images of transduced adult-born neurons at 17dpi. CAG-dnLEF-IRES-GFP (dnLEF, green) and CAG-RFP (control, red) double transduced cells (arrows) and CAG-RFP single transduced cells (arrowheads). Scale bar= 20 µm


C Representative reconstructions of control and dnLEF neurons . Scale bar = 20

C Representative reconstructions of control and dnLEF neurons. Scale bar= 20 µm


D Analysis of morphology showed a reduction in dendritic length in dnLEF transduced neurons [ p < 0 . 0001 ] , while the number of branch points remained comparable [ p = 0 . 7914 ] . Sholl analysis displayed a reduction in dendritic complexity in dnLEF transduced neurons and indicated decreased growth of terminal dendritic branches [ p < 0 . 0001 ] ( control : n = 20 cells from 3 animals , dnLEF : n = 38 cells from 6 animals ) .

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

D Analysis of morphology showed a reduction in dendritic length in dnLEF transduced neurons [p<0.0001], while the number of branch points remained comparable [p=0.7914]. Sholl analysis displayed a reduction in dendritic complexity in dnLEF transduced neurons and indicated decreased growth of terminal dendritic branches [p<0.0001] (control: n= 20 cells from 3 animals, dnLEF: n= 38 cells from 6 animals).


E DnLEF neurons displayed basal dendrites [ p < 0 . 0001 ] ( control : n = 20 cells from 3 animals , dnLEF : n = 38 cells from 6 animals ) .

O O O O O O O O O O O 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 DnLEF neurons displayed basal dendrites [p<0.0001] (control: n= 20 cells from 3 animals, dnLEF: n= 38 cells from 6 animals).