Figure 6b , Left : screening procedure for cellular components critical for the secretion of EV - TβRII ; Right : nanosight analysis of GFP positive EVs derived from MDA - MB - 231 cells expressing TβRII - GFP and pre - treated with anti - cancer compounds . n = 3 technical replicates . c , Immunoblot analysis of total cell lysates derived from MDA - MB - 231 breast cancer cell treated with USP8 inhibitor and TGF - β ( 2 . 5 ng / ml ) as indicated . d , Effect of USP8 inhibitor ( 5 μM ) on SMAD3 / SMAD4 - dependent CAGA12 - Luc transcriptional response induced by TGF - β ( 2 . 5 ng / ml ) for 16 h in HEK293T cells transfected with control vector ( Co . vec ) or USP8 wt / cs . n = 3 biological replicates . e , Heatmap shows differentially expressed genes in control DMSO - treated or USP8 inhibitor ( 5 μM ) - treated MDA - MB - 231 cells for 24 h ( left panel ) . Volcano plot of transcriptome proﬁles between control and USP8 inhibitor treated MDA - MB - 231 cells ( right panel ) . Red and green dots represent genes significantly up - regulated and down - regulated in control MDA - MB - 231 cells . f , qRT - PCR analysis of control ( DMSO - treated ) or USP8 inhibitor ( 5 μM for 24 h ) treated MDA - MB - 231 cells . Relative mRNA levels are shown as a heatmap . g , Gene signatures indicating the up - regulated epithelial - mesenchymal - transition ( EMT ) ( upper panel ) and breast cancer progenitors - related gene set ( bottom panel ) are significantly enriched in control cells ; shown by pre - ranked gene - set enrichment analysis ( GSEA ) . h , The down - regulated TGF - β signaling ( upper panel ) and metastasis - related gene sets ( bottom panel ) are significantly enriched in USP8 inhibitor - treated MDA - MB - 231 cells ; shown by pre - ranked gene - set enrichment analysis ( GSEA ) . j , The total concentration of EVs by nanosight analysis ( left ) and the percentage of TβRII + EVs by FACS ( right ) from 4T1 and MDA - MB - 231 cells treated with USP8 inhibitor ( 5 μM ) for 24 h . n = 3 technical replicates . k - n , Experimental procedure in vivo MDA - MB - 231 - Luc cells ( 1 × 105 per mouse ) were intracardially injected into nude mice ( n = 7 for each group ) . USP8 inhibitor ( 1 mg / kg ) was injected at day 1 via intraperitoneal injection every the other day for three weeks . Normalized BLI signals ( left panel ) and BLI imaging of three representive nude mice from each group at week 6 ( right panel ) were shown ( k ) . Percentage of metastasis - free survival in each experimental group followed in time ( l ) . Number of bone metastasis nodules ( m ) . Percentage of TβRII + crEVs in plasma samples from mice ( left panel ) and immunoblot analysis of metastatic tumor nodule in mice and circulating EVs from plasma ( right panel ) ( n ) .

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 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 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 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 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 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 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 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

Figure 6b, Left: screening procedure for cellular components critical for the secretion of EV-TβRII; Right: nanosight analysis of GFP positive EVs derived from MDA-MB-231 cells expressing TβRII-GFP and pre-treated with anti-cancer compounds. n = 3 technical replicates.c, Immunoblot analysis of total cell lysates derived from MDA-MB-231 breast cancer cell treated with USP8 inhibitor and TGF-β (2.5 ng/ml) as indicated.d, Effect of USP8 inhibitor (5 μM) on SMAD3/SMAD4-dependent CAGA12-Luc transcriptional response induced by TGF-β (2.5 ng/ml) for 16 h in HEK293T cells transfected with control vector (Co.vec) or USP8 wt/cs. n = 3 biological replicates.e, Heatmap shows differentially expressed genes in control DMSO-treated or USP8 inhibitor (5 μM)-treated MDA-MB-231 cells for 24 h (left panel). Volcano plot of transcriptome proﬁles between control and USP8 inhibitor treated MDA-MB-231 cells (right panel). Red and green dots represent genes significantly up-regulated and down-regulated in control MDA-MB-231 cells.f, qRT-PCR analysis of control (DMSO-treated) or USP8 inhibitor (5 μM for 24 h) treated MDA-MB-231 cells. Relative mRNA levels are shown as a heatmap.g, Gene signatures indicating the up-regulated epithelial-mesenchymal-transition (EMT) (upper panel) and breast cancer progenitors-related gene set (bottom panel) are significantly enriched in control cells; shown by pre-ranked gene-set enrichment analysis (GSEA).h, The down-regulated TGF-β signaling (upper panel) and metastasis-related gene sets (bottom panel) are significantly enriched in USP8 inhibitor-treated MDA-MB-231 cells; shown by pre-ranked gene-set enrichment analysis (GSEA).j, The total concentration of EVs by nanosight analysis (left) and the percentage of TβRII+ EVs by FACS (right) from 4T1 and MDA-MB-231 cells treated with USP8 inhibitor (5 μM) for 24 h. n = 3 technical replicates.k-n, Experimental procedure in vivo MDA-MB-231-Luc cells (1 × 105 per mouse) were intracardially injected into nude mice (n = 7 for each group). USP8 inhibitor (1 mg/kg) was injected at day 1 via intraperitoneal injection every the other day for three weeks. Normalized BLI signals (left panel) and BLI imaging of three representive nude mice from each group at week 6 (right panel) were shown (k). Percentage of metastasis-free survival in each experimental group followed in time (l). Number of bone metastasis nodules (m). Percentage of TβRII+ crEVs in plasma samples from mice (left panel) and immunoblot analysis of metastatic tumor nodule in mice and circulating EVs from plasma (right panel) (n).


Figure 1B , C , Representative images of pPLT3 : : erCFP ( cyan ) expressing and PI - stained ( red ) Arabidopsis roots in Col or wox5 background , respectively . D , Mean fluorescence intensities of the pPLT3 : : erCFP roots summarized in box and scatter plots . The mean fluorescence intensity of the CFP signal in Col roots was to set to 100 % . D , Box = 25 - 75 % of percentile , whisker = 1 . 5 interquartile range , − = median , □ = mean value , = minimum / maximum . The data was statistically analyzed by one - way ANOVA and Holm - Sidak post - hoc multiple comparisons test . Asterisks indicate statistically significant differences ( α = 0 . 01 ) . Number of analysed roots ( n ) ( biological replicates ) is indicated for each genotype and results from two technical replicates . B , C , Scale bars represent 10 µm . PI = propidium iodide CFP = cyan fluorescent protein . E , F , Representative images of pPLT3 : : PLT3 - YFP ( yellow ) expressing and FM4 - 64 - stained ( red ) Arabidopsis roots in Col or wox5 mutant background , respectively . g , Mean fluorescence intensities of the pPLT3 : : PLT3 - YFP expressing roots summarized in box and scatter plots . The mean fluorescence intensity of the YFP signal in Col roots was to set to 100 % . G , Box = 25 - 75 % of percentile , whisker = 1 . 5 interquartile range , − = median , □ = mean value , = minimum / maximum . The data was statistically analyzed by one - way ANOVA and Holm - Sidak post - hoc multiple comparisons test . Asterisks indicate statistically significant differences ( α = 0 . 01 ) . Number of analysed roots ( n ) ( biological replicates ) is indicated for each genotype and results from two technical replicates . , E , F , Scale bars represent 10 µm . YFP = yellow fluorescent

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 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

Figure 1B, C, Representative images of pPLT3::erCFP (cyan) expressing and PI-stained (red) Arabidopsis roots in Col or wox5 background, respectively. D, Mean fluorescence intensities of the pPLT3::erCFP roots summarized in box and scatter plots. The mean fluorescence intensity of the CFP signal in Col roots was to set to 100 %. D, Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, □ = mean value, = minimum/maximum. The data was statistically analyzed by one-way ANOVA and Holm-Sidak post-hoc multiple comparisons test. Asterisks indicate statistically significant differences (α = 0.01). Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from two technical replicates. B, C, Scale bars represent 10 µm. PI = propidium iodide CFP = cyan fluorescent protein.E, F, Representative images of pPLT3::PLT3-YFP (yellow) expressing and FM4-64-stained (red) Arabidopsis roots in Col or wox5 mutant background, respectively. g, Mean fluorescence intensities of the pPLT3::PLT3-YFP expressing roots summarized in box and scatter plots. The mean fluorescence intensity of the YFP signal in Col roots was to set to 100%. G, Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, □ = mean value, = minimum/maximum. The data was statistically analyzed by one-way ANOVA and Holm-Sidak post-hoc multiple comparisons test. Asterisks indicate statistically significant differences (α = 0.01). Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from two technical replicates. , E, F, Scale bars represent 10 µm. YFP = yellow fluorescent protein


Figure 2A - F , Representative FM4 - 64 - stained Arabidopsis roots ( grey ) expressing pWOX5 : : mVenus - NLS ( green ) in Col , plt2 , plt3 and plt2 , plt3 double mutant background in longitudinal ( A - D ) , or transversal ( E - F ) optical sections . E ' , F ' , Analysis of representative images in ( E ) and ( F ) in Imaris to detect and count individual expressing nuclei . E ' ' , F ' ' , Overlay of 10 roots ( biological replicates ) showing the area of detected fluorescence ( high levels in red , low levels in blue ) in Col and plt2 , plt3 double mutant roots . G , Number of nuclei ( biological replicates ) expressing pWOX5 : : mVenus - NLS in Col and plt2 , plt3 double mutant roots summarized in box and scatter plots . H , Area of WOX5 expression in µm2 in Col and plt2 , plt3 double mutant roots summarized in box and scatter plots . G , H Box = 25 - 75 % of percentile , whisker = 1 . 5 interquartile range , − = median , □ = mean value , = minimum / maximum . ( G , H ) Kruskal - Wallis ANOVA analysis with subsequent Dunns test ( G ) or one - way ANOVA and post - hoc Holm - Sidak multiple comparisons test was used to test for statistical significance ( H ) . Asterisks indicate statistically significant differences ( α = 0 . 01 ) . Number of analysed roots ( n ) ( biological replicates ) is indicated for each genotype and results from three technical replicates per genotype . Scale bars represent 10 µm ; NLS = nuclear localisation signal .

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

Figure 2A-F, Representative FM4-64-stained Arabidopsis roots (grey) expressing pWOX5::mVenus-NLS (green) in Col, plt2, plt3 and plt2, plt3 double mutant background in longitudinal (A-D), or transversal (E-F) optical sections. E', F', Analysis of representative images in (E) and (F) in Imaris to detect and count individual expressing nuclei. E'', F'', Overlay of 10 roots (biological replicates) showing the area of detected fluorescence (high levels in red, low levels in blue) in Col and plt2, plt3 double mutant roots. G, Number of nuclei (biological replicates) expressing pWOX5::mVenus-NLS in Col and plt2, plt3 double mutant roots summarized in box and scatter plots. H, Area of WOX5 expression in µm2 in Col and plt2, plt3 double mutant roots summarized in box and scatter plots. G, H Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, □ = mean value, = minimum/maximum. (G, H) Kruskal-Wallis ANOVA analysis with subsequent Dunns test (G) or one-way ANOVA and post-hoc Holm-Sidak multiple comparisons test was used to test for statistical significance (H). Asterisks indicate statistically significant differences (α = 0.01). Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from three technical replicates per genotype. Scale bars represent 10 µm; NLS = nuclear localisation signal.


Figure 3A , Schematic representation of a longitudinal section of an Arabidopsis RM . QC cells are marked in red , CSCs are marked in dark blue , CCs in light blue . Combined mPSPI ( grey ) and EdU ( red ) staining for 24 hours ( SCN staining ) to analyse the CSC ( A - I ) and QC division phenotype ( J - R ) within the same roots are shown . B - H , Representative images of the SCN staining in Col , and the indicated single , double , and triple mutant roots . QC positions are marked by yellow arrowhead . I , Analyses of the SCN staining for CSC phenotypes . Frequencies of roots showing 0 , 1 , 2 , or 3 CSC layers are plotted as bar graphs . J , Schematic representation of a transversal section of an Arabidopsis RM . QC cells are marked in red , CEI initials are marked in turquoise . K - Q , Representative images of transversal sections with QC cells outlined in yellow . R , Analyses of the SCN staining for QC division phenotypes . Frequencies of roots showing 0 , 1 , 2 , 3 or ≥ 4 dividing QC cells are plotted as bar graphs . Number of analysed roots ( n ) ( biological replicates ) is indicated for each genotype and results from 2 - 5 technical replicates per genotype . QC = quiescent center , CSC = columella stem cell , CEI = cortex endodermis initial , SCN = stem cell niche , mPSPI = modified pseudo - Schiff propidium iodide , EdU = 5 - ethynyl - 2 ' - deoxyuridine , scale bars represent 5 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

Figure 3A, Schematic representation of a longitudinal section of an Arabidopsis RM. QC cells are marked in red, CSCs are marked in dark blue, CCs in light blue. Combined mPSPI (grey) and EdU (red) staining for 24 hours (SCN staining) to analyse the CSC (A-I) and QC division phenotype (J-R) within the same roots are shown. B-H, Representative images of the SCN staining in Col, and the indicated single, double, and triple mutant roots. QC positions are marked by yellow arrowhead. I, Analyses of the SCN staining for CSC phenotypes. Frequencies of roots showing 0, 1, 2, or 3 CSC layers are plotted as bar graphs. J, Schematic representation of a transversal section of an Arabidopsis RM. QC cells are marked in red, CEI initials are marked in turquoise. K-Q, Representative images of transversal sections with QC cells outlined in yellow. R, Analyses of the SCN staining for QC division phenotypes. Frequencies of roots showing 0, 1, 2, 3 or ≥4 dividing QC cells are plotted as bar graphs. Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from 2-5 technical replicates per genotype. QC = quiescent center, CSC = columella stem cell, CEI = cortex endodermis initial, SCN = stem cell niche, mPSPI = modified pseudo-Schiff propidium iodide, EdU = 5-ethynyl-2'-deoxyuridine, scale bars represent 5 µm.


Figure 4The combined results of the SCN staining 3 are shown as 2D plots to visualise the correlation of the CSC layer and QC division phenotypes . Number of CSC layers are shown on the y axis and the QC division phenotype is shown on the x axis . The darker the colour , the more roots show the respective phenotype ( see colour gradient top right indicating the frequencies in percent ) . Col wild - type roots show one layer of CSCs and no EdU stained cells ( no QC division ) after 24 h EdU staining . Number of analysed roots ( n ) ( biological replicates ) is indicated for each genotype and results from 2 - 5 technical replicates per genotype .

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

Figure 4The combined results of the SCN staining 3 are shown as 2D plots to visualise the correlation of the CSC layer and QC division phenotypes. Number of CSC layers are shown on the y axis and the QC division phenotype is shown on the x axis. The darker the colour, the more roots show the respective phenotype (see colour gradient top right indicating the frequencies in percent). Col wild-type roots show one layer of CSCs and no EdU stained cells (no QC division) after 24 h EdU staining. Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from 2-5 technical replicates per genotype.


Figure 7A - H , Fluorescence Lifetime Imaging ( FLIM ) results of transiently expressing N . benthamiana leaf epidermal cells . A - H , Representative FLIM images of WOX5 - mVenus ( donor only ) plus the indicated acceptors after a pixel - wise mono - exponential fit of the mVenus fluorescence signal . The fluorescence lifetime of WOX5 - mVenus in ns is color - coded . Low lifetimes ( blue ) due to FRET indicate strong interaction of the two proteins and high lifetimes ( red ) indicate weaker or no interaction . Scale bars represent 5 µm . I , Fluorescence lifetimes in ns are summarized in combined jitter and box plots . The dashed line represents the fluorescence lifetime mean value of the WOX5 - mV co - expressed with free mCh as negative control . The one - way ANOVA and Holm - Sidak post - hoc multiple comparisons test was used to test for statistical significance . Samples with identical letters do not show significant differences ( α = 0 . 01 ) . Number of nuclei analyzed ( n ) ( biological replicates ) is indicated and results from 2 to 9 technical replicates . Box = 25 - 75 % of percentile , whisker = 1 . 5 interquartile range , − = median , ▪ = mean value . J , Representative FLIM images of WOX5 - mVenus ( donor only ) after a pixel - wise mono - exponential fit of the mVenus fluorescence signal . The fluorescence lifetime of WOX5 - mVenus in ns is color - coded . Low lifetimes ( blue ) due to FRET indicate strong interaction of the two proteins and high lifetimes ( red ) indicate weaker or no interaction . Scale bars represent 5 µm . K , Individual nuclei showing nuclear bodies during co - expression of WOX5 - mV and PLT3 - mCh were analysed for WOX5 - mV lifetime in the nuclear bodies or nucleoplasm separately ( n = 7 ( biological replicates ) , from four technical replicates . mCh = mCherry . mV = mVenus .

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 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 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 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

Figure 7A-H, Fluorescence Lifetime Imaging (FLIM) results of transiently expressing N. benthamiana leaf epidermal cells. A-H, Representative FLIM images of WOX5-mVenus (donor only) plus the indicated acceptors after a pixel-wise mono-exponential fit of the mVenus fluorescence signal. The fluorescence lifetime of WOX5-mVenus in ns is color-coded. Low lifetimes (blue) due to FRET indicate strong interaction of the two proteins and high lifetimes (red) indicate weaker or no interaction. Scale bars represent 5 µm.I, Fluorescence lifetimes in ns are summarized in combined jitter and box plots. The dashed line represents the fluorescence lifetime mean value of the WOX5-mV co-expressed with free mCh as negative control. The one-way ANOVA and Holm-Sidak post-hoc multiple comparisons test was used to test for statistical significance. Samples with identical letters do not show significant differences (α = 0.01). Number of nuclei analyzed (n) (biological replicates) is indicated and results from 2 to 9 technical replicates. Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, ▪ = mean value.J, Representative FLIM images of WOX5-mVenus (donor only) after a pixel-wise mono-exponential fit of the mVenus fluorescence signal. The fluorescence lifetime of WOX5-mVenus in ns is color-coded. Low lifetimes (blue) due to FRET indicate strong interaction of the two proteins and high lifetimes (red) indicate weaker or no interaction. Scale bars represent 5 µm.K, Individual nuclei showing nuclear bodies during co-expression of WOX5-mV and PLT3-mCh were analysed for WOX5-mV lifetime in the nuclear bodies or nucleoplasm separately (n = 7 (biological replicates), from four technical replicates. mCh = mCherry. mV = mVenus.


Figure 1 ( A ) Masson ' s trichrome representative images of pulmonary fibrosis and quantification . ( B ) Relative transcript expression of Fibronectin , ( C ) Collagen I and ( D ) Collagen III to GAPDH . ( E ) Masson ' s trichrome representative images of vascular wall thickening and quantification . ( F ) Masson ' s trichrome representative images and correlation between lung fibrosis and vascular wall thickness in PF and PF - PH patients . Out of the 14 PF and PF - PH patients , 3 of them had no non - fibrotic area and therefore were excluded for comparison between non - fibrotic and fibrotic areas ( n = 11 / group ; Ashcroft score non - fibrosis : 0 - 2 ; mild - fibrosis : 3 - 5 ; severe - fibrosis > 5 ) ( * compared to non - fibrotic areas of the same group of patients ) . ( G ) Masson ' s trichrome representative images of vascular wall thickening in non - fibrotic area ( Ashcroft score 0 - 2 ) and fibrotic area ( Ashcroft score > 2 ) and quantification in PF and PF - PH . The number of samples per group for each experiment are written within each bar graph .

O O B-PANEL_START 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 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 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

Figure 1(A) Masson's trichrome representative images of pulmonary fibrosis and quantification.(B) Relative transcript expression of Fibronectin, (C) Collagen I and (D) Collagen III to GAPDH.(E) Masson's trichrome representative images of vascular wall thickening and quantification.(F) Masson's trichrome representative images and correlation between lung fibrosis and vascular wall thickness in PF and PF-PH patients. Out of the 14 PF and PF-PH patients, 3 of them had no non-fibrotic area and therefore were excluded for comparison between non-fibrotic and fibrotic areas (n=11/group; Ashcroft score non-fibrosis: 0-2; mild-fibrosis: 3-5; severe-fibrosis >5) (* compared to non-fibrotic areas of the same group of patients).(G) Masson's trichrome representative images of vascular wall thickening in non-fibrotic area (Ashcroft score 0-2) and fibrotic area (Ashcroft score >2) and quantification in PF and PF-PH. The number of samples per group for each experiment are written within each bar graph.


Figure 2 ( A ) Representative images of Ki67 - positive pulmonary vascular endothelial cells ( EC , vWF in green ) and smooth muscle cells ( SMC , aSMA in white ) and quantification ( * vs PF , * p < 0 . 05 , * * * p < 0 . 001 ) . Relative expression of Slug mRNA ( B ) normalized to GAPDH in PF and PF - PH patients . Relative expression of Slug protein ( C ) normalized to GAPDH in PF and PF - PH patients . ( D ) Representative images of Slug ( red ) expression in bronchial epithelial cells ( EpC ) , fibroblasts ( Fib ) ( vimentin in green ) macrophages ( Mф , CD68 in green ) pulmonary vascular endothelial cells ( EC , von Willebrand Factor in green ) and pulmonary vascular smooth muscle cells ( SMC , aSMA in white ) ; and quantification . ( E ) Relative CD68 mRNA expression normalized to GAPDH ( F ) Representative images of Slug ( red ) expression in macrophages ( Mф , CD68 in green ) in non - fibrotic and fibrotic areas of the lung in PF and PF - PH patients and quantification . The number of samples per group for each experiment are included within each bar graph .

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 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 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 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

Figure 2(A) Representative images of Ki67-positive pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC, aSMA in white) and quantification (* vs PF, *p<0.05, ***p<0.001).Relative expression of Slug mRNA (B) normalized to GAPDH in PF and PF-PH patients.Relative expression of Slug protein (C) normalized to GAPDH in PF and PF-PH patients.(D) Representative images of Slug (red) expression in bronchial epithelial cells (EpC), fibroblasts (Fib) (vimentin in green) macrophages (Mф, CD68 in green) pulmonary vascular endothelial cells (EC, von Willebrand Factor in green) and pulmonary vascular smooth muscle cells (SMC, aSMA in white); and quantification.(E) Relative CD68 mRNA expression normalized to GAPDH(F) Representative images of Slug (red) expression in macrophages (Mф , CD68 in green) in non-fibrotic and fibrotic areas of the lung in PF and PF-PH patients and quantification. The number of samples per group for each experiment are included within each bar graph.


Figure 3 ( A ) Heatmap of the 20 genes up - regulated in GSE24988 microarray and known to be Slug transcriptionnal target . ( C ) Heatmap of the seven transcriptional targets of Slug known to promote proliferation and to be extracellular . Relative expression of Prolactin - induced protein ( PIP ) mRNA ( D ) normalized to GAPDH in PF and PF - PH patients . Relative expression of Prolactin - induced protein ( PIP ) expression normalized to GAPDH in PF and PF - PH patients . ( F ) Representative images of PIP immunohistochemistry showing its extracellular localization and quantification . Proliferation assay on healthy fibroblast ( G ) Pulmonary arterial EC ( H ) and on healthy pulmonary arterial SMC ( I in the presence or absence of PIP protein . The number of samples per group for each experiment are included within each bar graph . Proliferation assay on healthy pulmonary arterial SMC J ) in the presence or absence of PIP protein . The number of samples per group for each experiment are included within each bar graph .

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 B-PANEL_START 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 B-PANEL_START 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 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

Figure 3(A) Heatmap of the 20 genes up-regulated in GSE24988 microarray and known to be Slug transcriptionnal target.(C) Heatmap of the seven transcriptional targets of Slug known to promote proliferation and to be extracellular.Relative expression of Prolactin-induced protein (PIP) mRNA (D) normalized to GAPDH in PF and PF-PH patients.Relative expression of Prolactin-induced protein (PIP) expression normalized to GAPDH in PF and PF-PH patients.(F) Representative images of PIP immunohistochemistry showing its extracellular localization and quantification.Proliferation assay on healthy fibroblast (G) Pulmonary arterial EC (H) and on healthy pulmonary arterial SMC (I in the presence or absence of PIP protein. The number of samples per group for each experiment are included within each bar graph.Proliferation assay on healthy pulmonary arterial SMC J) in the presence or absence of PIP protein. The number of samples per group for each experiment are included within each bar graph.


Figure 4 ( A ) Masson ' s trichrome representative images of lung fibrosis and quantification two weeks after PBS or bleomycin instillation . Quantification of ( B ) fibronectin , ( C ) Collagen I and ( D ) Collagen III mRNA . ( F ) Right ventricular systolic pressure ( RVSP ) and ( G ) Right ventricular hypertrophy index following the experimental protocol . ( H ) Masson ' s trichrome representative images and quantification of Ashcroft score in the experimental model . Relative expression of fibronectin ( I ) , collagen I ( J ) and collagen III ( K ) mRNA normalized to GAPDH . ( L ) Masson ' s trichrome representative images and quantification of vascular remodeling . ( M ) Masson ' s trichrome representative images and quantification of the vascular remodeling between fibrotic and non - fibrotic areas in the experimental model . The number of samples per group for each experiment are included within each bar graph .

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Figure 4(A) Masson's trichrome representative images of lung fibrosis and quantification two weeks after PBS or bleomycin instillation.Quantification of (B) fibronectin, (C) Collagen I and (D) Collagen III mRNA.(F) Right ventricular systolic pressure (RVSP) and (G) Right ventricular hypertrophy index following the experimental protocol.(H) Masson's trichrome representative images and quantification of Ashcroft score in the experimental model.Relative expression of fibronectin (I), collagen I (J) and collagen III (K) mRNA normalized to GAPDH.(L) Masson's trichrome representative images and quantification of vascular remodeling.(M) Masson's trichrome representative images and quantification of the vascular remodeling between fibrotic and non-fibrotic areas in the experimental model. The number of samples per group for each experiment are included within each bar graph.


Figure 5 ( A ) Representative images and quantification of Ki67 - positive ( red ) pulmonary vascular endothelial cells ( EC , vWF in green ) and smooth muscle cells ( SMC ) in the experimental model . Relative expression of Slug mRNA ( B ) normalized to GAPDH . Relative expression of Slug protein ( C ) normalized to GAPDH . ( D ) Representative images of Slug ( red ) expression in bronchial epithelial cells ( EpC ) , fibroblasts ( Fib , vimentin in green ) macrophages ( Mф , CD68 in green ) pulmonary vascular endothelial cells ( EC , vWF ) and smooth muscle cells ( SMC , aSMA in white ) ; and quantification . ( * vs Bleo , p < 0 . 05 ) ( E ) CD68 mRNA quantification in Bleo and Bleo - MCT rats ( F ) Representative images of Slug ( red ) expression in macrophages ( Mф , CD68 in green ) in non - fibrotic and fibrotic areas of the lung in in Bleo and Bleo - MCT rats and quantification . The number of samples per group for each experiment are included within each bar graph .

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 B-PANEL_START 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 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 B-PANEL_START 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

Figure 5(A) Representative images and quantification of Ki67-positive (red) pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC) in the experimental model.Relative expression of Slug mRNA (B) normalized to GAPDH.Relative expression of Slug protein (C) normalized to GAPDH.(D) Representative images of Slug (red) expression in bronchial epithelial cells (EpC), fibroblasts (Fib, vimentin in green) macrophages (Mф, CD68 in green) pulmonary vascular endothelial cells (EC, vWF) and smooth muscle cells (SMC, aSMA in white); and quantification. (* vs Bleo, p<0.05)(E) CD68 mRNA quantification in Bleo and Bleo-MCT rats(F) Representative images of Slug (red) expression in macrophages (Mф , CD68 in green) in non-fibrotic and fibrotic areas of the lung in in Bleo and Bleo-MCT rats and quantification. The number of samples per group for each experiment are included within each bar graph.


Figure 6Relative expression of PIP mRNA ( A ) normalized to GAPDH . Relative expression of PIP protein ( B ) normalized to GAPDH . ( C ) Representative images and PIP quantification . The number of samples per group for each experiment are included within each bar graph .

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Figure 6Relative expression of PIP mRNA (A) normalized to GAPDH.Relative expression of PIP protein (B) normalized to GAPDH.(C) Representative images and PIP quantification. The number of samples per group for each experiment are included within each bar graph.


Figure 7Relative expression of Slug protein ( B ) normalized to GAPDH . Relative expression of Slug mRNA ( C ) normalized to GAPDH . ( D ) right ventricular systolic pressure ( RVSP ) and ( E ) right ventricular hypertrophy index in PF - PH rats treated with Si - Slug compared to Si - Scrm . Relative expression of fibronectin ( F ) , Collagen I ( G ) and Collagen III ( H ) normalized to GAPDH . ( I ) Masson ' s trichrome representative images and quantification of the Ashcroft score . ( J ) Masson ' s trichrome representative images and quantification of vascular wall thickness . ( K ) Masson ' s trichrome representative images and quantification of the vascular remodeling between non - fibrotic and fibrotic areas of the lung . ( L ) Representative images and quantification of Ki67 - positive ( red ) pulmonary vascular endothelial cells ( EC , vWF in green ) and smooth muscle cells ( SMC , aSMA in white ) . The number of samples per group for each experiment are included within each bar graph .

O B-PANEL_START 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 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 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 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 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

Figure 7Relative expression of Slug protein (B) normalized to GAPDH.Relative expression of Slug mRNA (C) normalized to GAPDH.(D) right ventricular systolic pressure (RVSP) and (E) right ventricular hypertrophy index in PF-PH rats treated with Si-Slug compared to Si-Scrm.Relative expression of fibronectin (F), Collagen I (G) and Collagen III (H) normalized to GAPDH.(I) Masson's trichrome representative images and quantification of the Ashcroft score. (J) Masson's trichrome representative images and quantification of vascular wall thickness.(K) Masson's trichrome representative images and quantification of the vascular remodeling between non-fibrotic and fibrotic areas of the lung.(L) Representative images and quantification of Ki67-positive (red) pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC, aSMA in white). The number of samples per group for each experiment are included within each bar graph.


Figure 8 ( A ) Representative images and quantification of Slug - positive macrophages ( slug , red ; CD68 green ) . ( B ) Relative expression of CD68 mRNA normalized to GAPDH . ( C ) Representative images and quantification of Slug - positive macrophages between non - fibrotic and fibrotic areas of the lung . Relative expression of PIP mRNA ( D ) normalized to GAPDH . Relative expression of PIP protein ( E ) normalized to GAPDH . ( F ) Representative images and quantification of PIP immunohistochemistry .

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Figure 8(A) Representative images and quantification of Slug-positive macrophages (slug, red; CD68 green).(B) Relative expression of CD68 mRNA normalized to GAPDH.(C) Representative images and quantification of Slug-positive macrophages between non-fibrotic and fibrotic areas of the lung.Relative expression of PIP mRNA (D) normalized to GAPDH.Relative expression of PIP protein (E) normalized to GAPDH.(F) Representative images and quantification of PIP immunohistochemistry.


Figure 1B Immunoblot showing CDK5RAP2 levels in Cdk5rap2 wild - type ( WT ) , heterozygous ( HET ) and null erythroid progenitors ( EPs ) isolated from fetal livers . Actin was used as loading control . * * indicates nonspecific band . C Quantification of mean protein levels from ( B ) . Numbers in brackets correspond to number of embryos analyzed . Data information : Bar graphs display mean ± s . d . Statistical analysis was based on the number of embryos ( C , Statistical significances were determined by One - way ANOVA test with Tukey ' s ( C , test * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . D Immunofluorescence images of Cdk5rap2 WT , HET and null erythroid progenitors isolated from fetal livers . Progenitors were stained for CDK5RAP2 ( grey ) , γ - tubulin ( magenta ) and DNA ( Hoechst , blue ) . Images are maximum intensity projections of deconvolved z - stacks . Scale bar , 3 μm . Insets show higher magnification of centrosomes . Scale bar , 1 μm . E Quantification of mean centrosomal signal intensities of CDK5RAP2 and γ - tubulin from ( D ) . Numbers in brackets correspond to number of embryos analyzed with a total number of 470 ( WT ) , 406 ( HET ) and 379 ( null ) progenitors . Data information : Bar graphs display mean ± s . d . Statistical analysis was based on the number of embryos E , Statistical significances were determined by One - way ANOVA test with Tukey ' s E , test * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . I ImageStream images of ex vivo cultured enucleating EBs and reticulocytes . Cells were stained for TER119 ( erythroid marker , green ) and DNA ( Hoechst , blue ) . BF : brightfield . Scale bar , 5 μm . J Quantification of enucleating EBs and reticulocytes after 48 hours ( T48 ) in ex vivo culture . Genotypes are as indicated . The numbers in brackets correspond to the number of embryos analyzed . K Quantification of enucleating EB size from ( I ) . The numbers in brackets refer to the number of embryos analyzed . Data information : Box plots show 5th , 95th ( whiskers ) and 25th , 50th and 75th percentile ( boxes ) . Statistical analysis was based on the number of embryos I and J ) Statistical significances were determined by One - way ANOVA test with Tukey ' s J ) or Kruskal Wallis test with Dunn ' s ( K ) multiple comparisons . * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 1B Immunoblot showing CDK5RAP2 levels in Cdk5rap2 wild-type (WT), heterozygous (HET) and null erythroid progenitors (EPs) isolated from fetal livers. Actin was used as loading control. ** indicates nonspecific band. C Quantification of mean protein levels from (B). Numbers in brackets correspond to number of embryos analyzed. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of embryos (C, Statistical significances were determined by One-way ANOVA test with Tukey's (C, test *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.D Immunofluorescence images of Cdk5rap2 WT, HET and null erythroid progenitors isolated from fetal livers. Progenitors were stained for CDK5RAP2 (grey), γ-tubulin (magenta) and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 3 μm. Insets show higher magnification of centrosomes. Scale bar, 1 μm. E Quantification of mean centrosomal signal intensities of CDK5RAP2 and γ-tubulin from (D). Numbers in brackets correspond to number of embryos analyzed with a total number of 470 (WT), 406 (HET) and 379 (null) progenitors. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of embryos E, Statistical significances were determined by One-way ANOVA test with Tukey's E, test *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.I ImageStream images of ex vivo cultured enucleating EBs and reticulocytes. Cells were stained for TER119 (erythroid marker, green) and DNA (Hoechst, blue). BF: brightfield. Scale bar, 5 μm. J Quantification of enucleating EBs and reticulocytes after 48 hours (T48) in ex vivo culture. Genotypes are as indicated. The numbers in brackets correspond to the number of embryos analyzed. K Quantification of enucleating EB size from (I). The numbers in brackets refer to the number of embryos analyzed. Data information: Box plots show 5th,95th (whiskers) and 25th, 50th and 75th percentile (boxes). Statistical analysis was based on the number of embryos I and J) Statistical significances were determined by One-way ANOVA test with Tukey's J) or Kruskal Wallis test with Dunn's (K) multiple comparisons.*P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


Figure 2A Deconvolved STED images of centrosomes in ex vivo cultured wild - type non - enucleating EBs ( non - enucl . ) or enucleating EBs / reticulocytes ( enucl . / RetiC ) . Cells were stained for γ - tubulin ( magenta ) and centriolar ( CP110 , CEP135 ) or PCM ( CDK5RAP2 , PCNT ) proteins ( cyan ) . Scale bar , 400 nm .

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Figure 2A Deconvolved STED images of centrosomes in ex vivo cultured wild-type non-enucleating EBs (non-enucl.) or enucleating EBs/reticulocytes (enucl./RetiC). Cells were stained for γ-tubulin (magenta) and centriolar (CP110, CEP135) or PCM (CDK5RAP2, PCNT) proteins (cyan). Scale bar, 400 nm.


Figure 3E ImageStream images showing EBs enucleating with 2N or 4N DNA content at different stages from ( B ) . EBs were stained for DNA ( Hoechst , blue ) and TER119 ( erythroid marker , green ) . Scale bar , 5 μm . BF : brightfield . F Quantification of enucleation stages according to DNA content from ( B ) . 4 ( WT ) , 14 ( HET ) and 8 ( null ) embryos were analyzed . Data information : Bar graphs display mean ± s . d . All statistical analysis were based on the number of embryos . Statistical significance was determined by Two - way ANOVA with Tukey ' s ( F ) multiple comparisons . * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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Figure 3E ImageStream images showing EBs enucleating with 2N or 4N DNA content at different stages from (B). EBs were stained for DNA (Hoechst, blue) and TER119 (erythroid marker, green). Scale bar, 5 μm. BF: brightfield. F Quantification of enucleation stages according to DNA content from (B). 4 (WT), 14 (HET) and 8 (null) embryos were analyzed. Data information: Bar graphs display mean ± s.d. All statistical analysis were based on the number of embryos. Statistical significance was determined by Two-way ANOVA with Tukey's (F) multiple comparisons. **P≤0.01, ***P≤0.001, ****P≤0.0001.


Figure 4B Immunofluorescence images of untreated ( untx ) or CB - treated erythroid progenitors at stated differentiation stages after 24 ( T24 ) and 48 ( T48 ) hours in ex vivo culture . Cells were stained for γ - tubulin ( magenta ) , TER119 ( erythroid marker , green ) and DNA ( Hoechst , blue ) . Scale bar , 3 μm . C Quantification of centrosome numbers ( marked by γ - tubulin ) upon CB treatment from ( B ) . For the left panel , in total 632 ( untx , T24 ) , 637 ( CB , T24 ) , 544 ( untx , T48 ) and 467 ( CB , T48 ) cells from two litters were analyzed . T24 = 24 hours . T48 = 48 hours . For the right panel , in total 799 ( untx , non - enucl ) , 142 ( untx , enucl ) , 235 ( untx , RetiC ) , 839 ( CB , non - enucl ) , 127 ( CB , enucl ) and 131 ( CB , RetiC ) cells from two litters were analysed . Data information : Bar graphs display mean ± s . d . Statistical analysis was based on the number of litters ( C * 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 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

Figure 4B Immunofluorescence images of untreated (untx) or CB-treated erythroid progenitors at stated differentiation stages after 24 (T24) and 48 (T48) hours in ex vivo culture. Cells were stained for γ-tubulin (magenta), TER119 (erythroid marker, green) and DNA (Hoechst, blue). Scale bar, 3 μm.C Quantification of centrosome numbers (marked by γ-tubulin) upon CB treatment from (B). For the left panel, in total 632 (untx, T24), 637 (CB, T24), 544 (untx, T48) and 467 (CB, T48) cells from two litters were analyzed. T24 = 24 hours. T48 = 48 hours. For the right panel, in total 799 (untx, non-enucl), 142 (untx, enucl), 235 (untx, RetiC), 839 (CB, non-enucl), 127 (CB, enucl) and 131 (CB, RetiC) cells from two litters were analysed. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of litters (C *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


Figure 5A Immunofluorescence images of untreated ( untx ) or centrinone - B ( CB ) - treated cells at 36 hours ( T36 ) of ex vivo culture . Representative examples for different mitotic spindle morphologies are shown . Cells were stained for α - tubulin ( magenta ) , γ - tubulin ( grey ) , phospho - Histone H3 ( pHH3 , green ) and DNA ( Hoechst , blue ) . Images are maximum intensity projections of deconvolved z - stacks . Scale bar , 2 μm . B Quantification of mitotic spindle morphologies in untreated ( untx ) or centrinone - B ( CB ) - treated cells at T24 or T36 of ex vivo culture . Graph depicts percentage of spindle phenotypes . In total 315 ( untx , T24 ) , 367 ( CB , T24 ) , 295 ( untx , T36 ) , 282 ( CB , T36 ) cells were analyzed from 3 litters . Data information : Bar graphs display mean ± s . d . Statistical analysis was based on the number of litters ( B ) Statistical significance was determined by One - way ANOVA with Tukey ' s multiple comparisons test ( B , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . C Immunofluorescence images of Cdk5rap2HET and Cdk5rap2null cells at 36 hours ( T36 ) of ex vivo culture . Representative examples for different mitotic spindle morphologies are shown . Cells were stained for α - tubulin ( magenta ) , γ - tubulin ( grey ) , pHH3 ( green ) and DNA ( Hoechst , blue ) . Images are maximum intensity projections of deconvolved z - stacks . Scale bar , 2 μm . D Quantification of mitotic spindle morphology at T24 or T36 of ex vivo culture . Graph depicts percentage of spindle phenotypes . At T24 , 3 Cdk5rap2WT ( 323 cells ) , 3 Cdk5rap2HET ( 296 cells ) and 2 Cdk5rap2null ( 197 cells ) embryos were analyzed . At T36 , 5 Cdk5rap2WT ( 387 cells ) , 6 Cdk5rap2HET ( 384 cells ) and 3 Cdk5rap2null ( 202 cells ) embryos were analyzed . Data information : Bar graphs display mean ± s . d . Statistical analysis was based on the number of embryos ( D , Statistical significance was determined by One - way ANOVA with Tukey ' s multiple comparisons test D , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . E Schematic ( left ) shows experimental outline for nocodazole treatment of ex vivo cultured erythroid progenitors at indicated time points . Representative ImageStream images of ex vivo cultured cells at different mitotic stages ( right ) . Cells were stained for phospho - Histone H3 ( pHH3 , green ) , TER119 ( erythroid marker , magenta ) and DNA ( Hoechst , blue ) . BF : brightfield . Scale bar , 10 μm . F and G Quantification of pHH3pos , TER119pos cells and mitotic stages ( see text for details ) after 36 hours ( T36 ) upon DMSO or nocodazole treatment according to ( E ) using ImageStream . Number of embryos analyzed is shown in brackets . Data information : Box plots show 5th , 95th ( whiskers ) and 25th , 50th and 75th percentile ( boxes ) . Statistical analysis was based on the number of embryos F and G ) . Statistical significance was determined by One - way ANOVA with Tukey ' s multiple comparisons test F and G ) . * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 5A Immunofluorescence images of untreated (untx) or centrinone-B (CB)-treated cells at 36 hours (T36) of ex vivo culture. Representative examples for different mitotic spindle morphologies are shown. Cells were stained for α-tubulin (magenta), γ-tubulin (grey), phospho-Histone H3 (pHH3, green) and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 2 μm. B Quantification of mitotic spindle morphologies in untreated (untx) or centrinone-B (CB)-treated cells at T24 or T36 of ex vivo culture. Graph depicts percentage of spindle phenotypes. In total 315 (untx, T24), 367 (CB, T24), 295 (untx, T36), 282 (CB, T36) cells were analyzed from 3 litters. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of litters (B) Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test (B, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.C Immunofluorescence images of Cdk5rap2HET and Cdk5rap2null cells at 36 hours (T36) of ex vivo culture. Representative examples for different mitotic spindle morphologies are shown. Cells were stained for α-tubulin (magenta), γ-tubulin (grey), pHH3 (green) and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 2 μm. D Quantification of mitotic spindle morphology at T24 or T36 of ex vivo culture. Graph depicts percentage of spindle phenotypes. At T24, 3 Cdk5rap2WT (323 cells), 3 Cdk5rap2HET (296 cells) and 2 Cdk5rap2null (197 cells) embryos were analyzed. At T36, 5 Cdk5rap2WT (387 cells), 6 Cdk5rap2HET (384 cells) and 3 Cdk5rap2null (202 cells) embryos were analyzed. Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of embryos (D, Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test D, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.E Schematic (left) shows experimental outline for nocodazole treatment of ex vivo cultured erythroid progenitors at indicated time points. Representative ImageStream images of ex vivo cultured cells at different mitotic stages (right). Cells were stained for phospho-Histone H3 (pHH3, green), TER119 (erythroid marker, magenta) and DNA (Hoechst, blue). BF: brightfield. Scale bar, 10 μm. F and G Quantification of pHH3pos, TER119pos cells and mitotic stages (see text for details) after 36 hours (T36) upon DMSO or nocodazole treatment according to (E) using ImageStream. Number of embryos analyzed is shown in brackets. Data information: Box plots show 5th,95th (whiskers) and 25th, 50th and 75th percentile (boxes). Statistical analysis was based on the number of embryos F and G). Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test F and G).*P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


Figure 6B Immunofluorescence images of cells in interphase ( I ) and mitosis ( M ) at 24 ( T24 ) and 36 ( T36 ) hours of ex vivo culture . Cells were stained for γ - tubulin ( magenta ) , protein of interest ( POI , CDK5RAP2 , PCNT or CEP192 in grey ) and DNA ( Hoechst , blue ) . Images are maximum intensity projections of deconvolved z - stacks . Scale bar , 2 μm . C Quantification of mean centrosomal signal intensities of PCM proteins from ( B ) . Three litters were analyzed with the following number of cells for I , T24 ; M , T24 ; I , T36 and M , T36 : 290 , 157 , 259 and 174 ( CDK5RAP2 ) ; 291 , 190 , 203 and 181 ( PCNT ) ; 314 , 122 , 283 and 152 ( CEP192 ) ; 895 , 465 , 720 and 493 ( γ - tubulin ) . Data information : Bar graphs display mean ± s . d . Statistical analysis was based on the number of litters ( C Statistical significance was determined by One - way ANOVA with Tukey ' s multiple comparisons test * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6B Immunofluorescence images of cells in interphase (I) and mitosis (M) at 24 (T24) and 36 (T36) hours of ex vivo culture. Cells were stained for γ-tubulin (magenta), protein of interest (POI, CDK5RAP2, PCNT or CEP192 in grey) and DNA (Hoechst, blue). Images are maximum intensity projections of deconvolved z-stacks. Scale bar, 2 μm. C Quantification of mean centrosomal signal intensities of PCM proteins from (B). Three litters were analyzed with the following number of cells for I, T24; M, T24; I, T36 and M, T36: 290, 157, 259 and 174 (CDK5RAP2); 291, 190, 203 and 181 (PCNT); 314, 122, 283 and 152 (CEP192); 895, 465, 720 and 493 (γ-tubulin). Data information: Bar graphs display mean ± s.d. Statistical analysis was based on the number of litters (C Statistical significance was determined by One-way ANOVA with Tukey's multiple comparisons test *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


Figure 7A Immunoblot analysis of TP53 , P27 and P21 levels in ex vivo cultured Cdk5rap2 wild - type ( WT ) , heterozygous ( HET ) and null EBs after 24 ( T24 ) and 48 ( T48 ) hours . GAPDH was used as loading control . * * indicates unspecific band . B Quantification of mean protein levels from ( A ) . Numbers in brackets represent number of embryos analyzed . Data information : Box plots show 5th , 95th ( whiskers ) and 25th , 50th and 75th percentile ( boxes ) . All statistical analysis were based on number of embryos . All statistical significances were determined by One - way ANOVA with Tukey ' s multiple comparisons test . * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunoblot analysis of TP53, P27 and P21 levels in ex vivo cultured Cdk5rap2 wild-type (WT), heterozygous (HET) and null EBs after 24 (T24) and 48 (T48) hours. GAPDH was used as loading control. ** indicates unspecific band. B Quantification of mean protein levels from (A). Numbers in brackets represent number of embryos analyzed. Data information: Box plots show 5th,95th (whiskers) and 25th, 50th and 75th percentile (boxes). All statistical analysis were based on number of embryos. All statistical significances were determined by One-way ANOVA with Tukey's multiple comparisons test.*P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.


Figure 1B . Analysis of cell cycle arrest ( in G2 ) in response to DSB induction assayed by flow cytometry . C . Rad53 phosphorylation ( Rad53 - P ) in response to DSB assayed by western blotting . D . Cell survival upon DSB induction . Average ± SD ( n = 4 ) is shown for each genotype .

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

Figure 1B. Analysis of cell cycle arrest (in G2) in response to DSB induction assayed by flow cytometry.C. Rad53 phosphorylation (Rad53-P) in response to DSB assayed by western blotting.D. Cell survival upon DSB induction. Average ± SD (n=4) is shown for each genotype.


Figure 2B . Srs2 requirement in de novo telomere addition in cells with and without functional HR . All strains are pif1 - m2 . Strains used : NK1264 ; NK2375 , NK2376 ; NK2014 , NK2015 ; NK2451 , NK2452 ; NK2012 , NK2013 ; NK2457 , NK2458 ; NK2363 , NK2364 ; NK2469 ; 2369 , NK2370 ; NK2473 - 2475 . D . Dynamics of ( TG1 - 3 ) n addition monitored by qPCR through a time - course experiment ( asynchronous populations ) . The Y axis shows a fold increase in de novo telomere - specific PCR product relative to the background levels at 0 h and normalised against an internal control ( ARO1 locus ) . Average ± SD ( n = 3 ) is shown for each time - point of each genotype . Strains used : NK3292 , NK3293 ; NK4670 , NK4671 ; NK4112 , NK4113 ; NK4114 , NK4115 ; NK3292 est2 ∆ , NK3293 est2 ∆ ; NK4232 , NK4233 . F . Comparative analysis of ssDNA / dsDNA at de novo telomere addition loci in SRS2 and srs2Δ during a time - course experiment ( synchronised populations ) . The Y axis shows a fold increase in de novo telomere - specific PCR product relative to the background levels at 0 h and normalised against an internal control ( ARO1 locus ) . Average ± SD ( n = 3 ) is shown for each time - point of each genotype . Top set of error bars represents SD in relative increase of the de novo telomere - specific PCR product ( as in panel D ) while the lower set of error bars corresponds to quantifications of ss / dsDNA fractions . Strains used : NK3292 , NK3293 ; NK4670 , NK4671 .

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 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 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 2B. Srs2 requirement in de novo telomere addition in cells with and without functional HR. All strains are pif1-m2. Strains used: NK1264; NK2375, NK2376; NK2014, NK2015; NK2451, NK2452; NK2012, NK2013; NK2457, NK2458; NK2363, NK2364; NK2469; 2369, NK2370; NK2473-2475.D. Dynamics of (TG1-3)n addition monitored by qPCR through a time-course experiment (asynchronous populations). The Y axis shows a fold increase in de novo telomere-specific PCR product relative to the background levels at 0 h and normalised against an internal control (ARO1 locus). Average ± SD (n=3) is shown for each time-point of each genotype. Strains used: NK3292, NK3293; NK4670, NK4671; NK4112, NK4113; NK4114, NK4115; NK3292 est2∆, NK3293 est2∆; NK4232, NK4233.F. Comparative analysis of ssDNA/dsDNA at de novo telomere addition loci in SRS2 and srs2Δ during a time-course experiment (synchronised populations). The Y axis shows a fold increase in de novo telomere-specific PCR product relative to the background levels at 0 h and normalised against an internal control (ARO1 locus). Average ± SD (n=3) is shown for each time-point of each genotype. Top set of error bars represents SD in relative increase of the de novo telomere-specific PCR product (as in panel D) while the lower set of error bars corresponds to quantifications of ss/dsDNA fractions. Strains used: NK3292, NK3293; NK4670, NK4671.


Figure 3B . Southern blot analysis of re - synthesis of resected DNA during BIR in SRS2 and srs2Δ corresponding to the data quantifications in C . DNA was digested with EcoRI and BamHI , resolved on 0 . 7 % agarose gels , transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes ( three RS probes and a reference probe , REF , hybridizing to an ARS522 - containing fragment on chr . V which is not involved in the repair ) . A representative image of one of the three repeats is shown . C . Re - synthesis of resected DNA on chr . VIIL in SRS2 and srs2Δ cells ( solid and dashed lines , respectively ) at the distance of 15 . 2 ( RS15 . 2 ) , 6 . 8 ( RS6 . 8 ) and 2 . 6 ( RS2 . 6 ) kb away from the homology region . Average ± SD ( n = 3 ) is shown for each time point . D . Southern blot analysis of BIR - dependent DNA synthesis in SRS2 and srs2Δ corresponding to the data quantifications in D . DNA was digested with EcoRI and BamHI , resolved on 0 . 7 % agarose gels , transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes ( three BIR probes and a reference probe , REF , hybridizing to an ARS522 - containing fragment on chr . V which is not involved in the repair ) . A representative image of one of the three repeats is shown . C - control strain NK3980 . E . BIR - dependent DNA synthesis in SRS2 and srs2Δ cells ( solid and dashed lines , respectively ) at the distance of 6 ( BIR6 ) , 36 ( BIR36 ) and 77 ( BIR77 ) kb away from the homology region . Average ± SD ( n = 3 ) is shown for each time point .

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 O O O O O O O O O O O O O O O O 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 3B. Southern blot analysis of re-synthesis of resected DNA during BIR in SRS2 and srs2Δ corresponding to the data quantifications in C. DNA was digested with EcoRI and BamHI, resolved on 0.7% agarose gels, transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes (three RS probes and a reference probe, REF, hybridizing to an ARS522-containing fragment on chr. V which is not involved in the repair). A representative image of one of the three repeats is shown.C. Re-synthesis of resected DNA on chr.VIIL in SRS2 and srs2Δ cells (solid and dashed lines, respectively) at the distance of 15.2 (RS15.2), 6.8 (RS6.8) and 2.6 (RS2.6) kb away from the homology region. Average ± SD (n=3) is shown for each time point.D. Southern blot analysis of BIR-dependent DNA synthesis in SRS2 and srs2Δ corresponding to the data quantifications in D. DNA was digested with EcoRI and BamHI, resolved on 0.7% agarose gels, transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes (three BIR probes and a reference probe, REF, hybridizing to an ARS522-containing fragment on chr. V which is not involved in the repair). A representative image of one of the three repeats is shown. C - control strain NK3980.E. BIR-dependent DNA synthesis in SRS2 and srs2Δ cells (solid and dashed lines, respectively) at the distance of 6 (BIR6), 36 (BIR36) and 77 (BIR77) kb away from the homology region. Average ± SD (n=3) is shown for each time point.


Figure 4B . Frequency of DSB repair via SSA in SRS2 and srs2Δ cells with and without functional HR in the assay based on the system shown in panel A . Average ± SD ( n = 4 ) is shown for each genotype . Strains used in B and D : NK4691 - 4693 ; NK4805 - 4808 ; NK5081 - 5084 ; NK5085 - 5091 . D . Fragment L formation in SRS2 and srs2Δ cells in the presence and absence of Rad51 . Also see Figure EV3 for blot images . Average ± SD ( n = 4 ) is shown for each time point .

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

Figure 4B. Frequency of DSB repair via SSA in SRS2 and srs2Δ cells with and without functional HR in the assay based on the system shown in panel A. Average ± SD (n=4) is shown for each genotype. Strains used in B and D: NK4691-4693; NK4805-4808; NK5081-5084; NK5085-5091.D. Fragment L formation in SRS2 and srs2Δ cells in the presence and absence of Rad51. Also see Figure EV3 for blot images. Average ± SD (n=4) is shown for each time point.


Figure 1A , B : serum levels of S1P ( A ) and apoM ( B ) in HLT ( n = 47 ) and COV ( n = 111 ) patients . The box - plots represent the interquartile range with median value ( central line ) ; the whiskers represent the measured range of HLT and COV . Each measurement was run in triplicate , and performed at least twice . Two - tailed Student ' s t - test was used for statistical analysis . * * * * p < 0 . 0001 . Pearson correlation between S1P and apoM ( C ) Scatter plots , together with the fitted regression line , are shown . Pearson correlation was performed for statistical analysis . Exact p values or * * * * p < 0 . 0001 are reported . Pearson correlation between S1P and RBC number ( D ) , HGB concentration ( E ) , HCT value ( F ) . Scatter plots , together with the fitted regression line , are shown . Pearson correlation was performed for statistical analysis . Exact p values or * * * * p < 0 . 0001 are reported .

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 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

Figure 1A, B: serum levels of S1P (A) and apoM (B) in HLT (n=47) and COV (n=111) patients. The box-plots represent the interquartile range with median value (central line); the whiskers represent the measured range of HLT and COV. Each measurement was run in triplicate, and performed at least twice. Two-tailed Student's t-test was used for statistical analysis. ****p < 0.0001.Pearson correlation between S1P and apoM (C) Scatter plots, together with the fitted regression line, are shown. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported.Pearson correlation between S1P and RBC number (D), HGB concentration (E), HCT value (F). Scatter plots, together with the fitted regression line, are shown. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported.


Figure 2A , B : correlations between S1P and HDL - C ( A ) , and albumin ( B ) . Data information : Scatter plots and fitted regression line are shown in each figure . Each S1P measurement was run in triplicate , and performed at least twice in independent assays . Pearson correlation was performed for statistical analysis . Exact p values or * * * * p < 0 . 0001 are reported in the figure . C , D : correlations between apoM and HDL - C ( C ) and albumin ( D ) . Data information : Scatter plots and fitted regression line are shown in each figure . Each apoM measurement was run in triplicate , and performed at least twice in independent assays . Pearson correlation was performed for statistical analysis . Exact p values or * * * * p < 0 . 0001 are reported in the figure .

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 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

Figure 2A, B: correlations between S1P and HDL-C (A), and albumin (B). Data information: Scatter plots and fitted regression line are shown in each figure. Each S1P measurement was run in triplicate, and performed at least twice in independent assays. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported in the figure.C, D: correlations between apoM and HDL-C (C) and albumin (D). Data information: Scatter plots and fitted regression line are shown in each figure. Each apoM measurement was run in triplicate, and performed at least twice in independent assays. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported in the figure.


Figure 3 ( A - D ) The serum concentrations of S1P ( A ) , apoM ( B ) , albumin ( C ) and HDL - C ( D ) in noICU ( n = 89 ) and ICU ( n = 22 ) patients are shown . The box - plots represent the interquartile range with median ( central line ) ; the whiskers represent the measured range of noICU and ICU patients . Each S1P and apoM measurement was run in triplicate , and performed at least twice in independent assays . Two - tailed Student ' s t - test was used for statistical analysis . * * * * p < 0 . 0001 .

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

Figure 3(A-D) The serum concentrations of S1P (A), apoM (B), albumin (C) and HDL-C (D) in noICU (n=89) and ICU (n= 22) patients are shown. The box-plots represent the interquartile range with median (central line); the whiskers represent the measured range of noICU and ICU patients. Each S1P and apoM measurement was run in triplicate, and performed at least twice in independent assays. Two-tailed Student's t-test was used for statistical analysis. ****p < 0.0001.


Figure 4Pearson correlation between S1P ( A - C ) PSI days from admission and NLR in COV ( n = 111 ) . Scatter plots and fitted regression line are shown in each figure . Exact p values or * * * * p < 0 . 0001 are reported . Pearson correlation between apoM ( D - F ) and PSI , days from admission and NLR in COV ( n = 111 ) . Scatter plots and fitted regression line are shown in each figure . Exact p values or * * * * p < 0 . 0001 are reported .

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Figure 4Pearson correlation between S1P (A-C) PSI days from admission and NLR in COV (n=111). Scatter plots and fitted regression line are shown in each figure. Exact p values or ****p < 0.0001 are reported.Pearson correlation between apoM (D-F) and PSI , days from admission and NLR in COV (n=111). Scatter plots and fitted regression line are shown in each figure. Exact p values or ****p < 0.0001 are reported.


Figure 5 ( A , B ) Cumulative risk for ICU admission ( A ) and cumulative survival ( B ) in COV patients ( n = 111 ) , grouped for cut - off value of S1P serum level of 0 . 60 μM . Statistical analysis was performed by Cox regression . Exact p values are reported .

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 5(A,B) Cumulative risk for ICU admission (A) and cumulative survival (B) in COV patients (n=111), grouped for cut-off value of S1P serum level of 0.60 μM. Statistical analysis was performed by Cox regression. Exact p values are reported.


figf1 ( b ) Suppression of Htt103Q toxicity in yeast by Gpx1 , Hyr1 ( Gpx3 ) and mGpx1 . The viability of the parental strain , Y258 , and of cells overexpressing yeast GPxs was determined using spotting assays . Equal numbers of cells were serially diluted fivefold and plated on medium containing glucose to assess cell numbers and containing galactose ( GAL ) to induce the expression of Htt103Q and the indicated ORF .

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

figf1(b) Suppression of Htt103Q toxicity in yeast by Gpx1, Hyr1 (Gpx3) and mGpx1. The viability of the parental strain, Y258, and of cells overexpressing yeast GPxs was determined using spotting assays. Equal numbers of cells were serially diluted fivefold and plated on medium containing glucose to assess cell numbers and containing galactose (GAL) to induce the expression of Htt103Q and the indicated ORF.


figf2a ) Total GPx activity was measured in mGpx1 - overexpressing ( mGpx1 ) or control ( WT ) cell lines with ( induced ) or without ( uninduced ) Htt103Q expression . Overexpression of mGpx1 increases total GPx activity in PC12 cells ( * * * * P 0 . 0001 compared to uninduced controls ; n = 7 per condition ) . ( b ) Quantification of caspase - 3 / 7 activation in response to Htt103Q expression in cells overexpressing mGpx1 . Overexpression of mGpx1 reduces Htt103Q - mediated caspase - 3 / 7 activation ( * * * * P 0 . 0001 ) . Values represent the ratio of mGpx1 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q ( n = 12 ( WT ) ; n = 9 ( monomeric red fluorescent protein ( mRFP ) ) ; n = 8 ( mGpx1 ) per condition ) . ( c ) Effect of the GPx mimetic ebselen on Htt103Q - mediated caspase activation . A single 10 μM dose of ebselen significantly reduced caspase - 3 / 7 activation in Htt103Q - expressing cells ( * * * * P 0 . 0001 ) . Values represent the ratio of caspase - 3 / 7 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q . Statistical analysis was performed using an unpaired , two - tailed Mann - Whitney test ( n = 12 per condition ) . ( d ) mGpx1 overexpression ( * * * * P 0 . 0001 ) and ebselen treatment ( * * * * P 0 . 0001 ) significantly reduce Htt103Q - mediated ROS production ( n = 9 per condition ) . Unless stated otherwise , all statistical analyses were performed using one - way analysis of variance ( ANOVA ) with post - hoc tests . NS , not significant . * * P 0 . 01 . All data are shown as the mean ± s . e . m .

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 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 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

figf2a) Total GPx activity was measured in mGpx1-overexpressing (mGpx1) or control (WT) cell lines with (induced) or without (uninduced) Htt103Q expression. Overexpression of mGpx1 increases total GPx activity in PC12 cells (****P 0.0001 compared to uninduced controls; n = 7 per condition).(b) Quantification of caspase-3/7 activation in response to Htt103Q expression in cells overexpressing mGpx1. Overexpression of mGpx1 reduces Htt103Q-mediated caspase-3/7 activation (****P 0.0001). Values represent the ratio of mGpx1 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q (n = 12 (WT); n = 9 (monomeric red fluorescent protein (mRFP)); n = 8 (mGpx1) per condition).(c) Effect of the GPx mimetic ebselen on Htt103Q-mediated caspase activation. A single 10 μM dose of ebselen significantly reduced caspase-3/7 activation in Htt103Q-expressing cells (****P 0.0001). Values represent the ratio of caspase-3/7 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q. Statistical analysis was performed using an unpaired, two-tailed Mann-Whitney test (n = 12 per condition).(d) mGpx1 overexpression (****P 0.0001) and ebselen treatment (****P 0.0001) significantly reduce Htt103Q-mediated ROS production (n = 9 per condition). Unless stated otherwise, all statistical analyses were performed using one-way analysis of variance (ANOVA) with post-hoc tests. NS, not significant. **P 0.01. All data are shown as the mean ± s.e.m.


figf3 ( a ) Pseudopupil images from wild - type flies , Huntington ' s disease flies and Huntington ' s disease flies overexpressing mGpx1 at day 10 . In wild - type flies , seven rhabdomeres are visible per ommatidium . Scale bar , ∼ 10 μM ( b ) Quantification of the average number of rhabdomeres per ommatidium in Huntington ' s disease flies with and without pan - neural mGpx1 overexpression at days 1 ( n = 14 ( Htt93Q ) ; n = 9 ( Htt93Q + mGpx1 ) ) , 7 ( n = 7 ( Htt93Q ) ; n = 9 ( Htt93Q + mGpx1 ) ) and 10 ( n = 8 ( Htt93Q ) ; n = 9 ( Htt93Q + mGpx1 ) ) after eclosion . Significant effects of genotype ( F1 , 50 = 82 . 3 , P = 3 . 82 × 10 − 12 ) and age ( F2 , 50 = 94 . 3 , P = 1 . 06 × 10 − 17 ) were observed , as well as a significant interaction between genotype and age ( F2 , 50 = 7 . 76 , P = 0 . 00116 ) , indicating that mGpx1 reduces the rate of neurodegeneration in Htt93Q flies . Post - hoc Newman - Keuls comparisons showed significant rescue of neurodegeneration in mGpx1 - expressing flies at days 1 ( * P 0 . 05 ) , 7 ( * * * P 0 . 001 ) and 10 ( * * * P 0 . 001 ) . Browne - Forsythe analyses for equality of variance revealed a significant inequality of variance ( P = 0 . 02 ) due to a single group ( Htt93Q + mGpx1 , day 1 ) . The nonparametric Mann - Whitney test confirmed a difference at day 1 between the two groups ( P = 0 . 03 ) , and between Htt93Q + mGpx1 flies at days 1 and 7 ( P 0 . 0001 ) . ( c ) Total locomotor activity was assessed as the average number of beam crossings per 30 - min bin per fly . Htt93Qex1 - expressing flies ( n = 29 ) have reduced locomotor activity compared to controls ( * * * P 0 . 001 ; n = 30 ( WT ( w118 ) ) ; n = 32 ( mGpx1 ) ) , which is restored by mGpx1 ( * * P 0 . 01 ; n = 32 ) . Comparisons were performed by Kruskal - Wallis one - way ANOVA . ( d ) Neurodegeneration of Pdf clock neurons was assessed using confocal microscopy in adult flies at day 7 . Flies expressing Htt93Qex1 driven by Pdf - GAL4 ( n = 16 brain hemispheres ) showed loss of s - LNVs compared to Canton S ( CS ) control flies ( n = 13 ) , which was ameliorated by coexpression of mGpx1 ( * * * * P 0 . 0001 ; n = 16 ) . Comparisons were performed by Kruskal - Wallis one - way ANOVA . ( e ) Quantification of the average number of rhabdomeres per ommatidium in Huntington ' s disease flies with ebselen or vehicle ( DMSO ; n = 10 ) treatment at day 7 after eclosion and in newly emerged day 0 adult flies ( untreated control ; n = 10 ) . Significant treatment effects were observed , both with the 100 μM ( * * P 0 . 01 ; n = 9 ) and 300 μM ( * * * P 0 . 001 ; n = 9 ) doses of ebselen . All data are shown as the mean ± s . e . m .

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

figf3(a) Pseudopupil images from wild-type flies, Huntington's disease flies and Huntington's disease flies overexpressing mGpx1 at day 10. In wild-type flies, seven rhabdomeres are visible per ommatidium. Scale bar, ∼10 μM(b) Quantification of the average number of rhabdomeres per ommatidium in Huntington's disease flies with and without pan-neural mGpx1 overexpression at days 1 (n = 14 (Htt93Q); n = 9 (Htt93Q + mGpx1)), 7 (n = 7 (Htt93Q); n = 9 (Htt93Q + mGpx1)) and 10 (n = 8 (Htt93Q); n = 9 (Htt93Q + mGpx1)) after eclosion. Significant effects of genotype (F1,50 = 82.3, P = 3.82 × 10−12) and age (F2,50 = 94.3, P = 1.06 × 10−17) were observed, as well as a significant interaction between genotype and age (F2,50 = 7.76, P = 0.00116), indicating that mGpx1 reduces the rate of neurodegeneration in Htt93Q flies. Post-hoc Newman-Keuls comparisons showed significant rescue of neurodegeneration in mGpx1-expressing flies at days 1 (*P 0.05), 7 (***P 0.001) and 10 (***P 0.001). Browne-Forsythe analyses for equality of variance revealed a significant inequality of variance (P = 0.02) due to a single group (Htt93Q + mGpx1, day 1). The nonparametric Mann-Whitney test confirmed a difference at day 1 between the two groups (P = 0.03), and between Htt93Q + mGpx1 flies at days 1 and 7 (P 0.0001).(c) Total locomotor activity was assessed as the average number of beam crossings per 30-min bin per fly. Htt93Qex1-expressing flies (n = 29) have reduced locomotor activity compared to controls (***P 0.001; n = 30 (WT (w118)); n = 32 (mGpx1)), which is restored by mGpx1 (**P 0.01; n = 32). Comparisons were performed by Kruskal-Wallis one-way ANOVA.(d) Neurodegeneration of Pdf clock neurons was assessed using confocal microscopy in adult flies at day 7. Flies expressing Htt93Qex1 driven by Pdf-GAL4 (n = 16 brain hemispheres) showed loss of s-LNVs compared to Canton S (CS) control flies (n = 13), which was ameliorated by coexpression of mGpx1 (****P 0.0001; n = 16). Comparisons were performed by Kruskal-Wallis one-way ANOVA.(e) Quantification of the average number of rhabdomeres per ommatidium in Huntington's disease flies with ebselen or vehicle (DMSO; n = 10) treatment at day 7 after eclosion and in newly emerged day 0 adult flies (untreated control; n = 10). Significant treatment effects were observed, both with the 100 μM (**P 0.01; n = 9) and 300 μM (***P 0.001; n = 9) doses of ebselen. All data are shown as the mean ± s.e.m.


figf4PC12 cells were treated with either 10 μM ebselen or 10 mM N - acetylcysteine ( NAc ) for 24 h , with the addition of 400 nM bafilomycin A1 for the final 4 h . Autophagy was induced in the indicated samples by the addition of 200 nM rapamycin for 24 h . ( a ) Autophagy was determined by measuring the amount of LC3 - II by immunoblot analysis . ( b ) Densitometric analysis of the bands was performed using ImageJ , and the amounts of LC3 - II are expressed relative to the untreated control . All statistical analyses were performed using one - way ANOVA with post - hoc tests ( * * * * P 0 . 0001 ) . All data are shown as the mean ± s . e . m . ; n = 7 per condition .

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

figf4PC12 cells were treated with either 10 μM ebselen or 10 mM N-acetylcysteine (NAc) for 24 h, with the addition of 400 nM bafilomycin A1 for the final 4 h. Autophagy was induced in the indicated samples by the addition of 200 nM rapamycin for 24 h. (a) Autophagy was determined by measuring the amount of LC3-II by immunoblot analysis. (b) Densitometric analysis of the bands was performed using ImageJ, and the amounts of LC3-II are expressed relative to the untreated control. All statistical analyses were performed using one-way ANOVA with post-hoc tests (****P 0.0001). All data are shown as the mean ± s.e.m.; n = 7 per condition.


Figure 1A . BMDMM were treated as indicated with either 60ng / mL ultra - pure ( UP ) LPS , 50ng / mL human derived recombinant GM - CSF , 40µM WEHD - FMK for 16 hours or 5µM nigericin for the last hour . Supernatants were then analysed for IL - 1β secretion by ELISA . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons or two - way ANOVA to compare across genotypes . p values are shown . B . HoxB8 macrophages were treated with the indicated dilutions of GM - CSF using supernatants from GM - CSF expressing cells . Shown are ELISA analysis of IL - 1β secretion in supernatants and western blots indicating IL - 1β expression in lysates . . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons or two - way ANOVA to compare across genotypes . p values are shown . C . HoxB8 macrophages of the indicated genotypes were treated as indicated with 60ng / mL UP - LPS , 1 % GM - CSF containing supernatant ( approximately 2ng / mL GM - CSF ) , 40µM WEHD . FMK , 0 , 5µM CRT - 0066101 for 16 hours , 5µM nigericin was added for the last hour . Supernatants were analysed by ELISA for IL - 1β . Inlay panels show western blots for levels of IL - 1β expression in the indicated knockouts . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons or two - way ANOVA to compare across genotypes . p values are shown . D . HoxB8 cells were treated with 60ng / mL of UP - LPS , 1 % GM - CSF supernatant , 50mM KCl , and 5µM nigericin as indicated for 16 hours before supernatants were analysed for IL - 1β by ELISA . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons or two - way ANOVA to compare across genotypes . p values are shown .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1A. BMDMM were treated as indicated with either 60ng/mL ultra-pure (UP) LPS, 50ng/mL human derived recombinant GM-CSF, 40µM WEHD-FMK for 16 hours or 5µM nigericin for the last hour. Supernatants were then analysed for IL-1β secretion by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.B. HoxB8 macrophages were treated with the indicated dilutions of GM-CSF using supernatants from GM-CSF expressing cells. Shown are ELISA analysis of IL-1β secretion in supernatants and western blots indicating IL-1β expression in lysates.. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.C. HoxB8 macrophages of the indicated genotypes were treated as indicated with 60ng/mL UP-LPS, 1% GM-CSF containing supernatant (approximately 2ng/mL GM-CSF), 40µM WEHD.FMK, 0,5µM CRT-0066101 for 16 hours, 5µM nigericin was added for the last hour. Supernatants were analysed by ELISA for IL-1β. Inlay panels show western blots for levels of IL-1β expression in the indicated knockouts. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.D. HoxB8 cells were treated with 60ng/mL of UP-LPS, 1% GM-CSF supernatant, 50mM KCl, and 5µM nigericin as indicated for 16 hours before supernatants were analysed for IL-1β by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.