( E ) ( Left panel ) Quantitation of the percentage of GFP - LC3 puncta positive cells . Cells were left untreated , exposed to nutrient starvation for 5 h or to metabolic stress for 18 h , with or without treatment with AO1 ( NAC ( 2 mM ) and L - ascorbic acid ( 2 mM ) ) for 24 h . U2OS cells stably over - expressing GFP - LC3 in the presence of either scrambled , TIGAR siRNA1 or TIGAR siRNA2 . ( Right panel ) Basal or nutrient starvation - induced ( 5 h ) ROS levels in U2OS cells in the presence of either scrambled , TIGAR siRNA1 or TIGAR siRNA2 with or without treatment with AO1 ( NAC ( 2 mM ) and L - ascorbic acid ( 2 mM ) ) for 24 h , measured by flow cytometry after DCF treatment . The results are expressed as the mean intensity of cell fluorescence ( and standard deviation ) . * represents significant difference from control conditions ( P < 0 . 05 ) ; # represents a lack of significant difference from control conditions ( P > 0 . 05 ) .

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

(E) (Left panel) Quantitation of the percentage of GFP-LC3 puncta positive cells. Cells were left untreated, exposed to nutrient starvation for 5 h or to metabolic stress for 18 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. U2OS cells stably over-expressing GFP-LC3 in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2. (Right panel) Basal or nutrient starvation-induced (5 h) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2 with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h, measured by flow cytometry after DCF treatment. The results are expressed as the mean intensity of cell fluorescence (and standard deviation). * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).


( A ) Western blot showing knockdown of ATG5 protein expression by ATG5 siRNA .

(A) Western blot showing knockdown of ATG5 protein expression by ATG5 siRNA.


( B ) RT - PCR showing knockdown of ATG10 mRNA expression by ATG10 siRNA .

(B) RT-PCR showing knockdown of ATG10 mRNA expression by ATG10 siRNA.


( C ) Quantitation of the percentage of GFP - LC3 puncta positive cells . U2OS cells stably over - expressing GFP - LC3 in the presence of either scrambled , TIGAR siRNA1 or TIGAR siRNA2 , and either scrambled , ATG5 siRNA or ATG10 siRNA . After 48 h , cells were left untreated or exposed to nutrient starvation for 5 h .

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

(C) Quantitation of the percentage of GFP-LC3 puncta positive cells. U2OS cells stably over-expressing GFP-LC3 in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, and either scrambled, ATG5 siRNA or ATG10 siRNA. After 48 h, cells were left untreated or exposed to nutrient starvation for 5 h.


( D ) ROS levels in U2OS cells in the presence of either scrambled , TIGAR siRNA1 or TIGAR siRNA2 , and scrambled , ATG5 siRNA or ATG10 siRNA , measured by flow cytometry after DCF treatment . After 48 h , cells were left untreated or exposed to nutrient starvation for 5 h . The results are expressed as the mean intensity of cell fluorescence ( and standard deviation ) . * represents significant difference from control conditions ( P < 0 . 05 ) ; # represents a lack of significant difference from control conditions ( P > 0 . 05 ) .

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 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

(D) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, and scrambled, ATG5 siRNA or ATG10 siRNA, measured by flow cytometry after DCF treatment. After 48 h, cells were left untreated or exposed to nutrient starvation for 5 h. The results are expressed as the mean intensity of cell fluorescence (and standard deviation). * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).


( A ) Quantitation of the percentage of GFP - LC3 puncta positive cells . U2OS cells stably over - expressing Flag - tagged - TIGAR ( clones TIGAR # 5 and TIGAR # 7 ) or control cells ( clones Cont # 1 and Cont # 3 ) were left untreated or exposed to nutrient starvation for 6 h , with or without treatment with Z - VAD - FMK for 24 h . The percentage of cells with GFP - LC3 puncta was calculated , and data are shown as the mean and standard deviation of the mean from three independent experiments .

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

(A) Quantitation of the percentage of GFP-LC3 puncta positive cells. U2OS cells stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3) were left untreated or exposed to nutrient starvation for 6 h, with or without treatment with Z-VAD-FMK for 24 h. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation of the mean from three independent experiments.


( B ) Apoptosis in U2OS cells stably over - expressing Flag - tagged - TIGAR ( clones TIGAR # 5 and TIGAR # 7 ) or control cells ( clones Cont # 1 and Cont # 3 ) , as measured by cells with a sub - G1 DNA content . Cells were transfected with either scrambled , TIGAR siRNA1 or TIGAR siRNA2 . Cells were left untreated , exposed to nutrient starvation for 6 h or to metabolic stress for 24 h .

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

(B) Apoptosis in U2OS cells stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3), as measured by cells with a sub-G1 DNA content. Cells were transfected with either scrambled, TIGAR siRNA1 or TIGAR siRNA2. Cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h.


( C ) Apoptosis in U2OS cells cotransfected with scrambled or TIGAR siRNAs , and scrambled , ATG5 siRNA or ATG10 siRNA , as measured by cells with a sub - G1 DNA content . After 48 h , cells were left untreated , exposed to nutrient starvation for 6 h or to metabolic stress for 24 h . Data are shown as the mean and standard deviation from three independent experiments . In each case , the increase in apoptosis after knockdown of ATG5 or ATG10 , compared with the matched control , was statistically significant ; * represents significant difference from control conditions ( P < 0 . 05 ) ; # represents a lack of significant difference from control conditions ( P > 0 . 05 ) .

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

(C) Apoptosis in U2OS cells cotransfected with scrambled or TIGAR siRNAs, and scrambled, ATG5 siRNA or ATG10 siRNA, as measured by cells with a sub-G1 DNA content. After 48 h, cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h. Data are shown as the mean and standard deviation from three independent experiments. In each case, the increase in apoptosis after knockdown of ATG5 or ATG10, compared with the matched control, was statistically significant; * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).


The central axial sections of the reconstructions . Note the C - terminal domain of gp104 and other low - density features that do not visualize well in the isosurface representation ( panels ( A - C ) ) . Letters G , H and I indicate the position of the cross - sections shown in the homonymously labeled panels below . A 69 Å - thick slice of the reconstruction of the extended sheath cryoEM map orthogonal to the sixfold axis . The proximal and distal parts of one of the three fibers that run at 82 ° to the sixfold axis are indicated with a semitransparent cyan and magenta lines , respectively . Sections of the spontaneously and urea contracted sheath structures that cut through the baseplate / proximal part of the fiber plane , whose position in the reconstructions is indicated with a red line in panels ( E and F ) . The putative direction of the distal part of the fiber that is completely disordered in the reconstructions is indicated with a solar cross symbol .

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

The central axial sections of the reconstructions. Note the C-terminal domain of gp104 and other low-density features that do not visualize well in the isosurface representation (panels (A-C)). Letters G, H and I indicate the position of the cross-sections shown in the homonymously labeled panels below. A 69 Å-thick slice of the reconstruction of the extended sheath cryoEM map orthogonal to the sixfold axis. The proximal and distal parts of one of the three fibers that run at 82° to the sixfold axis are indicated with a semitransparent cyan and magenta lines, respectively. Sections of the spontaneously and urea contracted sheath structures that cut through the baseplate/proximal part of the fiber plane, whose position in the reconstructions is indicated with a red line in panels (E and F). The putative direction of the distal part of the fiber that is completely disordered in the reconstructions is indicated with a solar cross symbol.


Percentage of phage particles in the extended , intermediate , and contracted states after 5 , 20 and 80 minutes of incubation with L . monocytogenes cell walls . The numbers above the bars show the number of particles contained in the tomograms . The average length of the tail and the standard deviation for each of the three states is given next to the color legend .

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

Percentage of phage particles in the extended, intermediate, and contracted states after 5, 20 and 80 minutes of incubation with L. monocytogenes cell walls. The numbers above the bars show the number of particles contained in the tomograms. The average length of the tail and the standard deviation for each of the three states is given next to the color legend.


The pre - attachment state . Here and elsewhere in this figure , the EM image in the lower panel is a slice through the cryoET reconstruction . Note that in the presence of the Listeria cell wall the gp108 fibers are no longer in their stowed conformation but instead are fully or partially unraveled . Gp108 fibers attach to the host cell wall but the sheath is not yet contracted . RBP / TF gp106 ( and possibly gp107 ) pyramids reorient to point toward the cell surface , change their conformation , and bind to the cell wall . The baseplate - proximal part of the sheath is in a contracted state , whereas the capsid - proximal part is still in an extended state . The baseplate - proximal part of the sheath contains a segment where the tail tube is clearly visible because it no longer interacts with the sheath ( lower panel ) . The post - attachment state . The tail is fully contracted and the tail tube has pierced through the cell wall . The capsid appears full indicating that DNA injection has not yet begun .

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

The pre-attachment state. Here and elsewhere in this figure, the EM image in the lower panel is a slice through the cryoET reconstruction. Note that in the presence of the Listeria cell wall the gp108 fibers are no longer in their stowed conformation but instead are fully or partially unraveled. Gp108 fibers attach to the host cell wall but the sheath is not yet contracted. RBP/TF gp106 (and possibly gp107) pyramids reorient to point toward the cell surface, change their conformation, and bind to the cell wall. The baseplate-proximal part of the sheath is in a contracted state, whereas the capsid-proximal part is still in an extended state. The baseplate-proximal part of the sheath contains a segment where the tail tube is clearly visible because it no longer interacts with the sheath (lower panel). The post-attachment state. The tail is fully contracted and the tail tube has pierced through the cell wall. The capsid appears full indicating that DNA injection has not yet begun.


B : Growth charts of 2 probands each showing typical growth patterns of self - limited DP , without compromised linear growth before puberty . Solid horizontal black lines connect green dots representing bone age to red dots at the equivalent chronological age .

O O O O O O O O O O O O O O O O O O O O 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: Growth charts of 2 probands each showing typical growth patterns of self-limited DP, without compromised linear growth before puberty. Solid horizontal black lines connect green dots representing bone age to red dots at the equivalent chronological age.


C : Biological consequences of the 2 identified N - terminal mutations . Both WT and mutant N - terminal protein fragments ( p . Arg156Leu and p . Glu161Lys ) were expressed in HEK293 cells as demonstrated by western blotting . The GFP - tagged protein products of both were not detected in the conditioned media of mammalian cells , as compared to wild type ( WT ) , and appear to be retained in the intracellular compartment ( mean ± s . e . m . ; n = 3 ) . Ponceau redstaining is shown to demonstrate equal protein loading for conditioned media . UT - untransfected negative control ; nd - not detected ; * : p = 0 . 01094 ( WT vs p . Arg156Leu ) and p = 0 . 04408 ( WT vs p . Glu161Lys ) by 2 - tailed t - test . It has to date not been possible to test cytoplasmic retention for the two C - terminal mutations due to difficulty expressing the full length or C - terminal protein fragment in mammalian cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Biological consequences of the 2 identified N-terminal mutations. Both WT and mutant N-terminal protein fragments (p.Arg156Leu and p.Glu161Lys) were expressed in HEK293 cells as demonstrated by western blotting. The GFP-tagged protein products of both were not detected in the conditioned media of mammalian cells, as compared to wild type (WT), and appear to be retained in the intracellular compartment (mean ± s.e.m.; n = 3). Ponceau redstaining is shown to demonstrate equal protein loading for conditioned media. UT - untransfected negative control; nd - not detected; *: p=0.01094 (WT vs p.Arg156Leu) and p=0.04408 (WT vs p.Glu161Lys) by 2-tailed t-test. It has to date not been possible to test cytoplasmic retention for the two C-terminal mutations due to difficulty expressing the full length or C-terminal protein fragment in mammalian cells.


A - J : Igsf10 expression was not observed at E10 . 5 ( A , sagittal section ) but readily detectable at E12 . 5 , in the NM ( B and C , frontal sections ) and E14 . 5 , along the migratory path of GnRH neurons ( D , frontal section ) . Igsf10 positive cells were negative for Isolectin b4 ( marker of vasculature ) at all developmental stages ( E , at E14 . 5 ) . Incubation with the sense probe resulted in no signal at all stages ( E14 . 5 shown in F ) . Peripherin - positive cells olfactory axons were extending within the Igsf10 - positive NM ( G and H ) . Cxcl12 mRNA expression is shown in I ( E14 . 5 , frontal section ) . GnRH neurons are located in the MPOA by E17 . 5 ( J , frontal section ) in an Igsf10 negative area .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J: Igsf10 expression was not observed at E10.5 (A, sagittal section) but readily detectable at E12.5, in the NM (B and C, frontal sections) and E14.5, along the migratory path of GnRH neurons (D, frontal section). Igsf10 positive cells were negative for Isolectin b4 (marker of vasculature) at all developmental stages (E, at E14.5). Incubation with the sense probe resulted in no signal at all stages (E14.5 shown in F). Peripherin-positive cells olfactory axons were extending within the Igsf10-positive NM (G and H). Cxcl12 mRNA expression is shown in I (E14.5, frontal section). GnRH neurons are located in the MPOA by E17.5 (J, frontal section) in an Igsf10 negative area.


K - N : In human 11pcw brains , IGSF10 expression pattern was similar to that observed in mouse , with GnRH neurons interspersed in an IGSF10 - positive NM ( K - N , frontal sections , see also schematic in P ) . IGSF10 positive cells were also detected in the olfactory bulb ( OB , panel O ) . Sense probe resulted in no specific signal ( panel N ) .

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

K-N: In human 11pcw brains, IGSF10 expression pattern was similar to that observed in mouse, with GnRH neurons interspersed in an IGSF10-positive NM (K-N, frontal sections, see also schematic in P). IGSF10 positive cells were also detected in the olfactory bulb (OB, panel O). Sense probe resulted in no specific signal (panel N).


A : Levels of Igsf10 expression in native NIH3T3 cells , and cells stably expressing a scrambled ( sh - scr ) or Igsf10 ( sh - Igsf10 ) shRNA .

O O O O O O O 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: Levels of Igsf10 expression in native NIH3T3 cells, and cells stably expressing a scrambled (sh-scr) or Igsf10 (sh-Igsf10) shRNA.


B and C : migration of GN11 cells from aggregates into NIH3T3 sh - scr ( B ) or sh - Igsf10 ( C ) . D : Analysis of the migration index ( see Methods ) .

O O O O O O O O O O O O O O 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 and C: migration of GN11 cells from aggregates into NIH3T3 sh-scr (B) or sh-Igsf10 (C).D: Analysis of the migration index (see Methods).


E : RT - PCR analysis in zebrafish of total embryos and larvae showing the expression of Igsf10 mRNA at different time points . Expected band size : 905 bp .

E: RT-PCR analysis in zebrafish of total embryos and larvae showing the expression of Igsf10 mRNA at different time points. Expected band size: 905 bp.


F : Representative time - lapse analysis of mispair control ( Control Sp - MO and Control ATG - MO ) and Igsf10 morpholinos ( Igsf10 Sp - MO and Igsf10 ATG - MO ) injected Tg ( gnrh3 : EGFP ) embryos and larvae , at different time - points . Images show lateral view of live larvaehead ( 48 and 72 hpf ) and dorsal view ( 4 and 5 dpf ) of live larvaehead ; anterior is left . White arrowheads indicate normal GnRH3 neuron clusters in the olfactory area of control embryos . Brackets show the extension of the projections towards the hypothalamus . White arrows indicate examples of abnormal GnRH3 neurons scattered in the olfactory area of morphants accompanied by lack of projections toward the hypothalamus , indicated with Δ ( hpf - hours post fertilisation ; dpf - days post fertilisation . ) Scale bar , 100 um ( panels at 48 and 72hpf in F ) , 150 um ( panels at 4 and 5dpf in F ) . Panel G : Effect of Igsf10 Splice - site morpholino injection observed at 48hrs . Quantification of the percentage of embryos showing an abnormal GnRH3 - neuron phenotype was significantly higher in Igsf10 Splice - site - MO ( Sp - MO ) injected embryos compared to relative controls . * : p = 0 . 02422 , * * : p = 0 . 00101 , * * * : p = 0 . 00020 , * * * * : p = 0 . 00001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Representative time-lapse analysis of mispair control (Control Sp-MO and Control ATG-MO) and Igsf10 morpholinos (Igsf10 Sp-MO and Igsf10 ATG-MO) injected Tg(gnrh3:EGFP) embryos and larvae, at different time-points. Images show lateral view of live larvaehead (48 and 72 hpf) and dorsal view (4 and 5 dpf) of live larvaehead; anterior is left. White arrowheads indicate normal GnRH3 neuron clusters in the olfactory area of control embryos. Brackets show the extension of the projections towards the hypothalamus. White arrows indicate examples of abnormal GnRH3 neurons scattered in the olfactory area of morphants accompanied by lack of projections toward the hypothalamus, indicated with Δ (hpf - hours post fertilisation; dpf - days post fertilisation.) Scale bar, 100 um (panels at 48 and 72hpf in F), 150 um (panels at 4 and 5dpf in F). Panel G: Effect of Igsf10 Splice-site morpholino injection observed at 48hrs. Quantification of the percentage of embryos showing an abnormal GnRH3-neuron phenotype was significantly higher in Igsf10 Splice-site-MO (Sp-MO) injected embryos compared to relative controls. *: p=0.02422, **: p=0.00101, ***: p=0.00020, ****: p=0.00001.


D . The relative abundance of valine ( Val ) and leucine - isoleucine ( Leu - Ile ) . N = 18 ; Boxplot , central band stands for median , boxes stand for 50 % of the data , whiskers stand for min or max of the data E . The relative abundance of 3 - hydroxybutyrate ( 3 - HOB ) and acetylcarnitine ( AC ) . N = 18 ; Boxplot , central band stands for median , boxes stand for 50 % of the data , whiskers stand for min or max of the data Data information : Error bars stand for SEM of biological repeats . The p value was calculated by two - tailed t - test with 2 - way ANOVA correction .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O 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. The relative abundance of valine (Val) and leucine-isoleucine (Leu-Ile). N=18; Boxplot, central band stands for median, boxes stand for 50% of the data, whiskers stand for min or max of the data E. The relative abundance of 3-hydroxybutyrate (3-HOB) and acetylcarnitine (AC). N=18; Boxplot, central band stands for median, boxes stand for 50% of the data, whiskers stand for min or max of the data Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.


F . The concentration of FGF21 in serum . Control , N = 18 ; POI , N = 20 ; Boxplot , central band stands for median , boxes stand for 50 % of the data , whiskers stand for min or max of the data G . The concentration of GDF15 in serum . Control , N = 14 ; POI , N = 9 ; Boxplot , central band stands for median , boxes stand for 50 % of the data , whiskers stand for min or max of the data . Data information : Error bars stand for SEM of biological repeats . The p value was calculated by two - tailed t - test with 2 - way ANOVA correction .

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

F. The concentration of FGF21 in serum. Control, N=18; POI, N=20; Boxplot, central band stands for median, boxes stand for 50% of the data, whiskers stand for min or max of the data G. The concentration of GDF15 in serum. Control, N=14; POI, N=9; Boxplot, central band stands for median, boxes stand for 50% of the data, whiskers stand for min or max of the data. Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.


A and B . The serum concentration of FSH and the changes of follicles in mice on low BCAA diet for 1 . 5 months . A . Control , N = 10 ; low BCAA , N = 8 ; B . Control , N = 4 ; low BCAA , N = 4 ; Data information : S1 , Primordial ; S2 , Primary ; S3 , Secondary ; S4 , Antral ; S5 , Atretic . Error bars stand for SEM of biological repeats . The p value was calculated by two - tailed t - test with 2 - way ANOVA correction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B. The serum concentration of FSH and the changes of follicles in mice on low BCAA diet for 1.5 months. A. Control, N=10; low BCAA, N=8; B. Control, N=4; low BCAA, N=4; Data information: S1, Primordial; S2, Primary; S3, Secondary; S4, Antral; S5, Atretic. Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.


C . The serum concentration of FSH in mice on a different batch of the low BCAA diet . N = 5 ; D . The serum concentration of FSH in mice on the low BCAA diet from Research Diet . N = 10 ; Data information : The p value was calculated by two - tailed t - test with 2 - way ANOVA correction .

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

C. The serum concentration of FSH in mice on a different batch of the low BCAA diet. N=5; D. The serum concentration of FSH in mice on the low BCAA diet from Research Diet. N=10; Data information: The p value was calculated by two-tailed t-test with 2-way ANOVA correction.


E and F . The serum concentration of AMH and E2 in mice on the low BCAA diet for 1 . 5 months . Control , N = 5 ; low BCAA , N = 3 Data information : The p value was calculated by two - tailed t - test with 2 - way ANOVA correction .

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

E and F. The serum concentration of AMH and E2 in mice on the low BCAA diet for 1.5 months. Control, N=5; low BCAA, N=3 Data information: The p value was calculated by two-tailed t-test with 2-way ANOVA correction.


A - B . Elevation of serum ceramide in POI patients from the Fudan Cohort or the Shandong Cohort . Left , the relative abundance of total ceramide ; right , the relative abundance of ceramide with specific acyl chain . A . N = 18 ; B . N = 10 ; Truncated violin plot , central band stands for median , dotted lines stand for the upper quartile or the lower quartile of the data ; Data information : The p value was calculated by two - tailed t - test with 2 - way ANOVA correction .

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 B-MEASURED_VAR 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

A-B. Elevation of serum ceramide in POI patients from the Fudan Cohort or the Shandong Cohort. Left, the relative abundance of total ceramide; right, the relative abundance of ceramide with specific acyl chain. A. N=18; B. N=10; Truncated violin plot, central band stands for median, dotted lines stand for the upper quartile or the lower quartile of the data; Data information: The p value was calculated by two-tailed t-test with 2-way ANOVA correction.


D . Elevation of ceramide in the ovaries of mice on a low BCAA diet . N = 10 ; Truncated violin plot , central band stands for median , dotted lines stand for the upper quartile or the lower quartile of the data Data information : The p value was calculated by two - tailed t - test with 2 - way ANOVA correction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Elevation of ceramide in the ovaries of mice on a low BCAA diet. N=10; Truncated violin plot, central band stands for median, dotted lines stand for the upper quartile or the lower quartile of the data Data information: The p value was calculated by two-tailed t-test with 2-way ANOVA correction.


F . The serum concentration of FSH in mice with ceramide treatment . N = 7 Data information : The p value was calculated by two - tailed t - test with 2 - way ANOVA correction .

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

F. The serum concentration of FSH in mice with ceramide treatment. N=7 Data information: The p value was calculated by two-tailed t-test with 2-way ANOVA correction.


H . The changes in follicle count from mice with ceramide treatment . N = 7 . Data information : S1 , Primordial ; S2 , Primary ; S3 , Secondary ; S4 , Antral ; S5 , Atretic . Error bars stand for SEM of biological repeats . The p value was calculated by two - tailed t - test with 2 - way ANOVA correction .

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

H. The changes in follicle count from mice with ceramide treatment. N=7. Data information: S1, Primordial; S2, Primary; S3, Secondary; S4, Antral; S5, Atretic. Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.


C , Relative expression of genes related to ROS in granulosa cells Data information : Error bars stand for SEM of biological repeats . The p value was calculated by two - tailed t - test with 2 - way ANOVA correction .

O O O O O O O O O O O O O O O O O O O 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, Relative expression of genes related to ROS in granulosa cells Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.


E - F . Relative abundance of GSH and GSSG . N = 3 ; Data information : Error bars stand for SEM of biological repeats . The p value was calculated by two - tailed t - test with 2 - way ANOVA correction .

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

E-F. Relative abundance of GSH and GSSG. N=3; Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.


G . The concentration of E2 secreted by KGN cells with ceramide treatment w / o NAC . N = 4 ; Data information : Error bars stand for SEM of biological repeats . The p value was calculated by two - tailed t - test with 2 - way ANOVA correction .

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

G. The concentration of E2 secreted by KGN cells with ceramide treatment w/o NAC. N=4; Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.


H - J . Serum concentration of FSH , AMH , and E2 . H . Control , N = 10 ; BCAA , N = 5 ; TG = 9 ; TG + BCAA = 8 ; I . Control , N = 5 ; BCAA , N = 5 ; TG = 6 ; TG + BCAA = 6 ; J . N = 5 ; Data information : Error bars stand for SEM of biological repeats . The p value was calculated by two - tailed t - test with 2 - way ANOVA correction .

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 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 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 O 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- J. Serum concentration of FSH, AMH, and E2. H. Control, N=10; BCAA, N=5; TG=9; TG+BCAA=8; I. Control, N=5; BCAA, N=5; TG=6; TG+BCAA=6; J. N=5; Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.


K . The changes in follicle count . Control , N = 5 ; BCAA , N = 4 ; TG = 5 ; TG + BCAA = 5 ; Data information : Error bars stand for SEM of biological repeats . The p value was calculated by two - tailed t - test with 2 - way ANOVA correction .

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

K. The changes in follicle count. Control, N=5; BCAA, N=4; TG=5; TG+BCAA=5; Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.


Fluorescence intensity of the FRET donor ( GFP ) is shown before and after photobleaching of the FRET acceptor ( mCherry ) in ImageJ Smart pseudocolor scheme ( for intensity values see bars on the right ) . FRET is observed as an increase of donor fluorescence after acceptor photobleaching . The indicated GFP - and mCherry - tagged endocytic proteins localize at the endocytic patches on the plasma membrane of live yeast cells . The enrichment of individual endocytic patches ( diameter of 50 - 80 nm ) on the plasma membrane , caused by the latA - induced block of their subsequent invagination and / or their higher abundance at incipient buds , resulted in their apparent clustering on the diffraction - limited fluorescence images ( pixel size , 178 nm ) . All raw acquisitions of GFP fluorescence intensities before and after photobleaching are shown

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

Fluorescence intensity of the FRET donor (GFP) is shown before and after photobleaching of the FRET acceptor (mCherry) in ImageJ Smart pseudocolor scheme (for intensity values see bars on the right). FRET is observed as an increase of donor fluorescence after acceptor photobleaching. The indicated GFP- and mCherry-tagged endocytic proteins localize at the endocytic patches on the plasma membrane of live yeast cells. The enrichment of individual endocytic patches (diameter of 50-80 nm) on the plasma membrane, caused by the latA-induced block of their subsequent invagination and/or their higher abundance at incipient buds, resulted in their apparent clustering on the diffraction-limited fluorescence images (pixel size, 178 nm). All raw acquisitions of GFP fluorescence intensities before and after photobleaching are shown


Fluorescence intensity of the FRET donor ( GFP ) is shown before and after photobleaching of the FRET acceptor ( mCherry ) in ImageJ Smart pseudocolor scheme ( for intensity values see bars on the right ) . FRET is observed as an increase of donor fluorescence after acceptor photobleaching . The indicated GFP - and mCherry - tagged endocytic proteins localize at the endocytic patches on the plasma membrane of live yeast cells . The enrichment of individual endocytic patches ( diameter of 50 - 80 nm ) on the plasma membrane , caused by the latA - induced block of their subsequent invagination and / or their higher abundance at incipient buds , resulted in their apparent clustering on the diffraction - limited fluorescence images ( pixel size , 178 nm ) . All raw acquisitions of GFP fluorescence intensities before and after photobleaching are

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


Partial loss of FRET between Ent1 - mTurquoise2 and Sla1 - mNeonGreen during endocytic membrane invagination . FRET values ( in % ) of individual endocytic patches ( n = 74 , 75 ; 69 , 73 for ( B ) and ( C ) , respectively ) sorted according to the absence / presence of Abp1 protein are shown as box plots . Center , top and bottom lines of box plots show the medians , the 25th and 75th percentiles of individual datasets , respectively . Whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Notches indicate 95 % confidence intervals of the medians .

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

Partial loss of FRET between Ent1-mTurquoise2 and Sla1-mNeonGreen during endocytic membrane invagination. FRET values (in %) of individual endocytic patches (n=74, 75; 69, 73 for (B) and (C), respectively) sorted according to the absence/presence of Abp1 protein are shown as box plots. Center, top and bottom lines of box plots show the medians, the 25th and 75th percentiles of individual datasets, respectively. Whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Notches indicate 95% confidence intervals of the medians.


presence of FRET between Ent1 - mTurquoise2 and Sla2 - mNeonGreen during endocytic membrane invagination . FRET values ( in % ) of individual endocytic patches ( n = 74 , 75 ; 69 , 73 for ( B ) and ( C ) , respectively ) sorted according to the absence / presence of Abp1 protein are shown as box plots . Center , top and bottom lines of box plots show the medians , the 25th and 75th percentiles of individual datasets , respectively . Whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Notches indicate 95 % confidence intervals of the medians .

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

presence of FRET between Ent1-mTurquoise2 and Sla2-mNeonGreen during endocytic membrane invagination. FRET values (in %) of individual endocytic patches (n=74, 75; 69, 73 for (B) and (C), respectively) sorted according to the absence/presence of Abp1 protein are shown as box plots. Center, top and bottom lines of box plots show the medians, the 25th and 75th percentiles of individual datasets, respectively. Whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Notches indicate 95% confidence intervals of the medians.


Ede1 , Apl1 , Yap1801 , Gts1 , Bzz1 , Lsb3 , Las17 , and Vrp1 proteins tagged with mNeonGreen were photobleached in individual endocytic sites of LatA - treated cells and fluorescence recovery was followed every 0 . 5 s for 60 s ( 40 s in case of the highly dynamic Bzz1 protein ) . The curves represent means ± 95 % confidence intervals ( n = 9 - 17 ) .

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

Ede1, Apl1, Yap1801, Gts1, Bzz1, Lsb3, Las17, and Vrp1 proteins tagged with mNeonGreen were photobleached in individual endocytic sites of LatA-treated cells and fluorescence recovery was followed every 0.5 s for 60 s (40 s in case of the highly dynamic Bzz1 protein). The curves represent means ± 95% confidence intervals (n = 9-17).


Ede1 , Apl1 , Yap1801 , Gts1 , Bzz1 , Lsb3 , Las17 , and Vrp1 proteins tagged with mNeonGreen were photobleached in individual endocytic sites

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

Ede1, Apl1, Yap1801, Gts1, Bzz1, Lsb3, Las17, and Vrp1 proteins tagged with mNeonGreen were photobleached in individual endocytic sites of


( B ) Percentage infectivity of mouse tail fibroblasts ( MTFs ) infected with WT , RH , RHEV and EV CHIKV ( MOI 10 , n = 3 per group ) harvested at 12 hpi . Data are presented as the means ± SEM .

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

(B) Percentage infectivity of mouse tail fibroblasts (MTFs) infected with WT, RH, RHEV and EV CHIKV (MOI 10, n=3 per group) harvested at 12 hpi. Data are presented as the means ± SEM.


( C ) Viremia progression in virus - infected mice was monitored over 2 weeks ( n = 11 per group ) . Viremia is detected with CHIKV nsP1 probe via qRT - PCR .

O O O O O O O O O O O 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) Viremia progression in virus-infected mice was monitored over 2 weeks (n=11 per group). Viremia is detected with CHIKV nsP1 probe via qRT-PCR. Statistical


( D ) joint inflammation in virus - infected mice was monitored over 2 weeks ( n = 11 per group ) .

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

(D) joint inflammation in virus-infected mice was monitored over 2 weeks (n=11 per group).


( E ) Compiled percentages of CHIKV - infected ( ZsGreen positive ) leukocytes at 2 days post infection ( dpi ) for the respective groups ( n = 5 per group ) . Background ZsGreen signals are indicated by the dotted line .

O O O O O O O O O O O O O O O O O O O O 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) Compiled percentages of CHIKV-infected (ZsGreen positive) leukocytes at 2 days post infection (dpi) for the respective groups (n=5 per group). Background ZsGreen signals are indicated by the dotted line.


( F ) Dot - plots of the localized type - I interferon ( IFN - α and IFN - β ) concentrations at 15 hpi . Data are expressed as the means ± SD .

O O O O O O O O O O O O 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) Dot-plots of the localized type-I interferon (IFN-α and IFN-β) concentrations at 15 hpi. Data are expressed as the means ± SD.


Cell lysates were collected from the joints of mice infected with WT CHIKV or CHIKV nsP - mutants at 15 hpi , 2 dpi or 6 dpi ( n = 5 per group ) . The concentrations of 36 immune mediators were quantified using a 36 - plex microbead - based immunoassay . Immune mediators are grouped based on function . Each color represents the relative concentration of a particular analyte . Blue and red indicates low and high concentration , respectively .

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

Cell lysates were collected from the joints of mice infected with WT CHIKV or CHIKV nsP-mutants at 15 hpi, 2 dpi or 6 dpi (n=5 per group). The concentrations of 36 immune mediators were quantified using a 36-plex microbead-based immunoassay. Immune mediators are grouped based on function. Each color represents the relative concentration of a particular analyte. Blue and red indicates low and high concentration, respectively.


Luminex analytes from WT CHIKV - infected joint cell lysates are categorized based on fold change states ( up , down at 2 dpi relative to mock , and 6 dpi relative to 2 dpi . The numbers in the heatmap represent Log2 fold changes of analytes or genes . Analytes in complete or partial agreement with corresponding gene expression values derived from RNA - sequencing ( RNA - seq ) data appeared predominantly in the ( A ) up then down state ( Mock to 2 dpi , then 2 dpi to 6 dpi , respectively ) ,

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

Luminex analytes from WT CHIKV-infected joint cell lysates are categorized based on fold change states (up, down at 2 dpi relative to mock, and 6 dpi relative to 2 dpi. The numbers in the heatmap represent Log2 fold changes of analytes or genes. Analytes in complete or partial agreement with corresponding gene expression values derived from RNA-sequencing (RNA-seq) data appeared predominantly in the (A) up then down state (Mock to 2 dpi, then 2 dpi to 6 dpi, respectively),


The study analytes and transcripts in the RNA - seq data from wild - type CHIKV - infected and mock - infected samples in the same states were used . ( 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

The study analytes and transcripts in the RNA-seq data from wild-type CHIKV-infected and mock-infected samples in the same states were used. (C,


Luminex analytes from WT CHIKV - infected joint cell lysates are categorized based on fold change states no difference ( ND ) ) at 2 dpi relative to mock , and 6 dpi relative to 2 dpi . The numbers in the heatmap represent Log2 fold changes of analytes or genes . Analytes in complete or partial agreement with corresponding gene expression values derived from RNA - sequencing ( RNA - seq ) data appeared predominantly in the ( D ) up then ND state ( Mock to 2 dpi , then 2 dpi to 6 dpi , respectively ) . † indicates analyte involvement in the canonical pathways . Differences in analyte concentrations during attenuated CHIKV infection are indicated in the checkbox . Yellow represents lower while green represents higher concentrations relative to WT CHIKV .

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

Luminex analytes from WT CHIKV-infected joint cell lysates are categorized based on fold change states no difference (ND)) at 2 dpi relative to mock, and 6 dpi relative to 2 dpi. The numbers in the heatmap represent Log2 fold changes of analytes or genes. Analytes in complete or partial agreement with corresponding gene expression values derived from RNA-sequencing (RNA-seq) data appeared predominantly in the (D) up then ND state (Mock to 2 dpi, then 2 dpi to 6 dpi, respectively). † indicates analyte involvement in the canonical pathways. Differences in analyte concentrations during attenuated CHIKV infection are indicated in the checkbox. Yellow represents lower while green represents higher concentrations relative to WT CHIKV.


The study analytes and transcripts in the RNA - seq data from wild - type CHIKV - infected and mock - infected samples in the same states were used . F )

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

The study analytes and transcripts in the RNA-seq data from wild-type CHIKV-infected and mock-infected samples in the same states were used. F)


Joints from WT - CHIKV , RH - CHIKV , RHEV - CHIKV , EV - CHIKV and mock - infected WT C57BL / 6 mice were harvested at 6 dpi . ( A ) Absolute numbers of leukocytes present in the joints as determined by immune - phenotyping . Data are presented as the means ± SD ( n = 5 per group ) . No significance was obtained between the groups by Kruskal - Wallis test with Dunn ' s multiple comparisons .

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

Joints from WT-CHIKV, RH-CHIKV, RHEV-CHIKV, EV-CHIKV and mock-infected WT C57BL/6 mice were harvested at 6 dpi. (A) Absolute numbers of leukocytes present in the joints as determined by immune-phenotyping. Data are presented as the means ± SD (n=5 per group). No significance was obtained between the groups by Kruskal-Wallis test with Dunn's multiple comparisons.


( B ) Jensen - Shannon divergence heatmap showing the level of divergence between infected groups .

(B) Jensen-Shannon divergence heatmap showing the level of divergence between infected groups.


( C ) Superimposed PhenoGraphs of UMAP transformed FACs data from immune - phenotyping of joint cells from WT - CHIKV , RH - CHIKV , RHEV - CHIKV , EV - CHIKV and mock - infected animals ( n = 5 per group ) .

(C) Superimposed PhenoGraphs of UMAP transformed FACs data from immune-phenotyping of joint cells from WT-CHIKV, RH-CHIKV, RHEV-CHIKV, EV-CHIKV and mock-infected animals (n=5 per group).


( E ) Cluster ID annotation with heatmap . Cluster IDs are indicated in rows , while surface marker expression levels are indicated by the columns . The color represents the relative mean fluorescence intensity ( MFI ) of a surface marker . Blue and red represents low and high MFI , respectively . Red square and ovals highlight differences of several PhenoGraph clusters in infected joints at 6 dpi .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cluster ID annotation with heatmap. Cluster IDs are indicated in rows, while surface marker expression levels are indicated by the columns. The color represents the relative mean fluorescence intensity (MFI) of a surface marker. Blue and red represents low and high MFI, respectively. Red square and ovals highlight differences of several PhenoGraph clusters in infected joints at 6 dpi.


( A ) Anti - CHIKV IgG titers from the vaccination phase were determined with a CHIKV virion - based ELISA . Sera from four animals were pooled and diluted 1 : 500 for the assay . Statistical analyses were performed using two - tailed paired T - test ( * P = 0 . 029 RH - CHIKV 6 dpv , * P = 0 . 029 RH - CHIKV 90 dpv , * P = 0 . 029 RHEV - CHIKV 6 dpv , * P = 0 . 029 RHEV - CHIKV 90 dpv , * P = 0 . 029 EV - CHIKV 90 dpv ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Anti-CHIKV IgG titers from the vaccination phase were determined with a CHIKV virion-based ELISA. Sera from four animals were pooled and diluted 1:500 for the assay. Statistical analyses were performed using two-tailed paired T-test (*P =0.029 RH-CHIKV 6 dpv, *P =0.029 RH-CHIKV 90 dpv, *P =0.029 RHEV-CHIKV 6 dpv, *P =0.029 RHEV-CHIKV 90 dpv, *P =0.029 EV-CHIKV 90 dpv).


( B ) Half maximum inhibitory concentration ( IC50 ) of pooled sera collected on 90 dpv were characterized in HEK293T cells infected with WT - CHIKV ( MOI 5 ) . Sera were serially diluted at 1 : 625000 , 1 : 125000 , 1 : 25000 , 1 : 16000 , 1 : 8000 , 1 : 5000 , 1 : 4000 , 1 : 2000 , 1 : 1000 , 1 : 500 , 1 : 250 . Sera dilution to obtain IC50 are shown in parenthesis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Half maximum inhibitory concentration (IC50) of pooled sera collected on 90 dpv were characterized in HEK293T cells infected with WT-CHIKV (MOI 5). Sera were serially diluted at 1:625000, 1:125000, 1:25000, 1:16000, 1:8000, 1:5000, 1:4000, 1:2000, 1:1000, 1:500, 1:250. Sera dilution to obtain IC50 are shown in parenthesis.


Viremia were monitored over 2 weeks . Viremia is detected with CHIKV nsP1 probe via qRT - PCR .

Viremia were monitored over 2 weeks. Viremia is detected with CHIKV nsP1 probe via qRT-PCR.


( E ) severity of joint inflammation were monitored over 2 weeks .

(E) severity of joint inflammation were monitored over 2 weeks.


( A ) Representative hematoxylin and eosin ( H & E ) images of inflamed joint footpad at 6 dpc . Ed , region of edema ; red arrow , synovitis ; black arrow , normal synovial membrane ; blue arrow , mild synovial hyperplasia ; D , degeneration of muscle ; N , necrosis of muscle ; R , regeneration of muscle .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 hematoxylin and eosin (H&E) images of inflamed joint footpad at 6 dpc. Ed, region of edema; red arrow, synovitis; black arrow, normal synovial membrane; blue arrow, mild synovial hyperplasia; D, degeneration of muscle; N, necrosis of muscle; R, regeneration of muscle.


( B ) Histopathological scoring of edema , inflammation in different regions of the joint footpad and muscle pathology of WT - CHIKV challenged animals ( n = 5 ) at 6 dpc , performed blind . Three sections from each joint footpad were scored . The data are expressed as the means ± SD . Data were analyzed by one - way ANOVA with Tukey post - test ; Edema ( * * * P = 0 . 000002 WT - CHIKV versus PBS , * * * P = 0 . 000004 RH - CHIKV versus PBS ) , Muscle necrosis ( * * P = 0 . 005521 WT - CHIKV versus PBS , * P = 0 . 02628 RH - CHIKV versus PBS ) , Muscle regeneration ( * * * P < 0 . 000001 WT - CHIKV versus PBS , * * * P < 0 . 000001 WT - CHIKV versus RH - CHIKV ) , Muscle inflammation ( * P = 0 . 040258 WT - CHIKV versus PBS , * P = 0 . 040258 RH - CHIKV versus PBS ) , Synovial membrane inflammation ( * P = 0 . 014457 WT - CHIKV versus PBS , * P = 0 . 014457 RH - CHIKV versus PBS ) , Subcutaneous area inflammation ( * * * P = 0 . 00004 WT - CHIKV versus PBS , * * * P = 0 . 000074 RH - CHIKV versus PBS ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Histopathological scoring of edema, inflammation in different regions of the joint footpad and muscle pathology of WT-CHIKV challenged animals (n=5) at 6 dpc, performed blind. Three sections from each joint footpad were scored. The data are expressed as the means ± SD. Data were analyzed by one-way ANOVA with Tukey post-test; Edema (***P =0.000002 WT-CHIKV versus PBS, ***P =0.000004 RH-CHIKV versus PBS), Muscle necrosis (**P =0.005521 WT-CHIKV versus PBS, *P =0.02628 RH-CHIKV versus PBS), Muscle regeneration (***P <0.000001 WT-CHIKV versus PBS, ***P <0.000001 WT-CHIKV versus RH-CHIKV), Muscle inflammation (*P =0.040258 WT-CHIKV versus PBS, *P =0.040258 RH-CHIKV versus PBS), Synovial membrane inflammation (*P =0.014457 WT-CHIKV versus PBS, *P =0.014457 RH-CHIKV versus PBS), Subcutaneous area inflammation (***P =0.00004 WT-CHIKV versus PBS, ***P =0.000074 RH-CHIKV versus PBS).


Three - week - old WT C57BL / 6 mice were infected with 1E6 PFU of WT - ONNV at the metatarsal region of the footpad . ( A ) Viremia progression in ONNV - infected mice were monitored over 2 weeks ( n = 5 ) . Viremia is detected with ONNV nsP1 probe via qRT - PCR .

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

Three-week-old WT C57BL/6 mice were infected with 1E6 PFU of WT-ONNV at the metatarsal region of the footpad. (A) Viremia progression in ONNV-infected mice were monitored over 2 weeks (n=5). Viremia is detected with ONNV nsP1 probe via qRT-PCR.


Three - week - old WT C57BL / 6 mice were infected with 1E6 PFU of WT - ONNV at the metatarsal region of the footpad . ( B ) joint inflammation in ONNV - infected mice were monitored over 2 weeks ( n = 5 ) .

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

Three-week-old WT C57BL/6 mice were infected with 1E6 PFU of WT-ONNV at the metatarsal region of the footpad. (B) joint inflammation in ONNV-infected mice were monitored over 2 weeks (n=5).


Three - week - old WT C57BL / 6 mice were vaccinated subcutaneously with 1E6 PFU of WT - CHIKV or attenuated CHIKV ( RH ) at the metatarsal region of the footpad . Challenge with 1E6 PFU of WT - ONNV was performed via the same route at 3 months post - vaccination . Viremia progression of the mice were monitored over 2 weeks ( n = 5 per group ) . Viremia is detected with ONNV nsP1 probe via qRT - PCR .

Three-week-old WT C57BL/6 mice were vaccinated subcutaneously with 1E6 PFU of WT-CHIKV or attenuated CHIKV (RH) at the metatarsal region of the footpad. Challenge with 1E6 PFU of WT-ONNV was performed via the same route at 3 months post-vaccination. Viremia progression of the mice were monitored over 2 weeks (n=5 per group). Viremia is detected with ONNV nsP1 probe via qRT-PCR.


D ) Three - week - old WT C57BL / 6 mice were vaccinated subcutaneously with 1E6 PFU of WT - CHIKV or attenuated CHIKV ( RH ) at the metatarsal region of the footpad . Challenge with 1E6 PFU of WT - ONNV was performed via the same route at 3 months post - vaccination . ( D ) joint inflammation of the mice were monitored over 2 weeks ( n = 5 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Three-week-old WT C57BL/6 mice were vaccinated subcutaneously with 1E6 PFU of WT-CHIKV or attenuated CHIKV (RH) at the metatarsal region of the footpad. Challenge with 1E6 PFU of WT-ONNV was performed via the same route at 3 months post-vaccination. (D) joint inflammation of the mice were monitored over 2 weeks (n=5 per group).


Tau phospho - variants generated by in vitro phosphorylation of non - phosphorylated Tau from E . coli with different kinases ( PKA , GSK3β , or Cdk5 ) show differences in phosphorylation at specific phospho - site ( pT205 , pS262 / pS356 , pS324 and pS404 ) tested by Western blot . Non - phosphorylated Tau and p - Tau from insect cells are loaded for comparison . Position of tested p - sites is indicated in the Tau sequence cartoon . Western blot for total Tau ( Tau13 antibody ) confirms equal loading ( 0 . 2 μg Tau per lane ) of the Tau preparations .

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

Tau phospho-variants generated by in vitro phosphorylation of non-phosphorylated Tau from E. coli with different kinases (PKA, GSK3β, or Cdk5) show differences in phosphorylation at specific phospho-site (pT205, pS262/pS356, pS324 and pS404) tested by Western blot. Non-phosphorylated Tau and p-Tau from insect cells are loaded for comparison. Position of tested p-sites is indicated in the Tau sequence cartoon. Western blot for total Tau (Tau13 antibody) confirms equal loading (0.2 μg Tau per lane) of the Tau preparations.


Phos - tag gel shows degree and heterogeneity of phosphorylation in the different Tau phospho - variants . Intensity along the lanes are plotted as colored lines next to gel image .

Phos-tag gel shows degree and heterogeneity of phosphorylation in the different Tau phospho-variants. Intensity along the lanes are plotted as colored lines next to gel image.


Tubulin co - condensation with Tau . Addition of tubulin - Alexa647 ( 5 μM , 10 : 1 unlabeled : labeled tubulin ) to a Tau - Dy488 solution ( 25 μM ) induces Tau condensation . Addition of tubulin to Tau : PEG , Tau : RNA ( polyA - Cy3 ) , and Tau : PEG : RNA condensates leads to partitioning of tubulin into existing condensates and the occurrence of additional small Tau : tubulin condensates . Condensates with high tubulin - HiLyte647 intensity have low polyA - Cy3 intensity , and vice versa . In Tau : PEG : polyA condensates , individual condensates host RNA - and tubulin - rich domains ( arrow heads ) at homogeneous Tau - Dy488 intensity . Scale bars = 20 μm . Average intensity of RNA ( top graph ) normalized to Tau intensity in individual condensates . Addition of tubulin reduces the average amount of RNA in condensates . Intensity of tubulin ( middle graph ) normalized to Tau intensity in individual condensates . The presence of RNA increases the amount of tubulin in the condensates . Data shown as mean ± SD . Data + / - tubulin and + / - polyA were compared by two - tailed Student ' s t - tests . Tubulin and RNA intensity correlate negatively on an individual condensate level ( bottom graph ) . Line represent linear regression to all data . N = 3 experiments . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Condensate size ( are on surface ) relative to tubulin ( yellow data points ) and RNA ( pink data points ) intensity . Smaller ( new ) condensates are high in tubulin , larger ( old ) condensates have more RNA . Lines represent linear regressions to tubulin ( yellow ) and RNA ( pink ) data .

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

Tubulin co-condensation with Tau. Addition of tubulin-Alexa647 (5 μM, 10:1 unlabeled:labeled tubulin) to a Tau-Dy488 solution (25 μM) induces Tau condensation. Addition of tubulin to Tau:PEG, Tau:RNA (polyA-Cy3), and Tau:PEG:RNA condensates leads to partitioning of tubulin into existing condensates and the occurrence of additional small Tau:tubulin condensates. Condensates with high tubulin-HiLyte647 intensity have low polyA-Cy3 intensity, and vice versa. In Tau:PEG:polyA condensates, individual condensates host RNA- and tubulin-rich domains (arrow heads) at homogeneous Tau-Dy488 intensity. Scale bars=20 μm. Average intensity of RNA (top graph) normalized to Tau intensity in individual condensates. Addition of tubulin reduces the average amount of RNA in condensates. Intensity of tubulin (middle graph) normalized to Tau intensity in individual condensates. The presence of RNA increases the amount of tubulin in the condensates. Data shown as mean±SD. Data +/- tubulin and +/- polyA were compared by two-tailed Student's t-tests. Tubulin and RNA intensity correlate negatively on an individual condensate level (bottom graph). Line represent linear regression to all data. N=3 experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Condensate size (are on surface) relative to tubulin (yellow data points) and RNA (pink data points) intensity. Smaller (new) condensates are high in tubulin, larger (old) condensates have more RNA. Lines represent linear regressions to tubulin (yellow) and RNA (pink) data.


MT polymerization out of Tau : PEG and Tau : PEG : RNA condensates . Addition of tubulin - Alexa647 ( 5 μM , 10 : 1 unlabeled : labeled tubulin ) and GTP to Tau - Dy488 ( 25 μM ) diluted in BRB80 polymerization buffer induces MT polymerization . Images taken 30 min after addition of tubulin show thin partially curved MT bundles in Tau : PEG system , and thick bundles attached to persisting Tau : RNA condensates in the Tau : PEG : RNA system . Scale bar = 5 μm . Cross - sectional intensity plots ( line plots ) of MT bundles differ between Tau : PEG ( yellow / red line plots ) and Tau : PEG : RNA ( blue / green line plots ) systems . Four line plots at random positions are shown per condition .

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

MT polymerization out of Tau:PEG and Tau:PEG:RNA condensates. Addition of tubulin-Alexa647 (5 μM, 10:1 unlabeled:labeled tubulin) and GTP to Tau-Dy488 (25 μM) diluted in BRB80 polymerization buffer induces MT polymerization. Images taken 30 min after addition of tubulin show thin partially curved MT bundles in Tau:PEG system, and thick bundles attached to persisting Tau:RNA condensates in the Tau:PEG:RNA system. Scale bar=5 μm. Cross-sectional intensity plots (line plots) of MT bundles differ between Tau:PEG (yellow/red line plots) and Tau:PEG:RNA (blue/green line plots) systems. Four line plots at random positions are shown per condition.


Nocodazole treatment ( 120 μM for 30 min ) of MTs formed from Tau : PEG : RNA and Tau : PEG condensates . MT formed in absence of Tau shown as control . Graphs show surface coverage ( % ) of fibrillar MT structures before and after nocodazole treatment . N = 3 experiments , 3 images analyzed per experiment and condition . Data shown as mean ± SD . Two - tailed Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Scale bar = 20 μm .

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

Nocodazole treatment (120 μM for 30 min) of MTs formed from Tau:PEG:RNA and Tau:PEG condensates. MT formed in absence of Tau shown as control. Graphs show surface coverage (%) of fibrillar MT structures before and after nocodazole treatment. N=3 experiments, 3 images analyzed per experiment and condition. Data shown as mean±SD. Two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Scale bar=20 μm.


FRAP experiments of Tau : PEG , Tau : polyA , and Tau : PEG : polyA condensates at 1h , 4h , and 24h after LLPS induction . Tau : PEG and Tau : PEG : polyA condensates show a decrease in recovery rate and mobile fraction over time . Tau : polyA coacervates maintain fast recovery and a large mobile fraction even after 24h . Time until half maximum recovery : t ( y1 / 2 ) polyA : 1h ≈ 2s , 4h ≈ 3 . 5s , 24h ≈ 4 . 5s ; t ( y1 / 2 ) PEG : 1h ≈ 6s , 4h ≈ 8s , 24h ≈ 11s ; t ( y1 / 2 ) PEG + polyA : 1h ≈ 4 . 5s , 4h ≈ 9 . 5s , 24h ≈ 9s ) . Scale bars = 5 μm for Tau : polyA and Tau : PEG , and 10 μm for Tau : PEG : polyA . Data presented as error bars ( SEM ) , N = 15 to 20 condensates per condition and time point .

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

FRAP experiments of Tau:PEG, Tau:polyA, and Tau:PEG:polyA condensates at 1h, 4h, and 24h after LLPS induction. Tau:PEG and Tau:PEG:polyA condensates show a decrease in recovery rate and mobile fraction over time. Tau:polyA coacervates maintain fast recovery and a large mobile fraction even after 24h. Time until half maximum recovery: t(y1/2)polyA: 1h≈2s, 4h≈3.5s, 24h≈4.5s; t(y1/2)PEG: 1h≈6s, 4h≈8s, 24h≈11s; t(y1/2)PEG+polyA: 1h≈4.5s, 4h≈9.5s, 24h≈9s). Scale bars=5 μm for Tau:polyA and Tau:PEG, and 10 μm for Tau:PEG:polyA. Data presented as error bars (SEM), N=15 to 20 condensates per condition and time point.


Microscopy of Tau : PEG : polyA condensates ( Tau - Dylight488 and polyA - Cy3 ) shows condensates with different Cy3 intensities . The bimodal fluorescence intensity distribution suggests two groups of condensates with high or low Cy3 fluorescence . Cy3 - intensities , normalized to Tau - DyLight488 intensities in the same condensates , were independent of condensate size . Scale bar = 10 μm .

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

Microscopy of Tau:PEG:polyA condensates (Tau-Dylight488 and polyA-Cy3) shows condensates with different Cy3 intensities. The bimodal fluorescence intensity distribution suggests two groups of condensates with high or low Cy3 fluorescence. Cy3-intensities, normalized to Tau-DyLight488 intensities in the same condensates, were independent of condensate size. Scale bar=10 μm.


FRAP of Tau - Dylight488 and polyA - Cy3 of 4h - old Tau : PEG : polyA condensates . The mean FRAP of polyA - Cy3 is lower than of Tau - Dy488 ( left ) . Data shown as mean ± SEM , n = 24 condensates . FRAP data of individual condensates ( bottom ; # 1 to # 3 ) shows a decrease of Tau = Dy488 ( middle ) and polyA - Cy3 ( right ) FRAP with an increase of polyA - Cy3 ( relative to Tau - Dy488 ) in the condensates . Scale bar = 5μm .

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

FRAP of Tau-Dylight488 and polyA-Cy3 of 4h-old Tau:PEG:polyA condensates. The mean FRAP of polyA-Cy3 is lower than of Tau-Dy488 (left). Data shown as mean±SEM, n=24 condensates. FRAP data of individual condensates (bottom; #1 to #3) shows a decrease of Tau=Dy488 (middle) and polyA-Cy3 (right) FRAP with an increase of polyA-Cy3 (relative to Tau-Dy488) in the condensates. Scale bar=5μm.


HEK sensor cell Tau aggregation assay . Cells were treated with 24 h - old condensates or aggregates ( 1 . 2 µM Tau in the presence of 0 . 8 % lipofectamine ) . The number of Tau aggregates was determined from low magnification ( 20x ) images taken in the green channel and normalized to the number of cells ( DAPI ) . Scale bar = 100 μm .

HEK sensor cell Tau aggregation assay. Cells were treated with 24 h-old condensates or aggregates (1.2 µM Tau in the presence of 0.8% lipofectamine). The number of Tau aggregates was determined from low magnification (20x) images taken in the green channel and normalized to the number of cells (DAPI). Scale bar=100 μm.


Quantification of seeding assay in ( C ) . Data were collected from three images of 3 wells for each condition . Data shown as mean ± SD , N = 3 experiments , 1 - 3 images per experiment and condition . One - way ANOVA with Tukey test for multiple comparison . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

Quantification of seeding assay in (C). Data were collected from three images of 3 wells for each condition. Data shown as mean±SD, N=3 experiments, 1-3 images per experiment and condition. One-way ANOVA with Tukey test for multiple comparison. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


Confocal microscopy of HEK sensor cells treated with 24h - old Tau condensates ( Tau : tRNA ) shows different types of intracellular CFP / YFP - Tau accumulations : spherical intranuclear Tau ( NUC ) , bright cytoplasmic accumulations ( CYT ) adjacent to the nucleus , and small , dimmer accumulations right at and along the nuclear envelope ( NE ) . Cells treated with Tau aggregates ( hepAGG ) had mainly large cytoplasmic ( CYT ) Tau inclusions . The different Tau accumulations coexisted in most cells , and also more than one accumulation of the same type could occur . Nuclei were stained with propidium iodide ( PI , pink ) . Scale bars = 10 μm .

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

Confocal microscopy of HEK sensor cells treated with 24h-old Tau condensates (Tau:tRNA) shows different types of intracellular CFP/YFP-Tau accumulations: spherical intranuclear Tau (NUC), bright cytoplasmic accumulations (CYT) adjacent to the nucleus, and small, dimmer accumulations right at and along the nuclear envelope (NE). Cells treated with Tau aggregates (hepAGG) had mainly large cytoplasmic (CYT) Tau inclusions. The different Tau accumulations coexisted in most cells, and also more than one accumulation of the same type could occur. Nuclei were stained with propidium iodide (PI, pink). Scale bars=10 μm.


Fluorescence intensity of NE and CYT Tau compared to soluble Tau in the cytosol of cells without accumulations . N = 3 experiments , and 5 - 6 images per experiment . Data shown as mean ± SD . One - way ANOVA with Tukey test for multiple comparison . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

Fluorescence intensity of NE and CYT Tau compared to soluble Tau in the cytosol of cells without accumulations. N=3 experiments, and 5-6 images per experiment. Data shown as mean±SD. One-way ANOVA with Tukey test for multiple comparison. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


Immunolabeling of lamin - B1 and nuclear pore complexes ( NPC ) shows the location of CYT and NE Tau in relation to the nuclear envelope in seeded HEK sensor cells . White arrows and boxes show position and width of cross - sectional profiles shown in ( D ) . Scale bar = 10 μm . Cross sectional profiles show the localization of Tau ( green ) in CYT accumulations adjacent to the nucleus in the cytosol , and the colocalization of NE Tau with lamin ( pink ) and NPCs ( blue ) . Notably , CYT - 1 also contains NPC signal . Dashed vertical lines indicate x - positions of highest intensity values for respective intensity profiles .

Immunolabeling of lamin-B1 and nuclear pore complexes (NPC) shows the location of CYT and NE Tau in relation to the nuclear envelope in seeded HEK sensor cells. White arrows and boxes show position and width of cross-sectional profiles shown in (D). Scale bar=10 μm. Cross sectional profiles show the localization of Tau (green) in CYT accumulations adjacent to the nucleus in the cytosol, and the colocalization of NE Tau with lamin (pink) and NPCs (blue). Notably, CYT-1 also contains NPC signal. Dashed vertical lines indicate x-positions of highest intensity values for respective intensity profiles.


Immunolabeling of phosphorylated Tau ( PHF1 = pS396 / 404 , AT8 = pS199 / pS202 / pT205 , AT180 = pT231 ) in AD brain section . Both 3 , 3 ′ - Diaminobenzidine ( DAB ) and fluorescence labeling show Tau accumulating in a layer at the nuclear envelope ( NE ) in non - tangle neurons with or without cytosolic ( CYT ) Tau accumulations . Control brain does not show Tau labeling in neuronal cell bodies . Scale bars = 20 and 10 μm , as indicated .

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

Immunolabeling of phosphorylated Tau (PHF1=pS396/404, AT8=pS199/pS202/pT205, AT180=pT231) in AD brain section. Both 3,3′-Diaminobenzidine (DAB) and fluorescence labeling show Tau accumulating in a layer at the nuclear envelope (NE) in non-tangle neurons with or without cytosolic (CYT) Tau accumulations. Control brain does not show Tau labeling in neuronal cell bodies. Scale bars= 20 and 10 μm, as indicated.


CFP - FLIM of seeded HEK sensor cells expressing only CFP - TauRDP301L . The dense packing of CFP - TauRD in Tau accumulations induces fluorophore quenching that can be detected as decrease in CFP - lifetime . Fit - free analysis of lifetime phasor plots enables the assignment of distinct lifetime clusters ( circles in phasor plot ) to different Tau accumulation types . LT - 2 = NE Tau , LT - 3 = CYT and NUC Tau , and LT - 1 = soluble CFP - TauRD in the cytosol of cells without accumulations . Graphs show lifetimes of CFP - lifetime components assigned to soluble , NE , and CYT + NUC Tau in 6 different images ( left graph , RM one - way ANOVA with Tukey post - test ) and lifetime reduction relative to LT - 1 in each respective image ( right graph , one - way ANOVA with Tukey post - test . ) . N = 2 experiments , 2 - 3 cells measured per experiments . Data shown as mean ± SD . Scale bars = 10 μm . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

CFP-FLIM of seeded HEK sensor cells expressing only CFP-TauRDP301L. The dense packing of CFP-TauRD in Tau accumulations induces fluorophore quenching that can be detected as decrease in CFP-lifetime. Fit-free analysis of lifetime phasor plots enables the assignment of distinct lifetime clusters (circles in phasor plot) to different Tau accumulation types. LT-2=NE Tau, LT-3=CYT and NUC Tau, and LT-1=soluble CFP-TauRD in the cytosol of cells without accumulations. Graphs show lifetimes of CFP-lifetime components assigned to soluble, NE, and CYT+NUC Tau in 6 different images (left graph, RM one-way ANOVA with Tukey post-test) and lifetime reduction relative to LT-1 in each respective image (right graph, one-way ANOVA with Tukey post-test.). N=2 experiments, 2-3 cells measured per experiments. Data shown as mean±SD. Scale bars=10 μm. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


FLIM of 1h - and 24h - old CFP - Tau ( full - length ) condensates and heparin induced CFP - Tau aggregates . Lifetime components are indicated in phasor plots . Graph shows lifetime reduction in condensates and aggregates relative to respective lifetime of soluble CFP - Tau . N = 2 experiments , 2 - 3 cells measured per experiments . Data shown as mean ± SD . One - way ANOVA with Tukey post - test . Scale bar = 5 μm . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Bottom right : Schematic of lifetime quenching in condensates and aggregates .

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

FLIM of 1h- and 24h-old CFP-Tau (full-length) condensates and heparin induced CFP-Tau aggregates. Lifetime components are indicated in phasor plots. Graph shows lifetime reduction in condensates and aggregates relative to respective lifetime of soluble CFP-Tau. N=2 experiments, 2-3 cells measured per experiments. Data shown as mean±SD. One-way ANOVA with Tukey post-test. Scale bar=5 μm. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Bottom right: Schematic of lifetime quenching in condensates and aggregates.


A 293FT cells were transfected with 3xFlag - Usp27x ( pFCMV7 . 1 vector backbone ) together with a construct driving expression of untagged BimEL or 3xHA - BimEL ( both pMIG vector backbone ) . Cells were lysed , and 3xHA - BimEL was immunoprecipitated with anti - HA - antibodies . IP products were tested by Western blotting for the presence of Bim and FLAG - Usp27x probing with antibodies against Bim or against the FLAG - peptide . See also Appendix Fig . S1B . Western blots show representative of n ≥ 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 293FT cells were transfected with 3xFlag-Usp27x (pFCMV7.1 vector backbone) together with a construct driving expression of untagged BimEL or 3xHA-BimEL(both pMIG vector backbone). Cells were lysed, and 3xHA-BimEL was immunoprecipitated with anti-HA-antibodies. IP products were tested by Western blotting for the presence of Bim and FLAG-Usp27x probing with antibodies against Bim or against the FLAG-peptide. See also Appendix Fig. S1B. Western blots show representative of n≥3 independent experiments.


B Usp27x binds a mutant of Bim incapable of binding to anti - apoptotic Bcl - 2 - proteins . 293FT cells transfected with constructs encoding 3xFLAG - Usp27x and 3xHA - tagged BimEL ( see A , 2µg each ) or 3xHA - tagged BimEL ∆ ∆ ( a mutant with two mutations in the BH3 - domain , incapable of binding antiapoptotic Bcl - 2proteins [ 50 ] ) were immunoprecipitated from whole cell extracts using anti - HA - resin . Bim and Usp27x were detected with anti - HA - and anti - FLAG - antibodies as indicated ; antiapoptotic proteins : Mcl - 1 , Bcl - XL , Bcl - 2 . The caspase - inhibitor Q - VD - OPh ( QVD ) was added to the cultures described in A and B to inhibit Bim - induced apoptosis . Western blots are representative of n = 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Usp27x binds a mutant of Bim incapable of binding to anti-apoptotic Bcl-2-proteins. 293FT cells transfected with constructs encoding 3xFLAG-Usp27x and 3xHA-tagged BimEL (see A, 2µg each) or 3xHA-tagged BimEL∆∆(a mutant with two mutations in the BH3-domain, incapable of binding antiapoptotic Bcl-2proteins [50]) were immunoprecipitated from whole cell extracts using anti-HA-resin. Bim and Usp27x were detected with anti-HA- and anti-FLAG-antibodies as indicated; antiapoptotic proteins: Mcl-1, Bcl-XL, Bcl-2. The caspase-inhibitor Q-VD-OPh (QVD) was added to the cultures described in A and B to inhibit Bim-induced apoptosis. Western blots are representative of n=3 independent experiments.


C Usp27x interacts with endogenous BimEL independently of its catalytic activity293FT cells either carrying 3xFlag - Usp27x ( 293FT - TetR - 3xFlag - usp27x ) or the catalytic inactive mutant 3xFlag - Usp27xC87A under the control of the Tet - repressor were treated for 24h with dox to induce expression of Usp27x or Usp27xC87A . In all conditions PMA ( to induce Bim - ubiquitination , 16 . 2 nM ) , and Q - VD - OPh ( to inhibit apoptosis , 10 µM , see Fig . 3 ) were added at the time of Usp27x - induction . MG132 ( to prevent Bim - degradation , 40 µM ) was added in all conditions 4h prior to cell lysis . 3xFlag tagged Usp27x or Usp27xC87A was immunoprecipitated from whole cell lysates using anti - Flag resin . Interaction with BimEL or β - TrCP was detected by Western blotting using anti - Bim or anti - β - TrCP antibodies . Western blots are representative of n ≥ 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Usp27x interacts with endogenous BimEL independently of its catalytic activity293FT cells either carrying 3xFlag-Usp27x (293FT-TetR-3xFlag-usp27x) or the catalytic inactive mutant 3xFlag-Usp27xC87A under the control of the Tet-repressor were treated for 24h with dox to induce expression of Usp27x or Usp27xC87A. In all conditions PMA (to induce Bim-ubiquitination, 16.2 nM), and Q-VD-OPh (to inhibit apoptosis, 10 µM, see Fig. 3) were added at the time of Usp27x-induction. MG132 (to prevent Bim-degradation, 40 µM) was added in all conditions 4h prior to cell lysis. 3xFlag tagged Usp27x or Usp27xC87A was immunoprecipitated from whole cell lysates using anti-Flag resin. Interaction with BimEL or β-TrCP was detected by Western blotting using anti-Bim or anti-β-TrCP antibodies. Western blots are representative of n≥3 independent experiments.


D Usp27x expression does not inhibit interaction of BimEL to β - TrCP293FT - TetR - 3xFlag - Usp27x cells were transfected with pMIG - 3xHA - BimEL in the presence of both PMA and QVD . At the same time dox ( to induce 3xFlag - Usp27x ) was added as indicated and 20h later cells were treated with MG132 [ 40µM ] for additional 4h . Cells were lysed and 3xHA - BimEL was immunoprecipitated as described above . As a control HA - matrix was used without lysate to rule out any unspecific signal that might come from the immobilized anti HA - antibody ( beads ) . Western blots show representative of n = 2 independent experiments .

D Usp27x expression does not inhibit interaction of BimEL to β-TrCP293FT-TetR-3xFlag-Usp27x cells were transfected with pMIG-3xHA-BimEL in the presence of both PMA and QVD. At the same time dox (to induce 3xFlag-Usp27x) was added as indicated and 20h later cells were treated with MG132 [40µM] for additional 4h.Cells were lysed and 3xHA-BimEL was immunoprecipitated as described above. As a control HA-matrix was used without lysate to rule out any unspecific signal that might come from the immobilized anti HA-antibody (beads). Western blots show representative of n=2 independent experiments.


E Binding of Usp27x to BimEL can be blocked by the MEK - inhibitor UO126293FT - TetR - 3xFlag - Usp27x cells ( see C ) were treated for 24h with dox to induce expression of Usp27x . In all conditions PMA [ 16 . 2 nM ] , and Q - VD - OPh [ 10 µM ] were added at the time of Usp27x - induction . As a control , cells were pretreated with UO126 [ 10µM ] for 30 minutes to block the PMA stimulated ERK pathway before the addition of dox plus PMA plus QVD . 3xFlag tagged Usp27x was immuno - precipitated from whole cell lysates using anti - Flag resin . Interaction with BimEL was detected by Western blotting using anti - Bim antibodies . As a control Flag - matrix ( beads ) was used alongside the IP under same conditions but without addition of protein lysates . Western blots are representative of n = 4 independent experiments ( see also Appendix Fig . S2B ) .

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

E Binding of Usp27x to BimEL can be blocked by the MEK-inhibitor UO126293FT-TetR-3xFlag-Usp27x cells (see C) were treated for 24h with dox to induce expression of Usp27x. In all conditions PMA [16.2 nM], and Q-VD-OPh [10 µM] were added at the time of Usp27x-induction. As a control, cells were pretreated with UO126 [10µM] for 30 minutes to block the PMA stimulated ERK pathway before the addition of dox plus PMA plus QVD. 3xFlag tagged Usp27x was immuno-precipitated from whole cell lysates using anti-Flag resin. Interaction with BimEL was detected by Western blotting using anti-Bim antibodies. As a control Flag-matrix (beads) was used alongside the IP under same conditions but without addition of protein lysates. Western blots are representative of n=4 independent experiments (see also Appendix Fig. S2B).


F Binding of Usp27xto BimEL does not require β - TrCP293FT - TetR - 3xFlag - Usp27x cells were transfected with control siRNA ( siCo3 ) or siRNA specific for β - TrCP . 48h later cells were stimulated with PMA plus dox plus QVD for additional 24h . After cell lysis , Flag - Usp27x was immuno - precipitated using anti - Flag - matrix , and BimEL and β - TrCP bound to Usp27x were identified by Western blotting . Blots are representative of n = 2 independent experiments .

F Binding of Usp27xto BimEL does not require β-TrCP293FT-TetR-3xFlag-Usp27x cells were transfected with control siRNA (siCo3) or siRNA specific for β-TrCP. 48h later cells were stimulated with PMA plus dox plus QVD for additional 24h. After cell lysis, Flag-Usp27x was immuno-precipitated using anti-Flag-matrix, and BimEL and β-TrCP bound to Usp27x were identified by Western blotting. Blots are representative of n=2 independent experiments.


G Usp27x binds specifically to BimEL293FT - TetR - 3xFlag - Usp27x cells or 293FT - 3xFlag - Usp27x cells with a specific deletion of the BimELprotein were treated with dox , PMA and QVD as in C . Cells were lysed , and lysates were subjected to anti - Flagimmunoprecipitation . 3xFlag - Usp27x was detected using anti - FLAG antibodies . Blots are representative of n = 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Usp27x binds specifically to BimEL293FT-TetR-3xFlag-Usp27x cells or 293FT-3xFlag-Usp27x cells with a specific deletion of the BimELprotein were treated with dox, PMA and QVD as in C. Cells were lysed, and lysates were subjected to anti-Flagimmunoprecipitation. 3xFlag-Usp27x was detected using anti-FLAG antibodies. Blots are representative of n=3 independent experiments.


A Usp27x inhibits the PMA - induced degradation of BimEL in 293FT cells . 293FT - TetR - 3xFlagUsp27x or TetR - 3xFlag - Usp27xC87A cells were treated with dox to induce 3xFlag - Usp27x or Usp27xC87A , or with PMA [ 16 . 2 nM ] ( to induce BimEL degradation ) , or with the combination of dox , PMA and QVD [ 10µM ] ( to inhibit apoptosis ) as indicated for 24 h ( left ) or 48 h ( right ) . Cell lysates were analysed by Western blotting . The blots are representative of n = 4 similar experiments . See also Appendix Fig . S5B . * Probably modified form of wild - type 3xFlag - Usp27x ( such as dimer , ubiquitinated Usp27x or a stable complex of Usp27x with another interacting partner ) ; this form is only seen if high expression levels of catalytically active Usp27x are reached ( see Appendix Fig . S5A where Usp27x was transfected into 293FT cells ) or if wt Usp27x is enriched during immunoprecipitation . * * Probably degradation product of 3xFlag - Usp27x . These forms of Usp27x were not seen in every experiment .

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 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Usp27x inhibits the PMA-induced degradation of BimEL in 293FT cells. 293FT-TetR-3xFlagUsp27x or TetR-3xFlag-Usp27xC87A cells were treated with dox to induce 3xFlag-Usp27x or Usp27xC87A, or with PMA [16.2 nM] (to induce BimEL degradation), or with the combination of dox, PMA and QVD [10µM] (to inhibit apoptosis) as indicated for 24 h (left) or 48 h (right). Cell lysates were analysed by Western blotting. The blots are representative of n=4 similar experiments. See also Appendix Fig. S5B. * Probably modified form of wild-type 3xFlag-Usp27x (such as dimer, ubiquitinated Usp27x or a stable complex of Usp27x with another interacting partner); this form is only seen if high expression levels of catalytically active Usp27x are reached (see Appendix Fig. S5A where Usp27x was transfected into 293FT cells) or if wt Usp27x is enriched during immunoprecipitation.** Probably degradation product of 3xFlag-Usp27x. These forms of Usp27x were not seen in every experiment.


B 293FT - TetR - 3xFlagUsp27x or TetR - 3xFlag - Usp27xC87A cells treated as in A were analysed using conditions of higher resolution . A band very likely corresponding to phosphorylated BimEL is detectable . Similar results were obtained in n = 2 separate experiments .

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

B 293FT-TetR-3xFlagUsp27x or TetR-3xFlag-Usp27xC87A cells treated as in A were analysed using conditions of higher resolution. A band very likely corresponding to phosphorylated BimEL is detectable. Similar results were obtained in n=2 separate experiments.


C Usp27x stabilizes expression of p - BimEL ( Ser69 ) in Bim - degrading conditions without reducing β - TrCP levels293FT - TetR - 3xFlagUsp27x were treated with dox , PMA [ 16 . 2 nM ] , UO126 [ 10µM ] or QVD [ 10µM ] as indicated for 24h , and levels of phosphorylated BimEL at serine69 were analysed by Western blotting using a phospho - Bim ( Ser69 ) specific antibody . Scans are from the same membrane and exposure times . Similar results were obtained in n = 2 separate experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 O O O O O O O O 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 Usp27x stabilizes expression of p-BimEL (Ser69) in Bim-degrading conditions without reducing β-TrCP levels293FT-TetR-3xFlagUsp27x were treated with dox, PMA [16.2 nM], UO126 [10µM] or QVD [10µM] as indicated for 24h, and levels of phosphorylated BimEL at serine69 were analysed by Western blotting using a phospho-Bim (Ser69) specific antibody. Scans are from the same membrane and exposure times. Similar results were obtained in n=2 separate experiments.


D UO126 reduces phosphorylation - associated shift of BimEL293FT - TetR - 3xFlagUsp27x were treated for 24h with the indicated drug combinations and Bim was detected by Western blotting . UO126 was added 30 minutes prior other stimulation . Similar results were obtained in n = 3 separate experiments ( see Appendix Fig . S5D ) .

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

D UO126 reduces phosphorylation-associated shift of BimEL293FT-TetR-3xFlagUsp27x were treated for 24h with the indicated drug combinations and Bim was detected by Western blotting. UO126 was added 30 minutes prior other stimulation. Similar results were obtained in n=3 separate experiments (see Appendix Fig. S5D).


E Usp27x - expression reduces ubiquitination of Bim293FT - TetR - 3xFlagUsp27x cells were transfected with a vector coding for 6His - ubiquitin ( left ) or GFP ( right ) . After 24h cells were treated with PMA and QVD , and 3xFlag - Usp27x was induced by dox at the same time . After additional 20h cells were treated for 4h with MG132 ( 40 µM ) to block proteasomal degradation of ubiquitinated proteins . Cells were lysed under denaturing conditions and His - ubiquitin - labelled proteins or proteins from only GFP expressing cells were purified by Ni2 + - NTAaffinity chromatography . Ubiquitinated Bim ( Bim - Ubn ) was detected by Western blot using anti - Bim antibodies ( left ) . Similar results were obtained in n = 3 separate experiments ( another experiment including a Coomassie stain is shown in Appendix Fig . S3E ) . * Indicates non - His - ubiquitinated BimEL that was bound unspecifically to the Ni2 + - agarose

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Usp27x-expression reduces ubiquitination of Bim293FT-TetR-3xFlagUsp27x cells were transfected with a vector coding for 6His-ubiquitin (left) or GFP (right). After 24h cells were treated with PMA and QVD, and 3xFlag-Usp27x was induced by dox at the same time. After additional 20h cells were treated for 4h with MG132 (40 µM) to block proteasomal degradation of ubiquitinated proteins. Cells were lysed under denaturing conditions and His-ubiquitin-labelled proteins or proteins from only GFP expressing cells were purified by Ni2+-NTAaffinity chromatography. Ubiquitinated Bim (Bim-Ubn) was detected by Western blot using anti-Bim antibodies (left). Similar results were obtained in n=3 separate experiments (another experiment including a Coomassie stain is shown in Appendix Fig. S3E). *Indicates non-His-ubiquitinated BimEL that was bound unspecifically to the Ni2+-agarose beads