Figure 2A 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A 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.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.


Figure 3A - 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A-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.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.


Figure 4C , 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4C, 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.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.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.


Figure 2 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 O O

Figure 2 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


Figure 4 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 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.


Figure 5 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 O O

Figure 5 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).


Figure 1 ( 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 . Statistical ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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. Statistical(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.(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.


Figure 2Cell 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Cell 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.


Figure 3Luminex 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 , 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Luminex 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,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)


Figure 4Joints 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Joints 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.(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.


Figure 5 ( 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 . 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 .

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

Figure 5(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.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.


Figure 6 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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).


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

Figure 7Three-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).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).


Figure 3Tau 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 .

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

Figure 3Tau 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.


Figure 4Tubulin 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 . 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 .

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

Figure 4Tubulin 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.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.


Figure 5FRAP 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5FRAP 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.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.


Figure 6HEK 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6HEK 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.


Figure 7Confocal 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Confocal 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.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.


Figure 8CFP - 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8CFP-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.


Figure 1A 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 . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A 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.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.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.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.


Figure 2A 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 . 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A 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.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).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


Figure 3A 293FT - TetR - 3xFlag - Usp27x or 3xFlag - Usp27xC87A , a polyclonal derivative where the Bim - locus had been targeted by CRISPR / Cas9 - and four single clones obtained by serial dilution from the polyclonal line were treated as indicated ( PMA [ 16 . 2 nM ] , QVD [ 10 µM ] ) . Apoptosis was measured after 24 h of treatment by staining for active caspase - 3 , followed by flow cytometric analysis . Data ( means / SEM ) are from n = 14 ( 3xFlag - Usp27x line and 3xFlag - Usp27xBim2KO polyclonal line ) , n = 5 ( 3xFlag - Usp27xC87A , and 3xFlag - Usp27xBim2KO clone # 14 ) , n = 7 ( 3xFlag - Usp27xBim2KO clone # 11 ) or n = 4 ( 3xFlag - Usp27xBim2KO clone # 4 and # 7 ) separate experiments . P - values ( t - test ) for statistically significant differences are shown . B The same cells as in A ( except Usp27xC87A mutant ) were treated as in A and stained for active Bax . Data ( means / SEM ) are from n = 8 ( 3xFlag - Usp27x line and 3xFlag - Usp27xBim2KO polyclonal line ) , n = 5 ( 3xFlag - Usp27xBim2KO clone # 11 and # 14 ) or n = 4 ( 3xFlag - Usp27xBim2KO clone # 4 and # 7 ) separate experiments . P - values ( t - test ) for statistically significant differences are shown . Expression levels of Bim , β - TrCP and Flag - Usp27x of the different cell lines are shown in Appendix Fig . S5E .

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Figure 3A 293FT-TetR-3xFlag-Usp27x or 3xFlag-Usp27xC87A, a polyclonal derivative where the Bim-locus had been targeted by CRISPR/Cas9- and four single clones obtained by serial dilution from the polyclonal line were treated as indicated (PMA [16.2 nM], QVD [10 µM]). Apoptosis was measured after 24 h of treatment by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=14 (3xFlag-Usp27x line and 3xFlag-Usp27xBim2KO polyclonal line), n=5 (3xFlag-Usp27xC87A, and 3xFlag-Usp27xBim2KO clone #14), n=7 (3xFlag-Usp27xBim2KO clone #11) or n=4 (3xFlag-Usp27xBim2KO clone #4 and #7) separate experiments. P-values (t-test) for statistically significant differences are shown.B The same cells as in A (except Usp27xC87A mutant) were treated as in A and stained for active Bax. Data (means/SEM) are from n=8 (3xFlag-Usp27x line and 3xFlag-Usp27xBim2KO polyclonal line), n=5 (3xFlag-Usp27xBim2KO clone #11 and #14) or n=4 (3xFlag-Usp27xBim2KO clone #4 and #7) separate experiments. P-values (t-test) for statistically significant differences are shown. Expression levels of Bim, β-TrCP and Flag-Usp27x of the different cell lines are shown in Appendix Fig. S5E.


Figure 4A Lines based on the human melanoma cell line 1205Lu ( BRAF - V600E - positive ) were made to carry GFP - Usp27x , GFP - Usp27xC87A or GFP - Usp22 under the control of a doxycycline ( dox ) - inducible promoter . GFP - Usp27x , GFP - Usp27xC87A or GFP - Usp22 were induced with dox for 24 or 48h . Levels of Bim were determined by Western blotting . The two left panels are from the same membrane and exposure times . Similar results were obtained in n = 4 separate experiments ( left ) and n = 3 experiments ( right ) . See also Appendix Fig . S6A where 3xFlag - Usp22 is also shown to be unable to increase Bim levels . B A cell line derived from the BRAF - V600E - positive human melanoma line WM1158 was generated to carry inducible GFP - Usp27x . GFP - Usp27x was induced , and Bim was detected as in A . Similar results were obtained in n = 3 separate experiments . C Derivatives of the NSCLC cell line HCC827 ( expressing constitutively active EGFR ) were generated that only express the TetR - repressor or carrying in addition either GFP - Usp27x or GFP - Usp27xC87A . Cells were treated with dox for 72h . Bim was detected by Western blotting . Similar results were obtained in n = 2 separate experiments . GFP - Usp27x , GFP - Usp27xC87A , or GFP - Usp22 expression ( A - C ) was detected using an antibody against GFP . D Caco2 cells carrying a dox inducible HA - BRAF - V600E [ 37 ] ( left two lanes ) or the same cells stably expressing 3xFlag - Usp27x were treated with dox for 48h to induce expression of HA - BRAF - V600E . Bim was detected by Western blotting . The amount of BimEL was quantified from the shown immunoblot using the expression levels of the uninduced control cells set to 100 % ( normalized to the tubulin signal for each condition ) . Similar results were obtained in n = 3 separate experiments with varying induction times . See also Appendix Fig . S6D .

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

Figure 4A Lines based on the human melanoma cell line 1205Lu (BRAF-V600E-positive) were made to carry GFP-Usp27x, GFP-Usp27xC87A or GFP-Usp22 under the control of a doxycycline (dox)-inducible promoter. GFP-Usp27x, GFP-Usp27xC87A or GFP-Usp22 were induced with dox for 24 or 48h. Levels of Bim were determined by Western blotting. The two left panels are from the same membrane and exposure times. Similar results were obtained in n=4 separate experiments (left) and n=3 experiments (right). See also Appendix Fig. S6A where 3xFlag-Usp22 is also shown to be unable to increase Bim levels.B A cell line derived from the BRAF-V600E-positive human melanoma line WM1158 was generated to carry inducible GFP-Usp27x. GFP-Usp27x was induced, and Bim was detected as in A. Similar results were obtained in n=3 separate experiments.C Derivatives of the NSCLC cell line HCC827 (expressing constitutively active EGFR) were generated that only express the TetR-repressor or carrying in addition either GFP-Usp27x or GFP-Usp27xC87A. Cells were treated with dox for 72h. Bim was detected by Western blotting. Similar results were obtained in n=2 separate experiments. GFP-Usp27x, GFP-Usp27xC87A, or GFP-Usp22 expression (A-C) was detected using an antibody against GFP.D Caco2 cells carrying a dox inducible HA-BRAF-V600E [37] (left two lanes) or the same cells stably expressing 3xFlag-Usp27x were treated with dox for 48h to induce expression of HA-BRAF-V600E. Bim was detected by Western blotting. The amount of BimEL was quantified from the shown immunoblot using the expression levels of the uninduced control cells set to 100% (normalized to the tubulin signal for each condition). Similar results were obtained in n=3 separate experiments with varying induction times. See also Appendix Fig. S6D.


Figure 5A Loss of endogenous Usp27x enhances destabilization of BimEL in response to BRAF - V600ECaco2 cells carrying inducible HA - BRAF - V600E were transfected with control siRNA ( siCo3 ) or siRNA directed against Usp27x ( combination of three siRNAs targeting Usp27xmRNA ) for 24h prior to BRAF - V600E expression with dox for 2h ( left ) or 4h ( right ) . BimEL was detected by Western blotting . Similar results were also observed in n = 2 more experiments after 3h induction ( not shown , see Appendix Fig . S6E for siRNA efficacy ) . B 293FT cells deficient for Usp27x show enhanced destabilization of BimEL in response to PMA293FT wt cells or 293FT - Usp27xKO ( clone 2 / 10 , generated using CRISPR - Cas - 9 ) were treated with PMA [ 16 . 2 nM ] for 16h or 24h to induce BimEL degradation . Western blots show protein levels of n = 3 independently performed experiments ( similar results were obtained in one more experiment , not shown ) . See Appendix Fig . S6G for verification of the Usp27xKO cell line by DNA sequence analysis .

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

Figure 5A Loss of endogenous Usp27x enhances destabilization of BimEL in response to BRAF-V600ECaco2 cells carrying inducible HA-BRAF-V600E were transfected with control siRNA (siCo3) or siRNA directed against Usp27x (combination of three siRNAs targeting Usp27xmRNA) for 24h prior to BRAF-V600E expression with dox for 2h (left) or 4h (right). BimEL was detected by Western blotting. Similar results were also observed in n=2 more experiments after 3h induction (not shown, see Appendix Fig. S6E for siRNA efficacy).B 293FT cells deficient for Usp27x show enhanced destabilization of BimEL in response to PMA293FT wt cells or 293FT-Usp27xKO (clone 2/10, generated using CRISPR-Cas-9) were treated with PMA [16.2 nM] for 16h or 24h to induce BimEL degradation. Western blots show protein levels of n=3 independently performed experiments (similar results were obtained in one more experiment, not shown). See Appendix Fig. S6G for verification of the Usp27xKO cell line by DNA sequence analysis.


Figure 6A 293FT - TetR - 3xFlagUsp27x cells were treated with PMA [ 16 . 2 nM ] plus QVD [ 10 µM ] and 3xFlag - Usp27x was induced by dox at the same time for 24h followed by addition of cycloheximide ( CHX ) . Cells were harvested at the indicated time points , and levels of BimEL were determined by Western blotting . Similar results were obtained in n = 3 separate experiments . Cut membranes shown were from one membrane with same exposure time . For each condition BimEL - levels were quantified and normalized to the tubulin - signal . Percent BimEL gives the expression relative to the starting point . B 1205Lu melanoma cells carrying dox - inducible GFP - Usp27x were treated with dox for 24h . Cycloheximide ( CHX , 1µg / ml ) was then added . Cells were harvested at the indicated time points , and levels of BimEL were determined by Western blotting . GFP - Usp27x was detected with anti - GFP - antibodies . Similar results were obtained in n = 3 separate experiments . A second experiment is shown in the right panel also including 1205Lu melanoma carrying dox - inducible GFP - Usp27xC87A mutant . For each condition BimEL - levels were quantified and normalized to the tubulin - signal . Percent BimEL gives the expression relative to the starting point set to 100 % . The starting point for the non - Usp27x / C87A inducing cells ( - dox ) is shown in lane 1 for experiment one and two ( - dox , - CHX ) . Cut membranes shown were from one membrane with same exposure time . Same results were obtained for WM1158 melanoma cells ( data not shown ) . C HCC827 cells were induced to express GFP - Usp27x with dox as indicated . 24h later cycloheximide ( CHX , 1µg / ml ) was added for the indicated time . BimEL was detected after the indicated times of treatment by Western blotting . For each condition BimEL - levels were quantified and normalized to the tubulin - signal . Percent BimEL gives the expression relative to the starting point . Similar results were obtained in n = 2 separate experiments . Cut membranes shown were from one membrane with same exposure time .

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

Figure 6A 293FT-TetR-3xFlagUsp27x cells were treated with PMA [16.2 nM] plus QVD [10 µM] and 3xFlag-Usp27x was induced by dox at the same time for 24h followed by addition of cycloheximide (CHX). Cells were harvested at the indicated time points, and levels of BimEL were determined by Western blotting. Similar results were obtained in n=3 separate experiments. Cut membranes shown were from one membrane with same exposure time. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point.B 1205Lu melanoma cells carrying dox-inducible GFP-Usp27x were treated with dox for 24h. Cycloheximide (CHX, 1µg/ml) was then added. Cells were harvested at the indicated time points, and levels of BimEL were determined by Western blotting. GFP-Usp27x was detected with anti-GFP-antibodies. Similar results were obtained in n=3 separate experiments. A second experiment is shown in the right panel also including 1205Lu melanoma carrying dox-inducible GFP-Usp27xC87A mutant. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point set to 100%. The starting point for the non-Usp27x/C87A inducing cells (-dox) is shown in lane 1 for experiment one and two (-dox, - CHX). Cut membranes shown were from one membrane with same exposure time. Same results were obtained for WM1158 melanoma cells (data not shown).C HCC827 cells were induced to express GFP-Usp27x with dox as indicated. 24h later cycloheximide (CHX, 1µg/ml) was added for the indicated time. BimEL was detected after the indicated times of treatment by Western blotting. For each condition BimEL-levels were quantified and normalized to the tubulin-signal. Percent BimEL gives the expression relative to the starting point. Similar results were obtained in n=2 separate experiments. Cut membranes shown were from one membrane with same exposure time.


Figure 7A 1205Lu melanoma cells carrying dox - inducible GFP , GFP - Usp27x or GFP - Usp27xC87A were treated with doxycycline ( dox ) as indicated . After 48 hUO126 ( 10µM ) was added for another 48 h . Apoptosis was measured by staining for active caspase - 3 , followed by flow cytometric analysis . Data ( means / SEM ) are from n = 3 ( GFP ) or from n ≥ 6 separate experiments ( GFP - Usp27x and GFP - Usp27xC87A ) . P - values ( t - test ) for statistically significant differences are shown . N . s . , p > 0 . 05 . The addition of QVD inhibited cells from active Caspase - 3 positive staining ( not shown ) . A stain for active Bax is shown in Appendix Fig . S7B . B HCC827 NSCLC cells carrying dox - inducible GFP - Usp27xC87A , GFP - Usp27x , or two separate lines established from these GFP - Usp27x - cells where the Bim - locus had been targeted for deletion using CRISPR / Cas9 ( Bim - KO ) , were treated with combinations of dox and the EGFR - inhibitor gefitinib as indicated . Apoptosis was measured after 72h of treatment by staining for active caspase - 3 , followed by flow cytometric analysis . Data ( means / SEM ) are from n = ≥ 15 ( maternal GFP - Usp27x line ) or from n ≥ 5 ( for Bim1KO ) or from n ≥ 6 ( for Bim2KO ) , or n = 4 ( for GFP - Usp27xC87A ) separate experiments . P - values ( t - test ) for statistically significant differences are shown . Again , the addition of QVD inhibited cells from active Caspase - 3 positive staining ( not shown ) . The inset shows Bim - knock - out - efficiency ( n = 2 ) .

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

Figure 7A 1205Lu melanoma cells carrying dox-inducible GFP, GFP-Usp27x or GFP-Usp27xC87A were treated with doxycycline (dox) as indicated. After 48 hUO126 (10µM) was added for another 48 h. Apoptosis was measured by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=3 (GFP) or from n≥6 separate experiments (GFP-Usp27x and GFP-Usp27xC87A). P-values (t-test) for statistically significant differences are shown. N.s., p>0.05. The addition of QVD inhibited cells from active Caspase-3 positive staining (not shown). A stain for active Bax is shown in Appendix Fig. S7B.B HCC827 NSCLC cells carrying dox-inducible GFP-Usp27xC87A, GFP-Usp27x, or two separate lines established from these GFP-Usp27x-cells where the Bim-locus had been targeted for deletion using CRISPR/Cas9 (Bim-KO), were treated with combinations of dox and the EGFR-inhibitor gefitinib as indicated. Apoptosis was measured after 72h of treatment by staining for active caspase-3, followed by flow cytometric analysis. Data (means/SEM) are from n=≥15 (maternal GFP-Usp27x line) or from n≥5 (for Bim1KO) or from n≥6 (for Bim2KO), or n=4 (for GFP-Usp27xC87A) separate experiments. P-values (t-test) for statistically significant differences are shown. Again, the addition of QVD inhibited cells from active Caspase-3 positive staining (not shown). The inset shows Bim-knock-out-efficiency (n=2).


Figure 8Figure 8 Loss of Usp27xreduces gefitinib - induced apoptosis in HCC827 NSCLCHCC827 NSCLC cells and two separate polyclonal lines established from these cells where the Usp27x - locus had been targeted for deletion using CRISPR / Cas9 with two different guide RNA against Usp27x ( Usp27xKO - 1 and Usp27xKO - 4 ) , were treated with gefitinib [ 10µM ] for 48h . Apoptosis was measured by staining for active caspase - 3 ( A ) or by staining for activated Bax ( antibody : 6A7 clone ) followed by flow cytometric analysis . Data shown in A , B ( means / SEM ) are from n = 4 separate experiments . P - values ( t - test ) for statistically significant differences are shown .

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Figure 8Figure 8 Loss of Usp27xreduces gefitinib-induced apoptosis in HCC827 NSCLCHCC827 NSCLC cells and two separate polyclonal lines established from these cells where the Usp27x-locus had been targeted for deletion using CRISPR/Cas9 with two different guide RNA against Usp27x (Usp27xKO-1 and Usp27xKO-4), were treated with gefitinib [10µM] for 48h. Apoptosis was measured by staining for active caspase-3 (A) or by staining for activated Bax (antibody: 6A7 clone) followed by flow cytometric analysis. Data shown in A, B (means/SEM) are from n=4 separate experiments. P-values (t-test) for statistically significant differences are shown.


Figure 1Number of EMPs per embryo equivalent ( e . e . ) of wild type and Sl / Sl E9 . 5 and E11 . 5 YS , determined by flow cytometry ( panels in B , D ) . E9 . 5 Kit + CD41 + CD16 / 32 + EMP numbers are the mean ± SD from 4 wild type and 3 Sl / Sl biological replicates , with each replicate consisting of single or 2 pooled YS of the same genotype . Total number of embryos analyzed : 7 + / + ( 14 - 23sp ) , 5 Sl / Sl ( 17 - 25sp ) . E11 . 5 Kit + CD41 + EMP numbers are the mean ± SD of 5 + / + and 6 Sl / Sl YS analyzed individually over 2 independent experiments . Total live cells per YS : 1 . 7 ± 0 . 3x105 ( wt ) , 1 . 7 ± 0 . 2x105 ( Sl / Sl ) at E9 . 5 ; 4 . 2 ± 1 . 4x105 ( wt ) , 4 . 0 ± 2 . 2x105 ( Sl / Sl ) at E11 . 5 . Example of flow cytometry analysis of E9 . 5 YS EMPs quantified in panel ( A ) . Analyses were performed on Ter119 - live cells . Number of CFU - C in wild type and Sl / Sl E9 . 5 and E11 . 5 YS . E9 . 5 data ( mean ± SD ) are from 3 wild type and 2 Sl / Sl biological replicates plated in duplicate , with each replicate consisting of single or 2 pooled YS of the same genotype . Total number of analyzed embryos : 5 + / + ( 17 - 23sp ) , 3 Sl / Sl ( 17 - 25sp ) over 2 independent experiments . For E11 . 5 , data are from 4 wild type and 6 Sl / Sl biological replicates plated in duplicate , with each replicate consisting of single or 2 pooled YSs of the same genotype . Total number of embryos analyzed : 6 + / + , 7 Sl / Sl over 4 independent experiments . GEMM : granulocyte , erythroid , monocyte / macrophage , megakaryocyte ; G / M / GM : granulocyte , monocyte / macrophage ; Ery : erythroid . Example of flow cytometry analysis of E11 . 5 YS EMPs quantified in panel ( A ) . Analyses were performed on Ter119 - live cells . Confocal WM - IF analysis of CD31 + pHH3 + Kit + proliferating EMPs ( arrowheads ) in E9 . 5 wild type and Sl / Sl YS . A single 2 . 5μm - thick Z section is shown . For each YS , CD31 + Kit + pHH3 + cells were counted in 3 to 5 fields using a 25x objective and the percentage of pHH3 + proliferating EMPs was calculated . Bar graphs represent the mean ± SD from 3 wild type ( 21 - 24sp ) and 3 Sl / Sl ( 22 - 23sp ) YS . Scale bars : 50μm . Cell cycle analysis of wild type and Sl / Sl E11 . 5 YS EMPs . The percentage of EMPs ( Ter119 - Kit + CD41 + ) in G0 / G1 or S / G2 / M was determined by flow cytometry on the basis of BrdU and 7 - AAD incorporation . a : apoptotic ( percentage not shown ) . Embryos were analyzed individually . N = 6 ( + / + ) , N = 5 ( Sl / Sl ) over 3 independent experiments . E11 . 5 embryos were staged according to tail somite counts as described in [ 76 ] and were : 12 - 17 ( + / + ) ; 12 - 17 ( Sl / Sl ) . Bar graphs show mean ± SD , and FACS plots representative results .

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Figure 1Number of EMPs per embryo equivalent (e.e.) of wild type and Sl/Sl E9.5 and E11.5 YS, determined by flow cytometry (panels in B,D). E9.5 Kit+ CD41+ CD16/32+ EMP numbers are the mean±SD from 4 wild type and 3 Sl/Sl biological replicates, with each replicate consisting of single or 2 pooled YS of the same genotype. Total number of embryos analyzed: 7 +/+ (14-23sp), 5 Sl/Sl (17-25sp). E11.5 Kit+ CD41+ EMP numbers are the mean±SD of 5 +/+ and 6 Sl/Sl YS analyzed individually over 2 independent experiments. Total live cells per YS: 1.7±0.3x105 (wt), 1.7±0.2x105 (Sl/Sl) at E9.5; 4.2±1.4x105 (wt), 4.0±2.2x105 (Sl/Sl) at E11.5.Example of flow cytometry analysis of E9.5 YS EMPs quantified in panel (A). Analyses were performed on Ter119- live cells.Number of CFU-C in wild type and Sl/Sl E9.5 and E11.5 YS. E9.5 data (mean±SD) are from 3 wild type and 2 Sl/Sl biological replicates plated in duplicate, with each replicate consisting of single or 2 pooled YS of the same genotype. Total number of analyzed embryos: 5 +/+ (17-23sp), 3 Sl/Sl (17-25sp) over 2 independent experiments. For E11.5, data are from 4 wild type and 6 Sl/Sl biological replicates plated in duplicate, with each replicate consisting of single or 2 pooled YSs of the same genotype. Total number of embryos analyzed: 6 +/+, 7 Sl/Sl over 4 independent experiments. GEMM: granulocyte, erythroid, monocyte/macrophage, megakaryocyte; G/M/GM: granulocyte, monocyte/macrophage; Ery: erythroid.Example of flow cytometry analysis of E11.5 YS EMPs quantified in panel (A). Analyses were performed on Ter119- live cells.Confocal WM-IF analysis of CD31+ pHH3+ Kit+ proliferating EMPs (arrowheads) in E9.5 wild type and Sl/Sl YS. A single 2.5μm-thick Z section is shown. For each YS, CD31+ Kit+ pHH3+ cells were counted in 3 to 5 fields using a 25x objective and the percentage of pHH3+ proliferating EMPs was calculated. Bar graphs represent the mean±SD from 3 wild type (21-24sp) and 3 Sl/Sl (22-23sp) YS. Scale bars: 50μm.Cell cycle analysis of wild type and Sl/Sl E11.5 YS EMPs. The percentage of EMPs (Ter119- Kit+ CD41+) in G0/G1 or S/G2/M was determined by flow cytometry on the basis of BrdU and 7-AAD incorporation. a: apoptotic (percentage not shown). Embryos were analyzed individually. N=6 (+/+), N=5 (Sl/Sl) over 3 independent experiments. E11.5 embryos were staged according to tail somite counts as described in [76] and were: 12-17 (+/+); 12-17 (Sl/Sl). Bar graphs show mean±SD, and FACS plots representative results.