Northern blot experiments ( A - C ) assessing GlmY * half - life under GlcN6P replete ( + GlcN ) depletion conditions ( - GlcN ) . Transcription was stopped by addition of rifampicin samples were harvested at indicated times for Northern . A of ∆ glmS Z1126 . GlcN6P depleted culture was split 8 min after rifampicin addition one of sub - cultures was resupplied with GlcN ( indicated by arrow ) . B ∆ glmS ∆ rapZ double mutant Z1127 was tested . C Semi - logarithmic plots of GlmY * decay for half - life determination .

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Northern blot experiments (A-C) assessing GlmY* half-life under GlcN6P replete (+GlcN) and depletion conditions (-GlcN). Transcription was stopped by addition of rifampicin and samples were harvested at indicated times for Northern analysis. A Analysis of the ∆glmS strain Z1126. The GlcN6P depleted culture was split 8 min after rifampicin addition and one of the sub-cultures was resupplied with GlcN (indicated by arrow). B The ∆glmS ∆rapZ double mutant Z1127 was tested. C Semi-logarithmic plots of GlmY* decay for half-life determination.


D EMSA experiments addressing role of GlcN6P for GlmY * / RapZ interaction . Radiolabelled GlmY * was incubated with incremental concentrations of RapZ ( left panel ) or RapZ - CTD ( right panel ) in absence or presence of 7 . 5 mM GlcN6P . Binding reactions were separated on native PAA gels analyzed by phospho - imaging . RapZ / GlmY * is indicated by an arrow .

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D EMSA experiments addressing the role of GlcN6P for GlmY*/RapZ interaction. Radiolabelled GlmY* was incubated with incremental concentrations of RapZ (left panel) or RapZ-CTD (right panel) in absence or presence of 7.5 mM GlcN6P. Binding reactions were separated on native PAA gels and analyzed by phospho-imaging. The RapZ/GlmY* complex is indicated by an arrow.


E EMSA following incubation of GlmY * with 1200 nM RapZ in presence of various GlcN6P concentrations ranging from 0 ( lane 2 ) to 8 mM ( lane 10 ) . fraction of GlmY * remaining in gel pocket is marked with an asterisk . RapZ / GlmY * is indicated by an arrow .

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E EMSA following incubation of GlmY* with 1200 nM RapZ in presence of various GlcN6P concentrations ranging from 0 (lane 2) to 8 mM (lane 10). The fraction of GlmY* remaining in the gel pocket is marked with an asterisk. The RapZ/GlmY* complex is indicated by an arrow.


F EMSA following incubation of GlmY * with 1200 nM RapZ in absence or presence of 7 . 5 mM of indicated metabolite . RapZ / GlmY * is indicated by an arrow .

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F EMSA following incubation of GlmY* with 1200 nM RapZ in absence or presence of 7.5 mM of the indicated metabolite. The RapZ/GlmY* complex is indicated by an arrow.


Western blot for phosphorylated total ERK in HEK293 transfected with native ΔA781 _ N783 ( left ) or P824R ( right ) CSF - 1R treated with CSF - 1 ( D , E ) or IL - 34 ( F , G ) for 10 minutes . Horizontal indicates untreated , with increasing concentrations 10 , 50 100 ng / ml . Corresponding densitometry displays of phospho - ERK to total ERK , normalised to of untreated control for native receptor ( * * * * p < 0 . 0001 , * * * p < 0 . 0005 , * * p < 0 . 01 , * p < 0 . 05 , one - way ANOVA with Sidak ' s post - for multiple comparison , n = 3 biological replicates , , error bars indicate ) .

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

Western blot for phosphorylated and total ERK in HEK293 cells transfected with native and ΔA781_N783 (left) or P824R (right) CSF-1R and treated with CSF-1 (D, E) or IL-34 (F, G) for 10 minutes. Horizontal line indicates untreated cells, with increasing concentrations 10, 50 and 100 ng/ml. Corresponding densitometry displays the ratio of phospho-ERK to total ERK, normalised to the ratio of the untreated control for the native receptor (**** p < 0.0001, *** p < 0.0005, ** p < 0.01, * p < 0.05, one-way ANOVA with Sidak's post-test for multiple comparison, n = 3 biological replicates, , error bars indicate SEM).


Western blot for CSF - 1R in HEK293 transfected with native ΔA781 _ N783 ( top ) or P824R ( bottom ) CSF - 1R treated with rapamycin . Horizontal indicates untreated , with concentrations 0 . 1 , 0 . 5 , 1 , 5 , 10μM . Corresponding densitometry ( below ) displays fold change of CSF - 1R relative to untreated control for each transfection ( * * * * p < 0 . 0001 , one - way ANOVA with Sidak ' s post - for multiple comparison to untreated , n = 3 biological replicates , error bars indicate ) . Western blot for CSF - 1R in HEK293 transfected with native ΔA781 _ N783 ( top ) or P824R ( bottom ) CSF - 1R treated with 3 - methyladenosine . Horizontal indicates untreated , with increasing concentrations 0 . 1 , 0 . 2 , 0 . 5 , 1mM . Corresponding densitometry ( below ) displays fold change of CSF - 1R relative to untreated control for each transfection ( * * * * p < 0 . 0001 , * p = 0 . 0114 , one - way ANOVA with Sidak ' s post - for multiple comparison to untreated , n = 3 biological replicates , error bars indicate ) . Western blot for CSF - 1R Actin in HEK293 transfected with native ΔA781 _ N783 ( top ) or P824R ( bottom ) CSF - 1R treated with MG132 or PLX3397 . Horizontal indicates untreated , 20μM PLX3397 , with increasing concentrations 0 . 1 , 0 . 5 , 0 . 1 , 5μM . Corresponding densitometry ( below ) displays fold change of CSF - 1R relative to untreated control for each transfection ( * * * p = 0 . 0002 , * * p = 0 . 002 , one - way ANOVA with Sidak ' s post - for multiple comparison to untreated , n = 3 biological replicates , error bars indicate ) .

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Western blot for CSF-1R in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with rapamycin. Horizontal line indicates untreated cells, with concentrations 0.1, 0.5, 1, 5, 10μM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (**** p < 0.0001, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM). Western blot for CSF-1R in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with 3-methyladenosine. Horizontal line indicates untreated cells, with increasing concentrations 0.1, 0.2, 0.5, 1mM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (**** p < 0.0001, * p = 0.0114, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM). Western blot for CSF-1R and Actin in HEK293 cells transfected with native and ΔA781_N783 (top) or P824R (bottom) CSF-1R and treated with MG132 or PLX3397. Horizontal line indicates untreated cells, 20μM PLX3397, with increasing concentrations 0.1, 0.5, 0.1, 5μM. Corresponding densitometry (below) displays the fold change of CSF-1R relative to the untreated control for each transfection (*** p = 0.0002, ** p = 0.002, one-way ANOVA with Sidak's post-test for multiple comparison to untreated, n = 3 biological replicates, error bars indicate SEM).


Immunocytochemistry of microglia isolated from Csf - 1rflx / wt ; Cx3cr1 - Cre - Csf - 1rflx / wt ; Cx3cr1 - Cre + P0 mouse pup brains . Microglia were stimulated with LPS exposed to opsonised latex beads for 1 hour before fixation of bead + via microscopy . were stained using MitoTracker ( red ) . Arrowheads ( ) indicate bead + . of mouse microglial phagocytic activity expressed as bead + ( n = 4 assays , error bars indicate ) .

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Immunocytochemistry of microglia isolated from Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ P0 mouse pup brains. Microglia were stimulated with LPS and exposed to fluorescent opsonised latex beads for 1 hour before fixation and quantification of bead+ cells via microscopy. Cells were stained using MitoTracker(red). Arrowheads (white) indicate bead+ cells. Quantification of mouse microglial phagocytic activity expressed as percentage bead+ cells (n = 4 assays, error bars indicate SEM).


L ) Immunocytochemistry of macrophages differentiated in vitro from Csf - 1rflx / wt ; Cx3cr1 - Cre - Csf - 1rflx / wt ; Cx3cr1 - Cre + mouse bone marrow . Macrophages were stimulated with LPS exposed to opsonised latex beads for 1 hour before fixation of bead + via microscopy . were stained using MitoTracker ( red ) DAPI ( blue ) . Arrowheads ( ) indicate bead + . M ) of mouse macrophage phagocytic activity expressed as bead + . ( * p = 0 . 024 , Student ' s t - with Welch ' s correction , n = 4 assays , error bars indicate ) .

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L) Immunocytochemistry of macrophages differentiated in vitro from Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mouse bone marrow. Macrophages were stimulated with LPS and exposed to fluorescent opsonised latex beads for 1 hour before fixation and quantification of bead+ cells via microscopy. Cells were stained using MitoTracker (red) and DAPI (blue). Arrowheads (white) indicate bead+ cells. M) Quantification of mouse macrophage phagocytic activity expressed as percentage bead+ cells. (* p = 0.024, Student's t-test with Welch's correction, n = 4 assays, error bars indicate SEM).


A ) Immunohistochemistry of Csf - 1rflx / wt ; Cx3cr1 - Cre - ( top ) Csf - 1rflx / wt ; Cx3cr1 - Cre + ( bottom ) mice unilaterally injected with Aβ1 - 42 in hippocampus stained for IB4 ( green ) , F4 / 80 ( ) Aβ ( red ) . Scale bars indicate 50 μm . B ) of F4 / 80 IB4 immunopositivity following intrahippocampal injection of Aβ1 - 42 . of immunopositivity in both injected uninjected hippocampi of Csf - 1rflx / wt ; Cx3cr1 - Cre - Csf - 1rflx / wt ; Cx3cr1 - Cre + mice . ( Two - way ANOVA with Sidak ' s for multiple comparisons . Asterisks ( * ) indicate comparison to immunopositivity values of uninjected hippocampus , obliques ( # ) indicate comparison between Csf - 1rflx / wt ; Cx3cr1 - Cre - Csf - 1rflx / wt ; Cx3cr1 - Cre + mice , # # or * * p < 0 . 003 , # # # # or * * * * p < 0 . 0001 , n = 5 mice per group , error bars indicate ) .

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A) Immunohistochemistry of Csf-1rflx/wt;Cx3cr1-Cre- (top) and Csf-1rflx/wt;Cx3cr1-Cre+ (bottom) mice unilaterally injected with Aβ1-42 in the hippocampus and stained for IB4 (green), F4/80 (white) and Aβ (red). Scale bars indicate 50 μm. B) Quantification of F4/80 and IB4 immunopositivity following intrahippocampal injection of Aβ1-42. Quantification of immunopositivity in both injected and uninjected hippocampi of Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mice. (Two-way ANOVA with Sidak's test for multiple comparisons. Asterisks (*) indicate comparison to immunopositivity values of the uninjected hippocampus, obliques (#) indicate comparison between Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mice, ## or ** p < 0.003, #### or **** p < 0.0001, n = 5 mice per group,error bars indicate SEM).


C ) Immunohistochemistry of Csf - 1rflx / wt ; Tie2 - Cre - ( top ) Csf - 1rflx / wt ; Tie2 - Cre + mice ( ottom ) mice unilaterally injected with Aβ1 - 42 in hippocampus stained for IB4 ( green ) , F4 / 80 ( ) Aβ ( red ) . D ) of F4 / 80 IB4 immunopositivity following intrahippocampal injection of Aβ1 - 42 . of immunopositivity in both injected uninjected hippocampi of Csf - 1rflx / wt ; Tie2 - Cre - Csf - 1rflx / wt ; Tie2 - Cre + mice . ( n = 5 mice per group . Two - way ANOVA with Sidak ' s for multiple comparisons . Asterisks ( * ) indicate comparison to immunopositivity values of uninjected hippocampus , * p < 0 . 01 , * * * p < 0 . 0005 , error bars indicate ) ) .

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C) Immunohistochemistry of Csf-1rflx/wt;Tie2-Cre- (top) and Csf-1rflx/wt;Tie2-Cre+ mice (ottom) mice unilaterally injected with Aβ1-42 in the hippocampus and stained for IB4 (green), F4/80 (white) and Aβ (red). D) Quantification of F4/80 and IB4 immunopositivity following intrahippocampal injection of Aβ1-42. Quantification of immunopositivity in both injected and uninjected hippocampi of Csf-1rflx/wt;Tie2-Cre- and Csf-1rflx/wt;Tie2-Cre+ mice. (n = 5 mice per group. Two-way ANOVA with Sidak's test for multiple comparisons. Asterisks (*) indicate comparison to immunopositivity values of the uninjected hippocampus, *p < 0.01, *** p < 0.0005, error bars indicate SEM)).


qPCR of wild type ( blue ) or Csf - 1r + / - ( red ) endothelial treated with control , Csf - 1r + / + MCM or Csf - 1r + / - MCM . Statistical analyses of inter - genotype changes . ( * * * * p < 0 . 0001 , * * p < 0 . 005 , Scatter plots represent technical replicates of n = 2 independent isolations microglia conditionings , Two - way ANOVA with multiple comparisons Sidak post - , error bars indicate ) ) .

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qPCR of wild type (blue) or Csf-1r+/- (red) endothelial cells treated with control, Csf-1r+/+ MCM or Csf-1r+/- MCM. Statistical analyses of inter-genotype changes. (**** p < 0.0001, ** p < 0.005, Scatter plots represent technical replicates of n = 2 independent primary cell isolations and microglia conditionings, Two-way ANOVA with multiple comparisons and Sidak post-test, error bars indicate SEM)).


of ALSP post mortem tissue for amyloid - β ( green ) CLD5 ( red ) , ( B ) human IgG ( green ) PDGFRβ ( red ) , ( C ) GFAP ( green ) CLD5 ( red ) . ΔA781 _ N783 P824R indicate CSF - 1R variant present .

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IHC of ALSP patient post mortem tissue for amyloid-β (green) and CLD5 (red), (B) human IgG (green) and PDGFRβ (red), (C) GFAP (green) and CLD5 (red). ΔA781_N783 and P824R indicate the CSF-1R variant present.


( D ) Iba1 ( green ) GFAP ( red ) , ( E ) amyloid - β ( green ) CD68 ( red ) , ( f ) CD163 ( green ) CLD5 ( red ) , imaging of an ALSP with ΔA781 _ N783 CSF - 1R variant . ΔA781 _ N783 P824R indicate CSF - 1R variant present .

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(D) Iba1 (green) and GFAP (red), (E) amyloid-β (green) and CD68 (red), (f) CD163 (green) and CLD5 (red), imaging of an ALSP patient with the ΔA781_N783 CSF-1R variant. ΔA781_N783 and P824R indicate the CSF-1R variant present.


( G ) Fibrinogen ( green ) CLD5 ( red ) ( H ) FLAIR imaging of an ALSP with ΔA781 _ N783 CSF - 1R variant . ΔA781 _ N783 P824R indicate CSF - 1R variant present .

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(G) Fibrinogen (green) and CLD5 (red) and (H) FLAIR imaging of an ALSP patient with the ΔA781_N783 CSF-1R variant. ΔA781_N783 and P824R indicate the CSF-1R variant present.


B : PK vs . PIPLC treatment to determine prion loads in infected in 384 - well plates . Both treatments discriminate between RML prion infected CAD5 non - infectious brain homogenate ( NBH ) treated CAD5 . * * * * = p - value < 0 . 0001 ( Student ' s t - ) . Shown are mean ± SD , n ≤ 30 individually cultured wells .

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B: PK vs. PIPLC treatment to determine prion loads in infected cells in 384-well plates. Both treatments discriminate between RML prion infected CAD5 and non-infectious brain homogenate (NBH) treated CAD5 cells. **** = p-value < 0.0001 (Student's t-test). Shown are mean ± SD, n ≤ 30 individually cultured wells.


C : infectivity of PIPLC PK - treated lysates was determined by infecting CAD5 with lysates as indicated . signal of highest dilution was measured compared to untreated RML CAD5 lysate . PIPLC - treated PK - treated lysates showed similar infectivity titers . Prion - infected NBH , as well as RML on CAD5ΔPrnp were used for control .

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C: The infectivity of PIPLC and PK-treated cell lysates was determined by infecting CAD5 cells with lysates as indicated. The signal intensity of the highest dilution was measured and compared to untreated RML CAD5 cell lysate. PIPLC-treated cells and PK-treated lysates showed similar infectivity titers. Prion-infected and NBH, as well as RML on CAD5ΔPrnp were used for control.


D : RML - infected CAD5 GT - 1 / 7 were transfected with non - targeting ( NT ) or Prnp targeting siRNAs in a 384 - well plate subjected to PK or PIPLC treatment . Z ' - factors were calculated for each condition . Shown are mean ± SD , n = 22 individually cultured wells .

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D: RML-infected CAD5 and GT-1/7 cells were transfected with non-targeting (NT) or Prnp targeting siRNAs in a 384-well plate and subjected to PK or PIPLC treatment. Z'-factors were calculated for each condition. Shown are mean ± SD, n = 22 individually cultured wells.


A : Western blot showing Hnrnpk siRNA transfection ( 96 hrs . ) decreases Hnrnpk while increases PrPSc in RML prion infected GT - 1 / 7 . Prnp siRNAs suppressed both PrPC PrPSc as expected . ⍺ : anti Quantifications are reported as normalized to Actin in comparison , to NT . PK - western blot is quantified relative to NT .

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A: Western blot showing Hnrnpk siRNA transfection (96 hrs.) decreases Hnrnpk protein levels while increases PrPSc in RML prion infected GT-1/7. Prnp siRNAs suppressed both PrPC and PrPSc as expected. ⍺: anti Quantifications are reported as normalized to Actin and in comparison, to NT. PK-western blot is quantified relative to NT.


B : Western Blot of PSA - treated uninfected infected GT - 1 / 7 . Increasing of PSA leads to a more prominent reduction of PrPSc in mouse . PK - western blot is quantified relative to DMSO .

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B: Western Blot of PSA-treated uninfected and infected GT-1/7 cells. Increasing concentration of PSA leads to a more prominent reduction of PrPSc in mouse cells. PK-western blot is quantified relative to DMSO.


C : mRNA of Hnrnpk Prnp following siRNA PSA treatment . FPKM : Fragments per kilobase of transcript per million mapped reads . Hnrnpk siRNAs lead to a decrease in Hnrnpk mRNA as well as an increase in Prnp mRNA . No difference is seen between DMSO treated PSA treated for either Hnrnpk of Prnp . n = 2 per treatment group .

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C: mRNA levels of Hnrnpk and Prnp following siRNA and PSA treatment. FPKM: Fragments per kilobase of transcript per million mapped reads. Hnrnpk siRNAs lead to a decrease in Hnrnpk mRNA levels as well as an increase in Prnp mRNA levels. No difference is seen between DMSO treated and PSA treated cells for either Hnrnpk levels of Prnp levels. n = 2 per treatment group.


D : of PrPSc in RML GT - 1 / 7 following treatment with 1 µM PSA in comparison to DMSO . Each dot represents an experiment . Shown are mean ± SD .

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D: Quantification of PrPSc levels in RML GT-1/7 cells following treatment with 1 µM PSA in comparison to DMSO. Each dot represents an experiment. Shown are mean ± SD.


E : Brightfield microscopy of effect of siRNA mediated HNRNPK downregulation on prion - induced vacuolation in PG127 hovS . Downregulation of HNRNPK leads to an enhanced cytopathological vacuolation phenotype when compared to NT siRNA . ovPRNP siRNA transfected , as well as uninfected were used as controls Downregulation of ovPRNP in infected hovS eliminates vacuoles ; Right panel : of vacuoles of NT , HNRNPK ovPRNP siRNA treated PG127 hovS . from pictures at three different positions in well were manually counted amount of vacuolated was normalized to total amount of . Values represent mean ± SD . * p = 0 . 0113 , * * * * p = < 0 . 0001 ( Dunnett ' s multiple comparisons ) . Scale bar = 100 µm . n = 3 technical replicates .

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

E: Brightfield microscopy images of the effect of siRNA mediated HNRNPK downregulation on prion-induced vacuolation in PG127 hovS cells. Downregulation of HNRNPK leads to an enhanced cytopathological vacuolation phenotype when compared to NT siRNA. ovPRNP siRNA transfected, as well as uninfected cells were used as controls Downregulation of ovPRNP in the infected hovS eliminates the vacuoles; Right panel: Quantification of vacuoles of NT, HNRNPK and ovPRNP siRNA treated PG127 hovS. Cells from pictures at three different positions in the well were manually counted and the amount of vacuolated cells was normalized to the total amount of cells. Values represent mean ± SD. * p = 0.0113, **** p = <0.0001 (Dunnett's multiple comparisons test). Scale bar = 100 µm. n = 3 technical replicates.


F : of PrPSc in PG127 hovS following treatment with 1 µM PSA in comparison to DMSO . Each dot represents an experiment . Shown are mean ± SD .

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F: Quantification of PrPSc levels in PG127 hovS cells following treatment with 1 µM PSA in comparison to DMSO. Each dot represents an experiment. Shown are mean ± SD.


G : Same as in E , using shRNAs instead of siRNA . Downregulation of HNRNPK leads to an enhanced cytopathological vacuolation phenotype when compared to NT shRNA . Uninfected were used as controls Right panel : of vacuoles of NT HNRNPK shRNA treated PG127 hovS . from pictures at three different positions in well were manually counted amount of vacuolated was normalized to total amount of . Values represent mean ± SD . * * * p = < 0 . 0009 ( Dunnett ' s multiple comparisons ) . Scale bar = 100 µm . n = 3 technical replicates .

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 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

G: Same as in E, using shRNAs instead of siRNA. Downregulation of HNRNPK leads to an enhanced cytopathological vacuolation phenotype when compared to NT shRNA. Uninfected cells were used as controls Right panel: Quantification of vacuoles of NT and HNRNPK shRNA treated PG127 hovS. Cells from pictures at three different positions in the well were manually counted and the amount of vacuolated cells was normalized to the total amount of cells. Values represent mean ± SD. *** p = <0.0009 (Dunnett's multiple comparisons test). Scale bar = 100 µm. n = 3 technical replicates.


H : of PrPSc in PG127 hovS following treatment with shRNA against HNRNPK 1 µM PSA in comparison to DMSO NT . Each dot represents an experiment . * = p - value < 0 . 027 ( Student ' s t - ) . Shown are mean ± SD .

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H: Quantification of PrPSc levels in PG127 hovS cells following treatment with shRNA against HNRNPK and 1 µM PSA in comparison to DMSO and NT. Each dot represents an experiment. *= p-value < 0.027 (Student's t-test). Shown are mean ± SD.


I : Gene set overrepresentation of differentially expressed genes ( log2FC - 0 . 25 ≥ or 0 . 25 ≤ FDR ≤ 0 . 05 ) for siRNA mediated Hnrnpk downregulation or PSA treatment in RML GT - 1 / 7 analyzed by RNAseq . Differentially regulated genes ( up in siRNA treatment down in PSA or vice versa ) were overlapped used for pathway . No significantly enriched pathways are detected for upregulated genes in Hnrnpk downregulated in PSA treatment . For opposing direction , an enrichment of genes involved in glucose metabolism was detected .

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

I: Gene set overrepresentation analysis of differentially expressed genes (log2FC -0.25 ≥ or 0.25 ≤ and FDR ≤ 0.05) for siRNA mediated Hnrnpk downregulation or PSA treatment in RML GT-1/7 cells analyzed by RNAseq. Differentially regulated genes (up in siRNA treatment and down in PSA or vice versa) were overlapped and used for pathway analysis. No significantly enriched pathways are detected for upregulated genes in Hnrnpk and downregulated in PSA treatment. For the opposing direction, an enrichment of genes involved in glucose metabolism was detected.


A : Western Blot of RML prion infected COCS treated with 1 µM PSA or DMSO . PSA treatment was started two after infection continued until lysis . PSA reduced amount of PrPSc . Right panel : of Western Blot ; n = 5 biological replicates . Values : mean ± SD . * p = 0 . 0211 ( Student ' s t - ) .

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A: Western Blot of RML prion infected COCS treated with 1 µM PSA or DMSO. PSA treatment was started two weeks after infection and continued until lysis. PSA reduced the amount of PrPSc. Right panel: Quantification of the Western Blot; n = 5 biological replicates. Values: mean ± SD. * p = 0.0211 (Student's t-test).


B : Western Blot of NBH - treated COCS . PSA treatment was identical to samples of A . PSA did not affect PrPC expression . Right panel : of Western Blot ; n = 5 biological replicates . Values represent mean ± SD . n . s . = not significant ( Student ' s t - ) . ⍺ :

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

B: Western Blot analysis of NBH-treated COCS. PSA treatment was identical to the samples of A. PSA did not affect PrPC expression. Right panel: Quantification of the Western Blot; n = 5 biological replicates. Values represent mean ± SD. n.s. = not significant (Student's t-test). ⍺: anti


C : Negative geotaxis climbing in prion infected Drosophila . Flies were treated with DMSO , 0 . 5 mM PSA , 0 . 75 mM PSA , or 1 mM PSA at larval stage during adulthood for duration of . Climbing ability was assessed on groups of flies ( n = 3 x 15 ) three times a week expressed as a performance index . Statistical on difference between PG127 prion infected versus control prion - free treatment group data in each graph was performed by an unpaired t : DMSO : p = 0 . 0002 ; PSA : 0 . 5 mM : p = 0 . 0186 ; 0 . 75 mM : p = n . s . ; 1 mM : p = n . s . n . s . = not significant . Shown are mean ±

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Negative geotaxis climbing assay in prion infected Drosophila. Flies were treated with DMSO, 0.5 mM PSA, 0.75 mM PSA, or 1 mM PSA at the larval stage and during adulthood for the duration of the assay. Climbing ability was assessed on groups of flies (n=3 x 15) three times a week and expressed as a performance index. Statistical analysis on the difference between PG127 prion infected versus control prion-free treatment group data in each graph was performed by an unpaired t test: DMSO: p = 0.0002; PSA: 0.5 mM: p = 0.0186; 0.75 mM: p = n.s.; 1 mM: p = n.s. n.s. = not significant. Shown are mean ± SD


D : RT - QuIC of whole - head homogenates of prion infected Drosophila . For each sample , 10 10 heads from same treatment group were homogenized , 1 : 20 diluted applied to RT - QuIC . Shown are RT - QuIC reactions of three independent homogenates per treatment group , each assessed in quadruplicates . For , lag - time of each reaction was calculated plotted in a graph . Shown are mean ± SD . assays were performed for 100 hours , samples not yielding a positive reaction are considered negative . * * * = p ≤ 0 . 0006 , n . s . = not significant , flies fed with NBH treated with DMSO were used as a negative control , a standard prion - free a prion - containing sample were used as controls . To control for potential interference of DMSO or PSA with RT - QuIC reaction , prion positive sample was spiked with 1 µM PSA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: RT-QuIC analysis of whole-head homogenates of prion infected Drosophila. For each sample, 10 male and 10 female heads from the same treatment group were homogenized, 1:20 diluted and applied to the RT-QuIC. Shown are the RT-QuIC reactions of three independent homogenates per treatment group, each assessed in quadruplicates. For quantification, the lag-time of each reaction was calculated and plotted in a graph. Shown are mean ± SD. The assays were performed for 100 hours, samples not yielding a positive reaction are considered negative. *** = p ≤ 0.0006, n.s. = not significant, flies fed with NBH and treated with DMSO were used as a negative control, a standard prion-free and a prion-containing sample were used as assay controls. To control for potential interference of DMSO or PSA with the RT-QuIC reaction, prion positive sample was spiked with 1 µM PSA.


SHAPE of complete sfRNAs of ZIKV ( D ) Nucleotides with SHAPE reactivity greater than 0 . 85 are labeled in red , moderately reactive site ( 0 . 4 - 0 . 85 ) are labeled in cyan . Unlabeled sites have low or no SHAPE reactivity ( < 0 . 4 ) . SHAPE reactivity were also annotated onto corresponding secondary structures in ( B ) Nucleotides which SHAPE reactivity was not determined are labeled in gray 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

SHAPE analysis of the complete sfRNAs of ZIKV (D) Nucleotides with SHAPE reactivity greater than 0.85 are labeled in red, and moderately reactive site (0.4-0.85) are labeled in cyan. Unlabeled sites have low or no SHAPE reactivity (<0.4). The SHAPE reactivity were also annotated onto the corresponding secondary structures in (B) Nucleotides which SHAPE reactivity was not determined are labeled in gray in (B)


SHAPE of complete sfRNAs of WNV ( E ) . Nucleotides with SHAPE reactivity greater than 0 . 85 are labeled in red , moderately reactive site ( 0 . 4 - 0 . 85 ) are labeled in cyan . Unlabeled sites have low or no SHAPE reactivity ( < 0 . 4 ) . SHAPE reactivity were also annotated onto corresponding secondary structures in ( C ) . Nucleotides which SHAPE reactivity was not determined are labeled in gray in ( C ) .

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

SHAPE analysis of the complete sfRNAs of WNV (E). Nucleotides with SHAPE reactivity greater than 0.85 are labeled in red, and moderately reactive site (0.4-0.85) are labeled in cyan. Unlabeled sites have low or no SHAPE reactivity (<0.4). The SHAPE reactivity were also annotated onto the corresponding secondary structures in (C). Nucleotides which SHAPE reactivity was not determined are labeled in gray in (C).


( A ) - ( D ) scattering profiles ( A ) , pair distribution functions ( PDDFs ) ( B ) , dimensionless Kratky plots ( C ) Porod - Debye plots ( D ) of complete sfRNAs of DENV2 ( cyan ) , ZIKV ( red ) WNV ( blue ) . fine features of P1 in scattering profiles in ( A ) arise from helical interstrand pair - correlation . PDDF profiles in ( B ) were calculated using GNOM ( qmax = 0 . 3 ) . dimensionless Kratky plot Porod - Debye plot for ZIKV xrRNA1 ( red dashed ) were included in ( C ) ( D ) for comparison , indicating that structures of sfRNAs are more extended open than that of ZIKV xrRNA1 .

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

(A)-(D) The scattering profiles (A), pair distance distribution functions (PDDFs) (B), dimensionless Kratky plots (C) and Porod-Debye plots (D) of the complete sfRNAs of DENV2 (cyan line), ZIKV (red line) and WNV (blue line). The fine features of P1 in the scattering profiles in (A) arise from helical interstrand pair-distance correlation. The PDDF profiles in (B) were calculated using GNOM (qmax=0.3). The dimensionless Kratky plot and Porod-Debye plot for ZIKV xrRNA1 (red dashed line) were included in (C) and (D) for comparison, indicating that the structures of sfRNAs are more extended and open than that of ZIKV xrRNA1.


( C , D ) scattering profiles ( left ) , PDDFs ( middle ) dimensionless Kratky plots ( right ) of xrRNA1s ( C ) xrRNA2s ( D ) of DENV2 ( cyan ) , ZIKV ( red ) , WNV ( blue ) MVEV ( green ) . insets in ( C ) ( D ) show Guinier regions of respective scattering profiles with a linear fit . back - calculated scattering profile of ZIKV - xrRNA1 crystal structure ( black , open circle ) can be nicely fitted onto its experimental SAXS data ( red ) in ( C ) , while back - calculated scattering profile of MVEV - xrRNA2 crystal structure ( black , open circle ) fits poorly to its experimental SAXS data ( green ) in ( D ) . asymmetric PDDFs of xrRNA1 ( C ) xrRNA2 ( D ) , indicate slightly elongated molecules with asymmetric shapes . dimensionless Kratky plots of xrRNA1 ( C ) xrRNA2 ( D ) of DENV2 , ZIKV , WNV MVEV suggest xrRNA1s xrRNA2s are well - folded of reduced flexibility in solution .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D) The scattering profiles (left), the PDDFs (middle) and the dimensionless Kratky plots (right) of individual xrRNA1s (C) and xrRNA2s (D) of DENV2 (cyan), ZIKV (red), WNV (blue) and MVEV (green). The insets in (C) and (D) show the Guinier regions of the respective scattering profiles with a linear fit line. The back-calculated scattering profile of ZIKV- xrRNA1 crystal structure (black, open circle) can be nicely fitted onto its experimental SAXS data (red line) in (C), while the back-calculated scattering profile of MVEV- xrRNA2 crystal structure (black, open circle) fits poorly to its experimental SAXS data (green line) in (D). The asymmetric PDDFs of xrRNA1 (C) and xrRNA2 (D), indicate slightly elongated molecules with asymmetric shapes. The dimensionless Kratky plots of xrRNA1 (C) and xrRNA2 (D) of DENV2, ZIKV, WNV and MVEV suggest all individual xrRNA1s and xrRNA2s are well-folded and of reduced flexibility in solution.


( A ) - ( C ) scattering profiles ( A ) , PDDFs ( B ) dimensionless Kratky plots ( C ) of xrRNA1 - 2s from DENV2 ( cyan ) , ZIKV ( red ) WNV ( blue ) . inset in ( A ) shows guinier regions of respective scattering profiles with linear fit lines . dimensionless Kratky plot of ZIKV xrRNA1 ( red dashed ) was included in ( C ) for comparison , indicating that structures of xrRNA1 - 2s are well folded .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The scattering profiles (A), the PDDFs (B) and the dimensionless Kratky plots (C) of xrRNA1-2s from DENV2 (cyan), ZIKV (red) and WNV (blue). The inset in (A) shows the guinier regions of the respective scattering profiles with linear fit lines. The dimensionless Kratky plot of ZIKV xrRNA1 (red dashed line) was included in (C) for comparison, indicating that the structures of xrRNA1-2s are all well folded.


( A ) - ( C ) scattering profiles ( A ) , PDDFs ( B ) dimensionless Kratky plots ( C ) of DB12s from DENV2 ( cyan ) , ZIKV ( red ) WNV ( blue ) . inset in ( A ) shows Guinier regions of respective scattering profiles with linear fit lines . dimensionless Kratky plot of ZIKV xrRNA1 ( red dashed ) was included in ( C ) for comparison , indicating that structures of DB12s of ZIKV WNV are well folded , but that of DB12 of DENV2 is partially folded . Kratky plots for two PK3 mutants of DB12s , ZIKV - DB12 - PK3 ∆ ( green ) WNV - DB12 - PK3 ∆ ( black ) , respectively , are also included for comparison , suggesting reduced compactness upon PK3 mutations .

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(A)-(C) The scattering profiles (A), the PDDFs (B) and the dimensionless Kratky plots (C) of DB12s from DENV2 (cyan), ZIKV (red) and WNV (blue). The inset in (A) shows the Guinier regions of the respective scattering profiles with linear fit lines. The dimensionless Kratky plot of ZIKV xrRNA1 (red dashed line) was included in (C) for comparison, indicating that the structures of DB12s of ZIKV and WNV are well folded, but that of DB12 of DENV2 is partially folded. The Kratky plots for the two PK3 mutants of DB12s, the ZIKV- DB12-PK3∆ (green) and WNV- DB12-PK3∆ (black), respectively, are also included for comparison, suggesting reduced compactness upon PK3 mutations.


( C ) E expression of ZIKV mutants showed by IFA .

(C) E protein expression of ZIKV and the mutants showed by IFA.


( D ) progeny in cultured supernatants of ZIKV mutants were detected by qRT - PCR in three technical replicates . Two - way ANOVA Dunnett ' s multiple comparison was used for statistical . error bars represent standard deviation . * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(D) The progeny virus RNA in cultured supernatants of ZIKV and the mutants were detected by qRT-PCR in three technical replicates. Two-way ANOVA and Dunnett's multiple comparison test was used for statistical analysis. The error bars represent the standard deviation. *** p<0.001, **** p<0.0001.


( E ) luciferase activity of ZIKV - Rep mutants were measured at different time points after transfection in three technical repelicates . error bars represent standard deviation .

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(E) The luciferase activity of the ZIKV-Rep and the mutants were measured at different time points after transfection in three technical repelicates. The error bars represent the standard deviation.


( A ) - ( C ) scattering profiles ( A ) , PDDFs ( B ) dimensionless Kratky plots ( C ) of 3 ' SLs from DENV2 ( cyan ) , ZIKV ( red ) , WNV ( blue ) WNV - 3 ' SLM ( 438UAG441A → 438GUC441U ) mutant ( green ) . inset in ( A ) shows guinier regions of respective scattering profiles with linear fit lines .

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(A)-(C) The scattering profiles (A), the PDDFs (B) and the dimensionless Kratky plots (C) of 3'SLs from DENV2 (cyan), ZIKV (red), WNV (blue) and WNV-3'SLM (438UAG441A→438GUC441U) mutant (green). The inset in (A) shows the guinier regions of the respective scattering profiles with linear fit lines.


( A ) - ( C ) fitting chi2 are plotted against ensemble for complete sfRNAs of DENV2 ( A ) , ZIKV ( B ) WNV ( C ) , suggesting optimized minimal structural ensembles with 3 , 3 , 3 conformers , respectively . respective insets show distribution of minimal ensembles initial pools of 10000 random conformers as a function of Rg .

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(A)-(C) The fitting chi2 are plotted against the ensemble size for complete sfRNAs of DENV2 (A), ZIKV (B) and WNV (C), suggesting optimized minimal structural ensembles with 3, 3 ,3 conformers, respectively. The respective insets show the distribution of the minimal ensembles and the initial pools of 10000 random conformers as a function of Rg.


( a ) Hiw - associated was purified by TAP ( see Online Methods ) analyzed by one - dimensional SDS - PAGE gel followed by Sypro Ruby . Mass spectrometry identified full - Hiw , β - tubulin , Rae1 Fsn , indicated by arrows .

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(a) The Hiw-associated complex was purified by TAP (see Online Methods) and analyzed by one-dimensional SDS-PAGE gel followed by Sypro Ruby staining. Mass spectrometry identified the full-length Hiw, β-tubulin, Rae1 and Fsn, indicated by arrows.


( b ) Rae1 - associated purified from fly brains by TAP was analyzed by one - dimensional SDS - PAGE gel followed by Coomassie blue G - 250 . Mass spectrometry identified Hiw , Nup98 Rae1 , indicated by arrows .

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(b) The Rae1-associated complex purified from fly brains by TAP was analyzed by one-dimensional SDS-PAGE gel followed by Coomassie blue G-250 staining. Mass spectrometry identified Hiw, Nup98 and Rae1, indicated by arrows.


( c ) Larval brain lysate of wild - type ( WT ) , hiw null mutant ( hiwΔN ) Fsn mutant ( Fsnf06595 / Df ( 2R ) 7872 ) flies was subject to co - immunoprecipitation with antibody to Hiw ( Hiw2b ) . Both input immunoprecipitated complexes were analyzed by western blots with antibodies to Hiw or Rae1 . was also blotted with antibody to β - tubulin to ensure equal amount of total in input samples . Full - blots are presented in Supplementary Figure 8 .

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(c) Larval brain lysate of wild-type (WT), hiw null mutant (hiwΔN) and Fsn mutant (Fsnf06595/Df(2R)7872) flies was subject to co-immunoprecipitation with antibody to Hiw (Hiw2b). Both the input and the immunoprecipitated complexes were analyzed by western blots with antibodies to Hiw or Rae1. The membrane was also blotted with antibody to β-tubulin to ensure equal amount of total proteins in the input samples. Full-length blots are presented in Supplementary Figure 8.


( a ) gene structure of Rae1 . black - arrowed boxes indicate exons . A transposable element , P { GawB } NP3499 , located ∼ 400 bp upstream of first exon of Rae1 was used to generate imprecise excision mutants of Rae1 . PCR indicates that ∼ 900 bp a ∼ 1 . 5 - k bp fragment in Rae1 promoter region are missing in Rae1EX28 Rae1EXB12 , respectively . ( b ) Western blot on Rae1 in brain lysate of wild - type , Df ( 2R ) 5764 / + , Rae1EX28 / + , or late second instar Rae1EX28 , Rae1EX28 / Df ( 2R ) 5764 ( / Df ) Rae1EXB12 / Df ( 2R ) 5764 larvae . β - tubulin was used as loading control . Full - blots are presented in Supplementary Figure 9 . ( c ) of Rae1 in Rae1 heterozygous homozygous mutant brains . Rae1 were first normalized to β - tubulin are presented as of wild - type level . * P 0 . 05 , * * P 0 . 001 . Error bars denote s . e . m .

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

(a) The gene structure of Rae1. The black-arrowed boxes indicate exons. A transposable element, P{GawB}NP3499, located ∼400 bp upstream of the first exon of Rae1 was used to generate imprecise excision mutants of Rae1. PCR analysis indicates that ∼900 bp and a ∼1.5-k bp DNA fragment in the Rae1 promoter region are missing in Rae1EX28 and Rae1EXB12, respectively. (b) Western blot analysis on Rae1 protein in the brain lysate of wild-type, Df(2R)5764/+, Rae1EX28/+, or late second instar Rae1EX28, Rae1EX28/Df(2R)5764(/Df) and Rae1EXB12 /Df(2R)5764 larvae. β-tubulin was used as the loading control. Full-length blots are presented in Supplementary Figure 9. (c) Quantification of Rae1 protein levels in the Rae1 heterozygous and homozygous mutant brains. Rae1 protein levels were first normalized to β-tubulin protein levels and are presented as percentage of wild-type level. *P 0.05, **P 0.001. Error bars denote s.e.m.


( a ) Representative confocal of segment A3 muscle 6 / 7 synapses stained for both DVGLUT ( green ) FasII ( red ) , in late 2nd / early 3rd instar wild - type , Rae1EX28 , Rae1EX28 presynaptic rescue ( Rae1EX28 ; elav - Gal4 / UAS - NTAP - Rae1 ) Rae1EX28 wallenda suppression ( Rae1EX28 ; wnd3 ) larvae . Scale bar represents 10 μm . ( b , c ) of bouton number ( b ) ( c ) in wild - type , Rae1EX28 / Df , Rae1EX28 , Rae1EX28 rescue , Rae1EX28 ; wnd3 suppression hiwΔN larval NMJs ( segment A3 muscle 6 / 7 ; n = 20 , 21 , 18 , 20 , 20 11 , respectively , for bouton number ; n = 271 , 465 , 548 , 726 , 486 826 , respectively , for bouton ) . There was no significant difference in either bouton number or bouton between Rae1EX28 Rae1EX28 / Df ( P > 0 . 1 for both comparisons ) . * P 0 . 001 . Error bars denote s . e . m .

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

(a) Representative confocal images of segment A3 muscle 6/7 synapses stained for both DVGLUT (green) and FasII (red), in late 2nd/early 3rd instar wild-type, Rae1EX28, Rae1EX28 presynaptic rescue (Rae1EX28; elav-Gal4/UAS-NTAP-Rae1) and Rae1EX28 wallenda suppression (Rae1EX28; wnd3) larvae. Scale bar represents 10 μm. (b,c) Quantification of bouton number (b) and size (c) in wild-type, Rae1EX28/Df, Rae1EX28, Rae1EX28 rescue, Rae1EX28; wnd3 suppression and hiwΔN larval NMJs (segment A3 muscle 6/7; n = 20, 21, 18, 20, 20 and 11, respectively, for bouton number; n = 271, 465, 548, 726, 486 and 826, respectively, for bouton size). There was no significant difference in either bouton number or bouton size between Rae1EX28 and Rae1EX28/Df (P > 0.1 for both comparisons). *P 0.001. Error bars denote s.e.m.


( a ) Representative confocal of muscle 4 synapses stained for both DVGLUT ( green ) FasII ( red ) in wild - type , Rae1EX28 / + , hiwND51 , hiwND51 ; Rae1EX28 / + , hiwΔN hiwΔN ; Rae1EX28 / + larvae . Scale bar represents 10 μm . ( b ) of bouton number at muscle 4 NMJs in wild - type , Rae1EX28 / + , hiwND51 , hiwND51 ; Rae1EX28 / + , hiwΔN hiwΔN ; Rae1EX28 / + larvae ( n = 27 , 23 , 42 , 46 , 23 25 , respectively ) . There was a significant increase in number of boutons formed in hiwND51 ; Rae1EX28 / + compared with hiwND51 3rd instar larval NMJs ( * P 0 . 001 ) . number of boutons in hiwΔN ; Rae1EX28 / + double mutant was not significantly different from that in hiwΔN ( * * P > 0 . 5 ) , demonstrating a lack of enhancement of hiwΔN phenotype by Rae1EX28 / + . Error bars denote s . e . m .

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

(a) Representative confocal images of muscle 4 synapses stained for both DVGLUT (green) and FasII (red) in wild-type, Rae1EX28/+, hiwND51, hiwND51; Rae1EX28/+, hiwΔN and hiwΔN; Rae1EX28/+ larvae. Scale bar represents 10 μm. (b) Quantification of bouton number at muscle 4 NMJs in wild-type, Rae1EX28/+, hiwND51, hiwND51; Rae1EX28/+, hiwΔN and hiwΔN; Rae1EX28/+ larvae (n = 27, 23, 42, 46, 23 and 25 cells, respectively). There was a significant increase in the number of boutons formed in hiwND51; Rae1EX28/+ compared with hiwND51 3rd instar larval NMJs (*P 0.001). The number of boutons in the hiwΔN; Rae1EX28/+ double mutant was not significantly different from that in hiwΔN (**P > 0.5), demonstrating a lack of enhancement of the hiwΔN phenotype by Rae1EX28/+. Error bars denote s.e.m.


( a ) Schematic presentation of NTAP - tagged ( NT - ) HM - tagged ( HM - ) hiw transgenes encoding full - , mutated or truncated Hiw . Hiw functional domains are marked with colored boxes . positions of - acid substitution in NT - HiwΔRING added acid residue necessary to maintain reading frame in NT - Hiw - RCC1 NT - Hiw - PHR are indicated . right column summarizes ability of given hiw transgene to rescue synaptic overgrowth phenotype in hiw mutants , to cause dominant negative overgrowth phenotype when expressed in wild - type background to bind to Rae1 . ( b ) Western blot of fly brain lysate using antibody to TAP ( PAP , Sigma ) or Myc revealed expression of wild - type mutant hiw transgenes in predicted .

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

(a) Schematic presentation of NTAP-tagged (NT-) and HM-tagged (HM-) hiw transgenes encoding full-length, mutated or truncated Hiw proteins. Hiw functional domains are marked with colored boxes. The positions of amino-acid substitution in NT-HiwΔRING and the added amino acid residue necessary to maintain the reading frame in NT-Hiw-RCC1 and NT-Hiw-PHR are indicated. The right column summarizes the ability of the given hiw transgene to rescue the synaptic overgrowth phenotype in hiw mutants, to cause dominant negative overgrowth phenotype when expressed in wild-type background and to bind to Rae1. (b) Western blot analysis of fly brain lysate using antibody to TAP (PAP, Sigma) or Myc revealed the expression of wild-type and mutant hiw transgenes in predicted size.


( c ) Representative confocal of muscle 4 synapses stained for DVGLUT ( green ) FasII ( red ) in wild - type ( + / hiwND8 ; + / + ; GS - elav - Gal4 / + ) , NT - Hiw overexpression ( + / hiwND8 ; UAS - NT - hiw / + ; GS - elav - Gal4 / + ) , NT - Hiw - NT overexpression ( + / hiwND8 ; UAS - NT - hiw - NT / + ; GS - elav - Gal4 / + ) , NT - Hiw - CT1000 overexpression ( + / hiwND8 ; + / + ; GS - elav - Gal4 / UAS - NT - hiw - CT1000 ) , NT - Hiw - PHR overexpression ( + / hiwND8 ; + / + ; GS - elav - Gal4 / UAS - NT - hiw - PHR ) , HM - Hiw - HindIII overexpression ( + / hiwND8 ; UAS - HW - hiw - HindIII / + ; GS - elav - Gal4 / + ) , NT - Hiw - RCC1 overexpression ( NT - hiw - RCC1 / hiwND8 ; + / + ; GS - elav - Gal4 / + ) , or NT - Hiw - CT overexpression ( + / hiwND8 ; + / + ; GS - elav - Gal4 / UAS - NT - hiw - CT ) 3rd larval NMJs . Overexpression is indicated by an up arrow . Scale bar represents 10 μm . ( d ) of number of boutons at muscle 4 NMJs in wild - type larvae or larvae overexpressing wild - type or mutant hiw transgenes ( n = 23 , 24 , 26 , 27 , 26 , 21 , 25 27 , respectively ; * P 0 . 001 ) . Error bars denote s . e . m .

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

(c) Representative confocal images of muscle 4 synapses stained for DVGLUT (green) and FasII (red) in wild-type (+/hiwND8; +/+; GS-elav-Gal4/+), NT-Hiw overexpression (+/hiwND8; UAS-NT-hiw/+; GS-elav-Gal4/+), NT-Hiw-NT overexpression (+/hiwND8; UAS-NT-hiw-NT/+; GS-elav-Gal4/+), NT-Hiw-CT1000 overexpression (+/hiwND8; +/+; GS-elav-Gal4/UAS-NT-hiw-CT1000), NT-Hiw-PHR overexpression (+/hiwND8; +/+; GS-elav-Gal4/UAS-NT-hiw-PHR), HM-Hiw-HindIII overexpression (+/hiwND8; UAS-HW-hiw-HindIII/+; GS-elav-Gal4/+), NT-Hiw-RCC1 overexpression (NT-hiw-RCC1/hiwND8; +/+; GS-elav-Gal4/+), or NT-Hiw-CT overexpression (+/hiwND8; +/+; GS-elav-Gal4/UAS-NT-hiw-CT) 3rd larval NMJs. Overexpression is indicated by an up arrow. Scale bar represents 10 μm. (d) Quantification of the number of boutons at muscle 4 NMJs in wild-type larvae or larvae overexpressing wild-type or mutant hiw transgenes (n = 23, 24, 26, 27, 26, 21, 25 and 27, respectively; *P 0.001). Error bars denote s.e.m.


( a ) Indicated NTAP - tagged hiw transgenes ( described in Fig . 5 ) a TAP only transgene were expressed in neurons under control of BG380 - Gal4 driver . Larval brain lysates from each sample were subject to IgG pulldown . Both inputs IgG pulldown complexes were analyzed by western blot using antibody to Rae1 . Rae1 was present in of pulldown complexes except for those from TAP only , NT - Hiw - CT1000 NT - Hiw - NT . Full - blots are presented in Supplementary Figure 10 . ( b ) of interaction between various Hiw transgenic Rae1 . Rae1 intensities in TAP pulldown blots were normalized to both intensities molecular weights of corresponding TAP pulldown .

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

(a) Indicated NTAP-tagged hiw transgenes (described in Fig. 5) and a TAP only transgene were expressed in neurons under the control of the BG380-Gal4 driver. Larval brain lysates from each sample were subject to IgG pulldown. Both the inputs and the IgG pulldown complexes were analyzed by western blot using antibody to Rae1. Rae1 was present in all of the pulldown complexes except for those from TAP only, NT-Hiw-CT1000 and NT-Hiw-NT. Full-length blots are presented in Supplementary Figure 10. (b) Quantification of the interaction between various Hiw transgenic proteins and Rae1. Rae1 intensities in TAP pulldown blots were normalized to both the intensities and molecular weights of the corresponding TAP pulldown proteins.


( c ) Representative confocal of muscle 4 synapses stained for both DVGLUT ( green ) FasII ( red ) in wild - type ( BG380 / Y ; + / + ; + / + ) , NT - Hiw - CT GAP - GFP ( BG380 / Y ; UAS - GAP - GFP / + ; UAS - NT - hiw - CT / + ) , NT - Hiw - CT Rae1 ( BG380 / Y ; + / + ; UAS - NT - hiw - CT / UAS - GFP - Rae1 ) larvae . Scale bar represents 10 μm . ( d ) of number of boutons at muscle 4 NMJs in wild - type , NT - Hiw - CT overexpression ( BG380 / Y ; + / + ; UAS - NT - hiw - CT / + ) , NT - Hiw - CT GAP - GFP , NT - Hiw - CT Rae1 ( n = 24 , 27 , 32 26 , respectively ) larvae . Error bars denote s . e . m . ; * P 0 . 001 .

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

(c) Representative confocal images of muscle 4 synapses stained for both DVGLUT (green) and FasII (red) in wild-type (BG380/Y;+/+; +/+), NT-Hiw-CT and GAP-GFP (BG380/Y; UAS-GAP-GFP/+; UAS-NT-hiw-CT/+), and NT-Hiw-CT and Rae1 (BG380/Y; +/+; UAS-NT-hiw-CT/UAS-GFP-Rae1) larvae. Scale bar represents 10 μm. (d) Quantification of number of boutons at muscle 4 NMJs in wild-type, NT-Hiw-CT overexpression (BG380/Y; +/+; UAS-NT-hiw-CT/+), NT-Hiw-CT and GAP-GFP, and NT-Hiw-CT and Rae1 (n = 24, 27, 32 and 26, respectively) larvae. Error bars denote s.e.m.; *P 0.001.


( a ) Neuronal expression of neither Hiw nor Rae1 rescues synaptic terminal overgrowth phenotype caused by loss of function of other gene . of number of boutons in wild - type , Rae1EX28 , Rae1EX28 ; Hiw rescue ( Res ) ( Rae1EX28 ; elav - Gal4 / UAS - GFP - hiw ) , hiwΔN hiwΔN ; Rae1 rescue ( hiwΔN ; elav - Gal4 / UAS - NTAP - Rae1 ) larval NMJs ( segment A3 muscle 6 / 7 ; n = 12 , 12 , 12 , 7 11 , respectively ) . ( b ) Representative confocal of wild - type ( BG380 - Gal4 / + ; + / + ; UAS - GFP - hiw / + ) or Rae1 mutant ( BG380 - Gal4 / + ; Rae1EX28 ; UAS - GFP - hiw / + ) ventral ganglions with UAS - GFP - hiw trangene expressed in neurons . larvae were stained with antibodies to horseradish peroxidase ( HRP , red ) GFP ( green ) . Scale bar represents 10 μm .

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

(a) Neuronal expression of neither Hiw nor Rae1 rescues the synaptic terminal overgrowth phenotype caused by the loss of function of the other gene. Quantification of the number of boutons in wild-type, Rae1EX28, Rae1EX28; Hiw rescue (Res) (Rae1EX28; elav-Gal4/UAS-GFP-hiw), hiwΔN and hiwΔN; Rae1 rescue (hiwΔN; elav-Gal4/UAS-NTAP-Rae1) larval NMJs (segment A3 muscle 6/7; n = 12, 12, 12, 7 and 11, respectively). (b) Representative confocal images of wild-type (BG380-Gal4/+; +/+; UAS-GFP-hiw/+) or Rae1 mutant (BG380-Gal4/+; Rae1EX28; UAS-GFP-hiw/+) ventral ganglions with UAS-GFP-hiw trangene expressed in neurons. The larvae were stained with antibodies to horseradish peroxidase (HRP, red) and GFP (green). Scale bar represents 10 μm.


( c ) Western blots of total extracted from wild - type , hiwΔN , Rae1EX28 Rae1EX28 rescue ( Rae1EX28 ; elav - Gal4 / UAS - NTAP - Rae1 ) larval brains probed with indicated antibodies . Full - blots are presented in Supplementary Figure 11 .

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

(c) Western blots of total proteins extracted from wild-type, hiwΔN, Rae1EX28 and Rae1EX28 rescue (Rae1EX28; elav-Gal4/UAS-NTAP-Rae1) larval brains probed with indicated antibodies. Full-length blots are presented in Supplementary Figure 11.


( d ) Western blot on total extracted from hiwΔN , wild - type ( C155 - Gal4 / + ) Rae1 expression ( C155 - Gal4 / + ; UAS - NTAP - Rae1 / + ) larval brains . Full - blots are presented in Supplementary Figure 12 . ( e ) of Hiw in wild - type Rae1 expression larval brains ( * P 0 . 001 , on seven independent experiments ) . Arrows indicate a 38 - kDa endogenous Rae1 arrowheads indicate 58 - kDa transgenic NTAP - Rae1 . Error bars denote s . e . m .

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

(d) Western blot analysis on total proteins extracted from hiwΔN, wild-type (C155-Gal4/+) and Rae1 expression (C155-Gal4/+; UAS-NTAP-Rae1/+) larval brains. Full-length blots are presented in Supplementary Figure 12. (e) Quantification of Hiw protein levels in the wild-type and the Rae1 expression larval brains (*P 0.001, based on seven independent experiments). Arrows indicate a 38-kDa endogenous Rae1 protein and the arrowheads indicate the 58-kDa transgenic NTAP-Rae1 protein. Error bars denote s.e.m.


( a ) Representative confocal of segment A3 muscle 6 / 7 synapses stained for both DVGLUT ( green ) FasII ( red ) , in wild - type , Rae1EX28 , Rae1EX28 ; atg1 / + , Rae1EX28 ; atg2 / + Rae1EX28 ; atg18 / + wandering larvae . Scale bar represents 10 μm . ( b ) of number of boutons in wild - type , atg1 / + , atg2 / + , atg18 / + , Rae1EX28 , Rae1EX28 ; atg1 / + , Rae1EX28 ; atg2 / + Rae1EX28 ; atg18 / + larval NMJs ( segment A3 muscle 6 / 7 ; n = 12 , 12 , 12 , 12 , 12 , 16 , 12 21 , respectively ) .

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

(a) Representative confocal images of segment A3 muscle 6/7 synapses stained for both DVGLUT (green) and FasII (red), in wild-type, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ wandering larvae. Scale bar represents 10 μm. (b) Quantification of the number of boutons in wild-type, atg1/+, atg2/+, atg18/+, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ larval NMJs (segment A3 muscle 6/7; n = 12, 12, 12, 12, 12, 16, 12 and 21, respectively).


( c ) Western blot on total extracted from wild - type , Rae1EX28 , Rae1EX28 ; atg1 / + , Rae1EX28 ; atg2 / + Rae1EX28 ; atg18 / + larval brains . neural form of β - catenin ( 82 kDa , see Online Methods ) was used as an internal control of neuronal . Full - blots are presented in Supplementary Figure 13 . ( d ) of Hiw in wild - type , Rae1EX28 , Rae1EX28 ; atg1 / + , Rae1EX28 ; atg2 / + Rae1EX28 ; atg18 / + larval brains ( on six independent experiments ) .

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

(c) Western blot analysis on total proteins extracted from wild-type, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ larval brains. The neural form of β-catenin (82 kDa, see Online Methods) was used as an internal control of neuronal proteins. Full-length blots are presented in Supplementary Figure 13. (d) Quantification of Hiw protein levels in wild-type, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ larval brains (based on six independent experiments).


( e ) Representative confocal of muscle 4synapses stained for both DVGLUT ( green ) FasII ( red ) in wandering larvae of wild - type ( C155 - Gal4 / + ) , Atg1Gap - GFP ( C155 - Gal4 / + ; UAS - Gap - GFP / + ; UAS - Atg16B / + ) , Atg1NTAP - Rae1 ( C155 - Gal4 / + ; UAS - NTAP - Rae1 / + ; UAS - Atg1 / + ) . Scale bar represents 10 μm . ( f ) of number of boutons at muscle 4synapses in wild - type , Rae1 expression ( C155 - Gal4 / + ; UAS - NTAP - Rae1 / + ) , Atg1Gap - GFP Atg1NTAP - Rae1 wandering larvae ( segment A2 - 4 , n = 28 , 27 , 33 34 , respectively ) . Error bars denote s . e . m . * P 0 . 001 , * * P 0 . 05 .

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

(e) Representative confocal images of muscle 4synapses stained for both DVGLUT (green) and FasII (red) in wandering larvae of wild-type (C155-Gal4/+), Atg1Gap-GFP (C155-Gal4/+; UAS-Gap-GFP/+; UAS-Atg16B/+), and Atg1NTAP-Rae1 (C155-Gal4/+; UAS-NTAP-Rae1/+; UAS-Atg1/+). Scale bar represents 10 μm. (f) Quantification of the number of boutons at muscle 4synapses in wild-type, Rae1 expression (C155-Gal4/+; UAS-NTAP-Rae1/+), Atg1Gap-GFP and Atg1NTAP-Rae1 wandering larvae (segment A2-4, n = 28, 27, 33 and 34, respectively). Error bars denote s.e.m. *P 0.001, **P 0.05.


a , TG in hepatocytes untreated ( None ) or treated with 3 - methyladenine ( 3MA ) cultured in regular medium ( RM ) or oleate ( OL ) ( * P 0 . 02 , * * P 0 . 002 , n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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, TG levels in hepatocytes untreated (None) or treated with 3-methyladenine (3MA) and cultured in regular medium (RM) or oleate (OL) (*P 0.02, **P 0.002, n = 3).


b , TG in vector - infected ( VEC ) siAtg5 in RM , OL or in MCDM ( * P 0 . 001 , n = 5 ) . OL values are in mM .

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

b, TG levels in vector-infected (VEC) and siAtg5 cells in RM, OL or in MCDM (*P  0.001, n = 5). OL values are in mM.


c , TG in wild - type ( WT ) or Atg5 knockout mice embryonic fibroblasts ( Atg5 - / - ) ( * P 0 . 01 or * * P 0 . 0001 , n = 5 ) .

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c, TG levels in wild-type (WT) or Atg5 knockout mice embryonic fibroblasts (Atg5-/-) (*P 0.01 or **P 0.0001, n = 5).


d f , from a b stained with BODIPY 493 / 503 ( d ) . Nuclei are highlighted with 4 , 6 - diamidino - 2 - phenylindole ( DAPI ; d ) . Error bars , 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

df, Cells from a and b stained with BODIPY 493/503 (d). Nuclei are highlighted with 4,6-diamidino-2-phenylindole (DAPI; d). Error bars, s.e.m.


d f , from a b stained with oil red O ( e ) or visualized by electron microscopy ( f ) . Right : quantifications of LD number ( * P 0 . 01 , * * P 0 . 001 with untreated wild - type ; # P 0 . 01 , # # P 0 . 001 with in RM ; n = 4 6 ) .

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

df, Cells from a and b stained with oil red O (e) or visualized by electron microscopy (f). Right: quantifications of LD number and size (*P  0.01, **P  0.001 with untreated wild-type cells; #P  0.01, # #P  0.001 with cells in RM; n = 46).


a , b , VEC siAtg5 cultured with oleate ( OL ) or in MCDM were examined for their rates of TG synthesis ( a ) β - oxidation ( b ) as compared to in regular medium alone ( * P 0 . 03 , * * P 0 . 004 with VEC in same medium , n = 3 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, VEC and siAtg5 cells cultured with oleate (OL) or in MCDM were examined for their rates of TG synthesis (a) and β-oxidation (b) as compared to cells in regular medium alone (*P  0.03, **P  0.004 with VEC cells in the same medium, n = 34).


c , d , Rates of TG decay in OL ( c ) MCDM ( d ) ( * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 as compared to VEC , n = 3 7 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, d, Rates of TG decay in OL (c) and MCDM (d) (*P  0.05, **P  0.01, ***P  0.001 as compared to VEC cells, n = 37).


e , TG in wild - type treated with dimethyl sulphoxide vehicle ( DMSO ) , 3 - methyladenine ( 3MA ) or diethylumbelliferyl phosphate ( DEUP ) ( * P 0 . 00001 with DMSO - treated , # P 0 . 003 with 3MA - treated , n = 6 ) . Error bars , 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

e, TG levels in wild-type cells treated with dimethyl sulphoxide vehicle (DMSO), 3-methyladenine (3MA) or diethylumbelliferyl phosphate (DEUP) (*P  0.00001 with DMSO-treated cells, #P  0.003 with 3MA-treated cells, n = 6). Error bars, s.e.m.


f , Co - localization of BODIPY 493 / 503 ( green ) with LAMP1 ( red ) or LC3 ( red ) in hepatocytes in MCDM .

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

f, Co-localization of BODIPY 493/503 (green) with LAMP1 (red) or LC3 (red) in hepatocytes in MCDM.


g , High - magnification regions of hepatocytes in MCDM alone ( none ) or treated with vinblastine stained as labelled . Arrows indicate co - localization .

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g, High-magnification regions of hepatocytes in MCDM alone (none) or treated with vinblastine and stained as labelled. Arrows indicate co-localization.


a , Electron of cultured hepatocytes . dm , double - vesicles . Arrows indicate membranes in LDs . Insets show double in nucleus ( N ) , lipid - containing vesicles ( L ) mitochondria ( mit ) , but not in LDs .

a, Electron micrographs of cultured hepatocytes. dm, double-membrane cytosolic vesicles. Arrows indicate membranes in LDs. Insets show the double membrane in nucleus (N), lipid-containing vesicles (L) and mitochondria (mit), but not in LDs.


b , Mouse liver LC3 immunogold . Insets show higher magnification . Arrowheads indicate gold particles ( black ) LC3 - labelled bilayer membranes ( ) . c , Percentages of autophagic vacuoles ( AVs ) containing only lipid ( Lipids , * ) , other cargo ( Other ) or mixed cargo ( Mixed ) in treated as in b ( * P 0 . 01 , * * P 0 . 001 with in RM , n = 4 6 ) . Left : representative examples . Error bars , s . e . m .

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

b, Mouse liver LC3 immunogold. Insets show higher magnification. Arrowheads indicate gold particles (black) and LC3-labelled bilayer membranes (white). c, Percentages of autophagic vacuoles (AVs) containing only lipid (Lipids, *), other cargo (Other) or mixed cargo (Mixed) in cells treated as in b (*P  0.01, **P  0.001 with cells in RM, n = 46). Left: representative examples. Error bars, s.e.m.


a , Immunoblots of homogenates ( Hom ) LDs from fed ( F ) or 24 - h starved ( S ) mice . IκB , inhibitor of factor of kappa polypeptide gene enhancer ; GPDH , glyceraldehyde - 3 - phosphate dehydrogenase .

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

a, Immunoblots of cellular homogenates (Hom) and LDs from fed (F) or 24-h starved (S) mice. IκB, inhibitor of the nuclear factor of kappa light polypeptide gene enhancer; GPDH, glyceraldehyde-3-phosphate dehydrogenase.


b , Percentages of autophagic vacuoles ( AVs ) containing only lipid , other cargo or mixed cargo calculated from samples processed as in Supplementary Fig . 16b ( * P 0 . 001 , * * P 0 . 0001 , n = 4 6 ) . ( Supplementary Figure 16 Effects of starvation on hepatic lipid droplets . a , Mouseliverelectron . Arrows : Lipid - containing double vesicles . b , Electron of autophagic vacuoles ( AVs ) isolated from mice fed or starved for indicated time periods . percentages of double vesicles containing lipid in isolated AVs from 3 - 4 mice per condition are shown ( * P < 0 . 01 as compared to fractions from fed ) . ( * ) indicate lipid )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Percentages of autophagic vacuoles (AVs) containing only lipid, other cargo or mixed cargo calculated from samples processed as in Supplementary Fig. 16b (*P  0.001, **P  0.0001, n = 46). (Supplementary Figure 16 Effects of starvation on hepatic lipid droplets. a, Mouseliverelectron micrographs. Arrows: Lipid-containing double membrane vesicles. b, Electron micrographs of autophagic vacuoles (AVs) isolated from mice fed or starved for the indicated time periods. The percentages of double membrane vesicles containing lipid in isolated AVs from 3-4 mice per condition are shown (*P<0.01 as compared to fractions from fed animals). (*) indicate lipid)


c , Immunoblots of Hom , AV lysosomes ( Lys ) . L - 1 , LAMP1 .

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c, Immunoblots of Hom, AV and lysosomes (Lys). L-1, LAMP1.


d , Immunoblots of liver Hom LDs from HFD - fed mice .

d, Immunoblots of liver Hom and LDs from HFD-fed mice.


e , Liver homogenate immunoblots . PDI , disulphide isomerase .

e, Liver homogenate immunoblots. PDI, protein disulphide isomerase.


f , Total hepatic TG cholesterol content ( * P 0 . 01 , * * P 0 . 00001 , n = 817 ) .

O O 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, Total hepatic TG and cholesterol content (*P 0.01, **P 0.00001, n = 817).


g, Hepatic TG concentration (*P  0.05, n = 3).


h , of cholesterol in lysosomes ( * P 0 . 02 , n = 4 ) .

h, Percentage of cellular cholesterol in lysosomes (*P  0.02, n = 4).


i , Immunoblots of homogenates isolated LDs . Error bars , s . e . m .

i, Immunoblots of homogenates and isolated LDs. Error bars, s.e.m.


rate of glucose uptake ( a , e ) , lactate released after 4 h of incubation ( b , f ) , glycolytic flux ( c , g ) pentose - phosphate flux ( d , h ) were assessed in MEF mouse cortical neurons in culture , as indicated , obtained from either WT or Pink1 KO ( Pink1 − / − ) 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 O O O O O 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 B-CONTROLLED_VAR O O O O O O

The rate of glucose uptake (a,e), lactate released after 4 h of incubation (b,f), glycolytic flux (c,g) and pentose-phosphate flux (d,h) were assessed in MEF and mouse cortical neurons in primary culture, as indicated, obtained from either WT or Pink1 KO (Pink1−/−) mice.


Blood concentrations of glucose ( i ) lactate ( j ) , rate of 14C - lactate appearance in blood from acutely injected [ U - 14C ] glucose in tail vein ( k ) of WT Pink1 KO mice were analysed . Data are expressed as mean ± s . e . m . * P0 . 05 ( Student ' s t - ; n = 3 - 4 independent experiments ) .

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Blood concentrations of glucose (i) and lactate (j), and the rate of 14C-lactate appearance in the blood from acutely injected [U-14C]glucose in the tail vein (k) of WT and Pink1 KO mice were analysed. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).


Quantitative real - time PCR ( RT - qPCR of relative mRNA abundances of Glut1 , Glut3 , Hk2 Gapdh in WT Pink1 KO miceMEF ( a ) ß - Actin mRNA was used for normalization , Pink1 KO data are expressed as change relative to WT .

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

Quantitative real-time PCR (RT-qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO miceMEF (a) ß-Actin mRNA was used for normalization, and the Pink1 KO data are expressed as the change relative to WT.


Western blot of abundances of PINK1 , GLUT1 , GLUT3 ( not detectable in MEF ) , HK2 , GAPDH HIF1α in WT Pink1 - null mice MEF ( b ) ß - Actin was used as loading control .

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

Western blot analysis of the protein abundances of PINK1, GLUT1, GLUT3 (not detectable in MEF), HK2, GAPDH and HIF1α in WT and Pink1-null mice MEF (b) ß-Actin was used as loading control.


Quantitative real - time PCR ( RT - qPCR of relative mRNA abundances of Glut1 , Glut3 , Hk2 Gapdh in WT Pink1 KO neurons ( c ) . ß - Actin mRNA was used for normalization , Pink1 KO data are expressed as change relative to WT .

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

Quantitative real-time PCR (RT-qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO neurons (c). ß-Actin mRNA was used for normalization, and the Pink1 KO data are expressed as the change relative to WT.


Western blot of abundances of PINK1 , GLUT1 , GLUT3 ( not detectable in MEF ) , HK2 , GAPDH HIF1α in WT Pink1 - null neurons ( d ) ; ß - Actin was used as loading control .

Western blot analysis of the protein abundances of PINK1, GLUT1, GLUT3 (not detectable in MEF), HK2, GAPDH and HIF1α in WT and Pink1-null neurons (d); ß-Actin was used as loading control.


( e ) Western blot against PINK1 , GLUT1 , GLUT3 HIF1α in WT Pink1 KO mice skeletal muscle samples ; α - Tubulin was used as loading control . Only representative western blots are shown per condition ; replicates semi - quantitative estimation of band intensities are shown in Supplementary Information . mean ± s . e . m . values of mRNA data were calculated from fold change of each ß - Actin - normalized transcript abundance in Pink1 KO samples versus that in WT . Thus , in cases , WT values were considered = 1 . 00 . * P0 . 05 versus corresponding WT ( Student ' s t - ; n = 3 - 4 independent experiments ) .

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

(e) Western blot against PINK1, GLUT1, GLUT3 and HIF1α in WT and Pink1 KO mice skeletal muscle samples; α-Tubulin was used as loading control. Only representative western blots are shown per condition; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information. The mean±s.e.m. values of the mRNA data were calculated from the fold change of each ß-Actin-normalized transcript abundance in the Pink1 KO samples versus that in the WT. Thus, in all cases, the WT values were considered=1.00. *P0.05 versus the corresponding WT (Student's t-test; n=3-4 independent experiments).


( a ) RT - qPCR of Hif1α mRNA abundance in MEF transfected with siHif1α , or siControl ( 100 nM ) , after 72 h .

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

(a) RT-qPCR analysis of Hif1α mRNA abundance in MEF transfected with siHif1α, or siControl (100 nM), after 72 h.


( b ) siHif1α effectively knockdowns expressed Hif1α cDNA WT , but not a Hif1α cDNA silent mutant form refractory to siHif1α ( Hif1α cDNA REFR . ) in mice MEF .

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

(b) siHif1α effectively knockdowns expressed Hif1α cDNA WT, but not a Hif1α cDNA silent mutant form refractory to siHif1α (Hif1α cDNA REFR.) in mice MEF.


( c ) RT - qPCR of relative mRNA abundances of Glut1 , Glut3 , Hk2 Gapdh in WT Pink1 KO mice MEF transfected with siControl or siHif1α . ß - Actin mRNA was used for normalization , data are expressed as change relative to WT - siControl .

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

(c) RT-qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO mice MEF transfected with siControl or siHif1α. ß-Actin mRNA was used for normalization, and all data are expressed as the change relative to WT-siControl.


( d ) Western blot against HIF1α in WT in Pink1 KO mice MEF transfected with siHif1α or siControl shows efficacy of siHif1α at knocking down endogenous HIF1α ; ß - actin was used as loading control . As shown , siHif1α partially prevented increase in GLUT1 , HK2 GAPDH abundances .

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

(d) Western blot against HIF1α in WT and in Pink1 KO mice MEF transfected with siHif1α or siControl shows the efficacy of the siHif1α at knocking down endogenous HIF1α; ß-actin was used as loading control. As shown, siHif1α partially prevented the increase in GLUT1, HK2 and GAPDH protein abundances.


increased rate of lactate released after 4 h of incubation ( e ) glycolytic flux ( f ) observed in Pink1 KO MEF was prevented by siHif1α .

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

The increased rate of lactate released after 4 h of incubation (e) and glycolytic flux (f) observed in the Pink1 KO MEF was prevented by siHif1α.


siHif1α was also effective at preventing endogenous increased HIF1α ( g ) , as well as increased glycolytic flux ( h ) observed in Pink1 KO neurons . Only representative western blots are shown ; replicates semi - quantitative estimation of band intensities are shown in Supplementary Information .

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 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

siHif1α was also effective at preventing the endogenous increased HIF1α protein (g), as well as the increased glycolytic flux (h) observed in Pink1 KO primary neurons. Only representative western blots are shown; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information.


( a ) Mitochondrial respiratory chain complexes activity values normalized to citrate synthase ( CS ) , ( b ) mitochondrial inner potential ( Δψm ) in WT Pink1 KO mice MEF .

O O O O 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

(a) Mitochondrial respiratory chain complexes activity values normalized to citrate synthase (CS), and (b) mitochondrial inner membrane potential (Δψm) in WT and Pink1 KO mice MEF.


( c ) Western blot against PDK1 HIF1α in WT Pink1 KO MEF transfected with siHif1α ( or its siControl ) ; ß - actin was used as loading control .

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

(c) Western blot against PDK1 and HIF1α in WT and Pink1 KO MEF transfected with siHif1α (or its siControl); ß-actin was used as loading control.


d ) PDH activity in WT Pink1 KO MEF transfected with siHif1α or siControl .

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

d) PDH complex activity in WT and Pink1 KO MEF transfected with siHif1α or siControl.


Rate of whole - H2O2 ( e ) mitochondrial O2 · − ( f ) detection in WT Pink1 KO

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

Rate of whole-cell H2O2 (e) and mitochondrial O2·− (f) detection in WT and Pink1 KO MEF


( g ) Detection of mitochondrial O2 · − using mitochondrial - specific probe MitoSox in MEF incubated in absence ( − ) or in presence ( + ) of plasma permeable GSH - EE , or by expressing mitochondrial - tagged forms of glutamate - cysteine ligase , ( + mitoGCL ; empty vector , pIRES2 - EGFP , denoted as − ) or catalase ( + mitoCAT ; empty vector denoted as − ) , shows effective mitochondrial ROS removal .

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

(g) Detection of mitochondrial O2·− using the mitochondrial-specific probe MitoSox in MEF incubated in the absence (−) or in the presence (+) of the plasma membrane permeable GSH-EE, or by expressing mitochondrial-tagged forms of glutamate-cysteine ligase, (+mitoGCL; empty vector, pIRES2-EGFP, denoted as −) or catalase (+ mitoCAT; empty vector denoted as −), shows effective mitochondrial ROS removal.


( h ) Western blot against HIF1α in Pink1 KO MEF incubated with ( or without ) GSH - EE , or transfected with empty vector , mitoGCL or mitoCAT , showing reduction in HIF1α abundance by treatments that remove mitochondrial ROS ; α - TUBULIN was used as loading control . Only representative western blots are shown ; replicates semi - quantitative estimation of band intensities are shown in Supplementary Information . Data are expressed as mean ± s . e . m . * P0 . 05 ( Student ' s t - ; n = 3 - 4 independent experiments ) .

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

(h) Western blot against HIF1α in Pink1 KO MEF incubated with (or without) GSH-EE, or transfected with empty vector, mitoGCL or mitoCAT, showing reduction in HIF1α protein abundance by the treatments that remove mitochondrial ROS; α-TUBULIN was used as loading control. Only representative western blots are shown; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).


( a ) Proportion of BrdU incorporation into cycle phases , ( b ) growth in WT Pink1 KO MEF after 72 h in absence ( denoted as - 2DG ) or in presence ( + 2DG ) of glucose - metabolizing inhibitor , 2 - deoxyglucose ( 2DG , 1 mM ) .

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

(a) Proportion of BrdU incorporation into DNA and cell cycle phases, and (b) cell growth in WT and Pink1 KO MEF after 72 h in the absence (denoted as -2DG) or in the presence (+2DG) of the glucose-metabolizing inhibitor, 2-deoxyglucose (2DG, 1 mM).


( c ) Proportions of BrdU incorporation into cycle phases , ( d ) growth in WT Pink1 KO MEF 72 h after transfection with siHif1α ( + siHif1α ) or siControl ( denoted as - siHif1α ) .

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

(c) Proportions of BrdU incorporation into DNA and cell cycle phases, and (d) cell growth in WT and Pink1 KO MEF 72 h after transfection with siHif1α (+siHif1α) or siControl (denoted as -siHif1α).


( e ) Ki67 , Sox2 DAPI immunostaining in subventricular zone of WT Pink1 KO mouse brain , corresponding . * P0 . 05 ; # P0 . 05 versus other conditions ( of variance followed by Bonferroni ; n = 3 - 4 independent experiments ) . data are expressed as mean ± s . e . m . Scale bar , 100 μm .

O O O B-MEASURED_VAR O B-MEASURED_VAR 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

(e) Ki67, Sox2 and DAPI immunostaining in the subventricular zone of WT and Pink1 KO mouse brain, and the corresponding quantification. *P0.05; #P0.05 versus all other conditions (analysis of variance followed by Bonferroni test; n=3-4 independent experiments). All data are expressed as mean±s.e.m. Scale bar, 100 μm.


a ) Rate of mitochondrial O2 · - detection in WT Pink1 KO mice neurons .

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

a) Rate of mitochondrial O2·- detection in WT and Pink1 KO mice primary neurons.


( b ) Proportion of BrdU incorporation into of WT Pink1 KO neurons .

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

(b) Proportion of BrdU incorporation into DNA of WT and Pink1 KO neurons.


( c ) Proportion of apoptosis in WT Pink1 KO neurons , as assessed by proportion of annexin V - positive / 7 - AAD - negative , determined by flow cytometry . Data are expressed as mean ± s . e . m . * P0 . 05 ( Student ' s t - ; n = 3 - 4 independent experiments ) .

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

(c) Proportion of apoptosis in WT and Pink1 KO neurons, as assessed by the proportion of annexin V-positive/7-AAD-negative cells, determined by flow cytometry. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).


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

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

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 ) SYCP1 ( pseudocoloured in pink ) on spread spermatocytes at ( F ) zygotene , ( G ) pachytene , ( H ) diplotene .

O O O O O O O B-MEASURED_VAR 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

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 ) HORMAD1 ( pseudocoloured in blue ) on spread spermatocytes at ( I ) zygotene , ( J ) pachytene , ( K ) diplotene . sex bivalent ( XY ) their pseudoautosomal regions ( arrows ) , as well as round ( arrowheads ) are indicated . Scale bar : 10 µm .

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

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

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

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.