( D ) Aneusomy index of chromosomes 7 , 12 and 18 measured by interphase FISH analysis of n = cells . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed chi - square tests .

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

(D) Aneusomy index of chromosomes 7, 12 and 18 measured by interphase FISH analysis of n=cells. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by two tailed chi-square tests.


( E ) Percentage of cytochalasin D - induced binucleated ( BN ) n = cells with chromosomes 7 , 12 , and 18 mis - segregation ( MS ) . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed chi - square tests .

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

(E) Percentage of cytochalasin D-induced binucleated (BN) n=cells with chromosomes 7, 12, and 18 mis-segregation (MS). Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by two tailed chi-square tests.


( F ) Percentage of micronuclei in n = cells scored when treated with DMSO or UMK57 . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed chi - square tests .

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

(F) Percentage of micronuclei in n=cells scored when treated with DMSO or UMK57. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by two tailed chi-square tests.


( G ) Percentage of n = cells staining positive for double immunostaining of Cdkn1a / p21 ( cell cycle inhibitor ) and 53BP1 ( ≥ 1 foci ; DNA damage ) senescence biomarkers . ( H ) Percentage of n = cells staining positive for SA - β - galactosidase ( SA - β - gal ) activity . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed chi - square tests .

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

(G) Percentage of n=cells staining positive for double immunostaining of Cdkn1a/p21 (cell cycle inhibitor) and 53BP1 (≥1 foci; DNA damage) senescence biomarkers. (H) Percentage of n=cells staining positive for SA-β-galactosidase (SA-β-gal) activity. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 by two tailed chi-square tests.


( A ) Mitotic duration of n = 100 neonatal ( N ) and elderly ( 87y ) human dermal fibroblasts ( HDF ) treated with DMSO ( - ) or UMK57 ( + ) for 96 hrs . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed ( A Mann - Whitney tests .

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

(A) Mitotic duration of n=100 neonatal (N) and elderly (87y) human dermal fibroblasts (HDF) treated with DMSO (-) or UMK57 (+) for 96 hrs. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, *** p<0.001, **** p<0.0001 by two tailed (A Mann-Whitney tests.


( B ) Relative calcium - stable k - fiber intensity levels scored by immunofluorescence analysis of n ≥ 29 tubulin - stained metaphase cells of neonatal and elderly samples treated with DMSO and UMK57 for 96 hrs . Levels were normalized to neonatal DMSO - treated condition . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed B , Mann - Whitney tests .

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

(B) Relative calcium-stable k-fiber intensity levels scored by immunofluorescence analysis of n≥29 tubulin-stained metaphase cells of neonatal and elderly samples treated with DMSO and UMK57 for 96 hrs. Levels were normalized to neonatal DMSO-treated condition. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, *** p<0.001, **** p<0.0001 by two tailed B, Mann-Whitney tests.


( C ) Aneusomy index of chromosomes 7 , 12 and 18 measured by interphase FISH analysis of neonatal and elderly n = cells treated for 96 hrs . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed chi - square tests .

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

(C) Aneusomy index of chromosomes 7, 12 and 18 measured by interphase FISH analysis of neonatal and elderly n=cells treated for 96 hrs. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, *** p<0.001, **** p<0.0001 by two tailed chi-square tests.


( D ) Cytochalasin D - induced binucleated ( BN ) n = cells with mis - segregation ( MS ) of chromosomes 7 , 12 and 18 in neonatal and elderly samples treated for 96 hrs . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed chi - square tests .

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

(D) Cytochalasin D-induced binucleated (BN) n=cells with mis-segregation (MS) of chromosomes 7, 12 and 18 in neonatal and elderly samples treated for 96 hrs. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, *** p<0.001, **** p<0.0001 by two tailed chi-square tests.


( E ) Percentage of n = cells staining positive for double immunostaining of Cdkn1a / p21 ( cell cycle inhibitor ) and 53BP1 ( ≥ 1 foci ; DNA damage ) senescence biomarkers after 96hrs of treatment . ( F ) Percentage of n = cells staining positive for SA - β - gal activity when treated for 96 hrs with DMSO or UMK57 . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed chi - square tests .

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

(E) Percentage of n=cells staining positive for double immunostaining of Cdkn1a/p21 (cell cycle inhibitor) and 53BP1 (≥1 foci; DNA damage) senescence biomarkers after 96hrs of treatment. (F) Percentage of n=cells staining positive for SA-β-gal activity when treated for 96 hrs with DMSO or UMK57. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, *** p<0.001, **** p<0.0001 by two tailed chi-square tests.


( G , Representative images of phospho - Aurora B Thr232 ( pAuroraB T232 ) levels at kinetochores / centromeres in n ≥ 75 neonatal and elderly metaphase cells treated with UMK57 for 24 hrs or 96 hrs . Intensity levels were normalized to ACA . Scale bar , 5µm .

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

(G, Representative images of phospho-Aurora B Thr232 (pAuroraB T232) levels at kinetochores/centromeres in n≥75 neonatal and elderly metaphase cells treated with UMK57 for 24 hrs or 96 hrs. Intensity levels were normalized to ACA. Scale bar, 5µm.


H ) quantification ( H ) of phospho - Aurora B Thr232 ( pAuroraB T232 ) levels at kinetochores / centromeres in n ≥ 75 neonatal and elderly metaphase cells treated with UMK57 for 24 hrs or 96 hrs . Intensity levels were normalized to ACA . Scale bar , 5µm . Data Information : All values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed H ) Mann - Whitney tests .

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

H) quantification (H) of phospho-Aurora B Thr232 (pAuroraB T232) levels at kinetochores/centromeres in n≥75 neonatal and elderly metaphase cells treated with UMK57 for 24 hrs or 96 hrs. Intensity levels were normalized to ACA. Scale bar, 5µm. Data Information: All values are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, *** p<0.001, **** p<0.0001 by two tailed H) Mann-Whitney tests.


A Immunostaining for the mitochondrial marker ATPβ in N2a cells expressing mito - QC . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 0001 for simple effects . Scale bar = 5 µm in ( A )

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

A Immunostaining for the mitochondrial marker ATPβ in N2a cells expressing mito-QC. Data information: Data are given as mean and SEM, * = p < 0.05, **= p <0.01, **** = p < 0.0001 for simple effects. Scale bar = 5 µm in (A)


B N2a cells expressing mito - QC and myc - Parkin together with hTau - V5 , hP301L - V5 or an empty vector control . Treatment with CCCP ( 8 µM , 17 h ) induces mitophagy in control cells , as indicated by a decrease in the GFP / mCherry fluorescence ratio , but not in hTau or hP301L cells . Deconvolution ( Classic Maximum Likelihood Estimation ) was applied to images for display only . C Quantification of GFP / mCherry fluorescence intensity per cell . Data were analysed by 2 - way ANOVA , showing significant main effects of CCCP treatment , F ( 1 , 314 ) = 15 . 66 , p < 0 . 0001 , and tau expression , F ( 2 , 314 ) = 12 . 26 , p < 0 . 0001 , and a significant interaction effect , F ( 2 , 314 ) = 6 . 137 , p = 0 . 0024 , n = 40 - 73 cells / group . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 0001 for simple effects . Scale bar = 10

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

B N2a cells expressing mito-QC and myc-Parkin together with hTau-V5, hP301L-V5 or an empty vector control. Treatment with CCCP (8 µM, 17 h) induces mitophagy in control cells, as indicated by a decrease in the GFP/mCherry fluorescence ratio, but not in hTau or hP301L cells. Deconvolution (Classic Maximum Likelihood Estimation) was applied to images for display only. C Quantification of GFP/mCherry fluorescence intensity per cell. Data were analysed by 2-way ANOVA, showing significant main effects of CCCP treatment, F (1, 314) = 15.66, p < 0.0001, and tau expression, F (2, 314) = 12.26, p < 0.0001, and a significant interaction effect, F (2, 314) = 6.137, p = 0.0024, n = 40-73 cells/group. Data information: Data are given as mean and SEM, * = p < 0.05, **= p <0.01, **** = p < 0.0001 for simple effects. Scale bar = 10 µm


D Cells expressing mito - QC and myc - Parkin together with hTau - V5 , hP301L - V5 or an empty vector control , treated with a mixture of antimycin and oligomycin ( AO , 15 µM , 17 h ) . E Quantification of GFP / mCherry fluorescence intensity per cell . Data were analysed by 2 - way ANOVA , showing no significant main effects of AO treatment , F ( 1 , 212 ) = 0 . 564 , p = 0 . 4534 , or tau expression F ( 2 , 212 ) = 1 . 579 , p = 0 . 2087 , but a significant interaction effect , F ( 2 , 212 ) = 9 . 854 , p < 0 . 0001 , n = 16 - 50 cells / group . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 0001 for simple effects . Scale bar = 10

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

D Cells expressing mito-QC and myc-Parkin together with hTau-V5, hP301L-V5 or an empty vector control, treated with a mixture of antimycin and oligomycin (AO, 15 µM, 17 h). E Quantification of GFP/mCherry fluorescence intensity per cell. Data were analysed by 2-way ANOVA, showing no significant main effects of AO treatment, F (1, 212) = 0.564, p = 0.4534, or tau expression F (2, 212) = 1.579, p = 0.2087, but a significant interaction effect, F (2, 212) = 9.854, p < 0.0001, n = 16-50 cells/group. Data information: Data are given as mean and SEM, * = p < 0.05, **= p <0.01, **** = p < 0.0001 for simple effects. Scale bar = 10 µm


A Illustration of the image analysis process . The inverted " Union Jack " look - up table ( LUT ) was used to better visualise GFP - Parkin clusters , which were then manually selected as regions of interest ( ROIs ) and the corresponding mitochondrial fluorescence intensity was measured . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 μm .

A Illustration of the image analysis process. The inverted "Union Jack" look-up table (LUT) was used to better visualise GFP-Parkin clusters, which were then manually selected as regions of interest (ROIs) and the corresponding mitochondrial fluorescence intensity was measured. Data information: Data are given as mean and SEM, *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Scale bar = 10 μm.


B Representative images of N2a cells expressing GFP - Parkin and hTau , hP301L or empty vector ( Control ) for 24 h , that were then treated with 10 μM CCCP for 4 h . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 μm .

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

B Representative images of N2a cells expressing GFP-Parkin and hTau, hP301L or empty vector (Control) for 24 h, that were then treated with 10 μM CCCP for 4 h. Data information: Data are given as mean and SEM, *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Scale bar = 10 μm.


C Quantification of Parkin clusters / cell at 24 h post - transfection . Data were analysed with a Kruskal - Wallis test , χ2 ( 2 ) = 8 . 18 , p = 0 . 0168 , followed by Dunn ' s post - hoc analysis , n = 33 - 38 cells / group . D Quantification of Parkin clusters / cell at 48 h post - transfection . Data were analysed with a Kruskal - Wallis test , χ2 ( 2 ) = 19 . 2 , p < 0 . 0001 , followed by Dunn ' s post - hoc analysis , n = 37 - 55 cells / group cells / group . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 μm .

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

C Quantification of Parkin clusters/cell at 24 h post-transfection. Data were analysed with a Kruskal-Wallis test, χ2 (2) = 8.18, p = 0.0168, followed by Dunn's post-hoc analysis, n = 33-38 cells/group. D Quantification of Parkin clusters/cell at 48 h post-transfection. Data were analysed with a Kruskal-Wallis test, χ2 (2) = 19.2, p < 0.0001, followed by Dunn's post-hoc analysis, n = 37-55 cells/group cells/group. Data information: Data are given as mean and SEM, *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Scale bar = 10 μm.


E To assess colocalisation of Parkin clusters and mitochondria at 24 h post - transfection , Pearson correlations between the mean normalised fluorescence intensity of the Parkin clusters and the mitochondrial signal following CCCP treatment were calculated . Control r = 0 . 616 , p = 0 . 0001 ; hTau r = 0 . 217 , p = 0 . 2771 ; hP301L r = 0 . 548 , p = 0 . 0014 ( n = 27 - 34 cells / group ) . F Pearson correlations between the mean normalised fluorescence intensity of the Parkin clusters and the mitochondrial signal following CCCP treatment at 48 h post - transfection . Control r = 0 . 434 , p = 0 . 0167 ; hTau r = 0 . 128 , p = 0 . 4941 ; hP301L r = - 0 . 299 , p = 0 . 2794 ( n = 15 - 31 cells / group ) . Note the different intensity scales between ( E ) and ( F ) reflect different bit depths of the images . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 μm .

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

E To assess colocalisation of Parkin clusters and mitochondria at 24 h post-transfection, Pearson correlations between the mean normalised fluorescence intensity of the Parkin clusters and the mitochondrial signal following CCCP treatment were calculated. Control r = 0.616, p = 0.0001; hTau r = 0.217, p = 0.2771; hP301L r = 0.548, p = 0.0014 (n = 27-34 cells/ group). F Pearson correlations between the mean normalised fluorescence intensity of the Parkin clusters and the mitochondrial signal following CCCP treatment at 48 h post-transfection. Control r = 0.434, p = 0.0167; hTau r = 0.128, p = 0.4941; hP301L r = -0.299, p = 0.2794 (n = 15-31 cells/ group). Note the different intensity scales between (E) and (F) reflect different bit depths of the images. Data information: Data are given as mean and SEM, *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Scale bar = 10 μm.


A FACS analysis of cells loaded with Rhodamine 123 . The geometric mean fluorescence was calculated and expressed as % of the empty vector control . A CCCP - treated cohort was included as a positive control , but not included in the statistical analysis . Data were analysed by one - way ANOVA , F ( 2 , 14 ) = 0 . 196 , p = 0 . 8245 . B FACS analysis of cells treated with CCCP after 24 h of tau expression . Data were analysed by one - way ANOVA , F ( 2 , 15 ) = 0 . 434 , p = 0 . 6555 . The DMSO negative control is shown but was not analysed statistically . Data information : Data are given as mean and SEM ( n = 5 - 6 samples / experimental group from two independent experiments ) .

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

A FACS analysis of cells loaded with Rhodamine 123. The geometric mean fluorescence was calculated and expressed as % of the empty vector control. A CCCP-treated cohort was included as a positive control, but not included in the statistical analysis. Data were analysed by one-way ANOVA, F (2, 14) = 0.196, p = 0.8245. B FACS analysis of cells treated with CCCP after 24 h of tau expression. Data were analysed by one-way ANOVA, F (2, 15) = 0.434, p = 0.6555. The DMSO negative control is shown but was not analysed statistically. Data information: Data are given as mean and SEM (n = 5-6 samples/ experimental group from two independent experiments).


C Representative dot plots of cells loaded with Rhodamine 123 . The gates ( black line ) were set in unstained cells to distinguish Rhodamine - positive cells ( right half ) from Rhodamine - negative cells ( left half ) . Data information : Data are given as mean and SEM ( n = 5 - 6 samples / experimental group from two independent experiments ) .

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

C Representative dot plots of cells loaded with Rhodamine 123. The gates (black line) were set in unstained cells to distinguish Rhodamine-positive cells (right half) from Rhodamine-negative cells (left half). Data information: Data are given as mean and SEM (n = 5-6 samples/ experimental group from two independent experiments).


A Immunostaining for microtubules after transient expression of hTau and hP301L . Data information : Data are given as mean and SEM , * * * * = p < 0 . 0001 . Scale bar = 10 µm .

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

A Immunostaining for microtubules after transient expression of hTau and hP301L. Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.


B Taxol induces microtubule bundling to a similar extent to tau expression . Data information : Data are given as mean and SEM , * * * * = p < 0 . 0001 . Scale bar = 10 µm .

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

B Taxol induces microtubule bundling to a similar extent to tau expression. Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.


C Representative images and quantification of Parkin clusters in N2a cells following CCCP and Taxol treatment ( Mann - Whitney test , U = 22448 , p < 0 . 0001 , n = 224 , 273 cells / group for CCCP only and CCCP + Taxol , respectively ) . Data information : Data are given as mean and SEM , * * * * = p < 0 . 0001 . Scale bar = 10 µm .

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

C Representative images and quantification of Parkin clusters in N2a cells following CCCP and Taxol treatment (Mann-Whitney test, U = 22448, p < 0.0001, n = 224, 273 cells/group for CCCP only and CCCP + Taxol, respectively). Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.


D Immunoblot analysis of acetylated tubulin ( Ace - Tubulin ) following expression of hTau and hP301L for 24 h ( One - way ANOVA , F ( 2 , 9 ) = 0 . 0432 , p = 0 . 9579 ) . Data information : Data are given as mean and SEM , * * * * = p < 0 . 0001 . Scale bar = 10 µm .

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

D Immunoblot analysis of acetylated tubulin (Ace-Tubulin) following expression of hTau and hP301L for 24 h (One-way ANOVA, F(2, 9) = 0.0432, p = 0.9579). Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.


E Immunoblot analysis of tyrosinated tubulin ( Tyr - Tubulin ) following expression of hTau and hP301L for 24 h ( One - way ANOVA , F ( 2 , 9 ) = 0 . 0402 , p = 0 . 9608 ) . Data information : Data are given as mean and SEM , * * * * = p < 0 . 0001 . Scale bar = 10 µm .

E Immunoblot analysis of tyrosinated tubulin (Tyr-Tubulin) following expression of hTau and hP301L for 24 h (One-way ANOVA, F(2, 9) = 0.0402, p = 0.9608). Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.


F Representative images and quantification of Parkin clusters in N2a cells following treatment with CCCP and the actin stabiliser Jasplakinolide ( JPL ) ( Mann - Whitney test , U = 16069 , p < 0 . 0001 , n = 224 , 199 cells / group for CCCP only and CCCP + JPL , respectively ) . Data information : Data are given as mean and SEM , * * * * = p < 0 . 0001 . Scale bar = 10 µm .

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

F Representative images and quantification of Parkin clusters in N2a cells following treatment with CCCP and the actin stabiliser Jasplakinolide (JPL) (Mann-Whitney test, U = 16069, p <0.0001, n = 224, 199 cells/group for CCCP only and CCCP + JPL, respectively). Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.


A Co - immunoprecipitations of V5 - tagged tau and GFP - Parkin . The V5 tag was used to pull down tau and the membrane was probed for V5 ( using an antibody raised in a different species ) and GFP . IP = immunoprecipitation , Flow = flow - through lysate following removal of beads . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

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

A Co-immunoprecipitations of V5-tagged tau and GFP-Parkin. The V5 tag was used to pull down tau and the membrane was probed for V5 (using an antibody raised in a different species) and GFP. IP = immunoprecipitation, Flow = flow-through lysate following removal of beads. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.


B Maximum projection images of proximity ligation assays ( PLAs ) for tau and endogenous Parkin in cells expressing hTau - GFP , hP301L - GFP or GFP . Inset = negative control ( antibodies to tau and histone H3 , which are not known binding partners ) . C Quantification of PLA signals in maximum projections normalised to the GFP signal . Results were analysed with a Kruskal - Wallis test , χ2 ( 2 ) = 27 . 2 , p < 0 . 0001 , followed by Dunn ' s post hoc analysis to test for differences from the GFP control , n = 17 cells in GFP condition , 14 cells in hTau condition and 27 cells in hP301L condition . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

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

B Maximum projection images of proximity ligation assays (PLAs) for tau and endogenous Parkin in cells expressing hTau-GFP, hP301L-GFP or GFP. Inset = negative control (antibodies to tau and histone H3, which are not known binding partners). C Quantification of PLA signals in maximum projections normalised to the GFP signal. Results were analysed with a Kruskal-Wallis test, χ2 (2) = 27.2, p < 0.0001, followed by Dunn's post hoc analysis to test for differences from the GFP control, n = 17 cells in GFP condition, 14 cells in hTau condition and 27 cells in hP301L condition. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.


D PLA for Parkin and hP301L in an aged pR5 mice and a littermate control . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

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

D PLA for Parkin and hP301L in an aged pR5 mice and a littermate control. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.


F , G PLA for Parkin and ∆ Tau , revealing interactions via the projection domain of tau . Data were analysed with a Mann - Whitney test ( U = 185 , p = 0 . 9224 , n = 21 , 18 cells / group for hTau and ∆ Tau , respectively ) . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

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

F, G PLA for Parkin and ∆Tau, revealing interactions via the projection domain of tau. Data were analysed with a Mann-Whitney test (U = 185, p = 0.9224, n = 21, 18 cells/group for hTau and ∆Tau, respectively). Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.


H , I PLA for Parkin and the microtubule - binding domain construct ( MTBD ) . Data were analysed with a t - test ( t = 5 . 226 , p < 0 . 0001 , n = 16 , 17 cells / group for hTau and MTBD , respectively ) . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

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

H, I PLA for Parkin and the microtubule-binding domain construct (MTBD). Data were analysed with a t-test (t = 5.226, p < 0.0001, n = 16, 17 cells/group for hTau and MTBD, respectively). Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.


J , K Cells expressing tau constructs together with GFP - Parkin ( 24 h ) with and without CCCP treatment . Parkin clusters were counted , illustrating that Parkin translocation was impaired by ∆ Tau , but not MTBD . Data were analysed with a Kruskal - Wallis test χ2 ( 2 ) = 10 . 4 , p = 0 . 0056 , followed by Dunn ' s post - hoc , n = 67 , 42 , 53 cells / group for for hTau , ∆ Tau , and MTBD , respectively . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

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

J, K Cells expressing tau constructs together with GFP-Parkin (24 h) with and without CCCP treatment. Parkin clusters were counted, illustrating that Parkin translocation was impaired by ∆Tau, but not MTBD. Data were analysed with a Kruskal-Wallis test χ2 (2) = 10.4, p = 0.0056, followed by Dunn's post-hoc, n = 67, 42, 53 cells/ group for for hTau, ∆Tau, and MTBD, respectively. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.


L The V5 fluorescence intensity in each cell analysed in ( K ) ( Mann - Whitney test , U = 953 , p = 0 . 2332 , n = 42 , 53 cells / group for ∆ Tau and MTBD , respectively ) . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

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

L The V5 fluorescence intensity in each cell analysed in (K) (Mann-Whitney test, U = 953, p = 0.2332, n = 42, 53 cells/group for ∆Tau and MTBD, respectively). Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.


M Cells were transfected with GFP - Parkin and hTau - V5 , hP301L - V5 or an empty vector control , and treated with CCCP . PLAs were performed for tau and Parkin . Notably , at Parkin clusters ( arrow ) , no interactions between tau and Parkin were evident . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

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

M Cells were transfected with GFP-Parkin and hTau-V5, hP301L-V5 or an empty vector control, and treated with CCCP. PLAs were performed for tau and Parkin. Notably, at Parkin clusters (arrow), no interactions between tau and Parkin were evident. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.


N 3D reconstructions in IMARIS , illustrating a lack of PLA signals in Parkin clusters . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

N 3D reconstructions in IMARIS, illustrating a lack of PLA signals in Parkin clusters. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.


Expression pattern of the mito - Rosella biosensor in the nervous system .

Expression pattern of the mito-Rosella biosensor in the nervous system.


B Analysis pipeline of mitochondria in the C . elegans ventral nerve cord . Mitochondria were selected by choosing a threshold in the dsRed channel . This threshold was used to create ROIs around single mitochondria and the corresponding GFP fluorescence intensity was then measured .

O O O O O O O O O O O O O O O O O O O O O O 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 Analysis pipeline of mitochondria in the C. elegans ventral nerve cord. Mitochondria were selected by choosing a threshold in the dsRed channel. This threshold was used to create ROIs around single mitochondria and the corresponding GFP fluorescence intensity was then measured.


C , D Analysis of mitophagy in worms transgenic for mito - Rosella but not tau ( WT ) , that were treated with NaN3 ( 8 mM , 1 h ) or CCCP ( 15 µM , 2 h ) . Data were analysed by unpaired t test , NaN3 t = 5 . 56 , p < 0 . 0001 ; CCCP t = 3 . 92 p = 0 . 0002 . Data information : n = 16 - 25 worms / group for NaN3 and 23 - 39 worms / groups for CCCP experiments . Data are given as mean and SEM . * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 µm . Images are high - magnification of a small number of mitochondria in the ventral

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

C, D Analysis of mitophagy in worms transgenic for mito-Rosella but not tau (WT), that were treated with NaN3 (8 mM, 1 h) or CCCP (15 µM, 2 h). Data were analysed by unpaired t test, NaN3 t = 5.56, p < 0.0001; CCCP t = 3.92 p = 0.0002. Data information: n = 16-25 worms/group for NaN3 and 23-39 worms/groups for CCCP experiments. Data are given as mean and SEM. *** = p < 0.001, **** = p < 0.0001. Scale bar = 10 µm. Images are high-magnification of a small number of mitochondria in the ventral cord


E , F Analysis of mitophagy in worms expressing hTau in response to NaN3 treatment ( unpaired t test , t = 1 . 62 , p = 0 . 1126 ) and CCCP treatment ( Mann - Whitney test , U = 113 , p = 0 . 002 ) . Data information : n = 16 - 25 worms / group for NaN3 and 23 - 39 worms / groups for CCCP experiments . Data are given as mean and SEM . * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 µm . Images are high - magnification of a small number of mitochondria in the ventral

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

E, F Analysis of mitophagy in worms expressing hTau in response to NaN3 treatment (unpaired t test, t = 1.62, p = 0.1126) and CCCP treatment (Mann-Whitney test, U = 113, p = 0.002). Data information: n = 16-25 worms/group for NaN3 and 23-39 worms/groups for CCCP experiments. Data are given as mean and SEM. *** = p < 0.001, **** = p < 0.0001. Scale bar = 10 µm. Images are high-magnification of a small number of mitochondria in the ventral cord


G , H Analysis of mitophagy in worms expressing hP301L in response to either NaN3 ( Mann - Whitney test , U = 126 , p = 0 . 2711 ) or CCCP ( Mann - Whitney test , U = 375 , p = 0 . 3697 ) treatment . Data information : n = 16 - 25 worms / group for NaN3 and 23 - 39 worms / groups for CCCP experiments . Data are given as mean and SEM . * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 µm . Images are high - magnification of a small number of mitochondria in the ventral

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

G, H Analysis of mitophagy in worms expressing hP301L in response to either NaN3 (Mann-Whitney test, U = 126, p = 0.2711) or CCCP (Mann-Whitney test, U = 375, p = 0.3697) treatment. Data information: n = 16-25 worms/group for NaN3 and 23-39 worms/groups for CCCP experiments. Data are given as mean and SEM. *** = p < 0.001, **** = p < 0.0001. Scale bar = 10 µm. Images are high-magnification of a small number of mitochondria in the ventral cord


I Tau expression in worm lysates , as detected using the human tau - specific antibody HT7 .

I Tau expression in worm lysates, as detected using the human tau-specific antibody HT7.


Survival of age - and sex - matched ( 8 - 10 week old ) Spata2 + / + ( WT ) and Spata2 - / - ( KO ) mice injected intraperitoneally ( i . p . ) with LPS ( 20 mg / kg bodyweight ) . Data pooled from three independent experiments are shown . Statistical analysis was performed using long - rank test .

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

Survival of age- and sex-matched (8-10 week old) Spata2+/+ (WT) and Spata2-/- (KO) mice injected intraperitoneally (i.p.) with LPS (20 mg/kg bodyweight). Data pooled from three independent experiments are shown. Statistical analysis was performed using long-rank test.


ELISA of serum IL - 1β , IL - 18 , TNFα and IL - 6 at 12 h after injection of WT or Spata2 - KO mice with 20 mg / kg LPS . Circles and triangles represent individual mice . Horizontal bars indicate mean values . * * P < 0 . 01 ; * * * * P < 0 . 0001 . Statistical analysis was performed using unpaired two - tailed Student ' s t - test .

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

ELISA of serum IL-1β, IL-18, TNFα and IL-6 at 12 h after injection of WT or Spata2-KO mice with 20 mg/kg LPS. Circles and triangles represent individual mice. Horizontal bars indicate mean values. **P < 0.01; ****P < 0.0001. Statistical analysis was performed using unpaired two-tailed Student's t-test.


Immunoblot analysis of the indicated phosphorylated ( P ) and total proteins in lysates of LPS - stimulated BMDMs derived from WT or Spata2 - KO mice . Molecular weights in kDa are indicated to the right .

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

Immunoblot analysis of the indicated phosphorylated (P) and total proteins in lysates of LPS-stimulated BMDMs derived from WT or Spata2-KO mice. Molecular weights in kDa are indicated to the right.


qRT - PCR analysis of the indicated mRNAs in untreated or LPS - stimulated WT or Spata2 - KO BMDMs . Bars and error bars represent the mean ± S . D . of triplicate experiments . Statistical analysis was performed using unpaired two - tailed Student ' s t - test .

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

qRT-PCR analysis of the indicated mRNAs in untreated or LPS-stimulated WT or Spata2-KO BMDMs. Bars and error bars represent the mean ± S.D. of triplicate experiments. Statistical analysis was performed using unpaired two-tailed Student's t-test.


ELISA of TNFα and IL - 6 in the supernatants from untreated or LPS - stimulated WT or Spata2 - KO BMDMs . Bars and error bars represent the mean ± S . D . of triplicate experiments . Statistical analysis was performed using unpaired two - tailed Student ' s t - test .

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

ELISA of TNFα and IL-6 in the supernatants from untreated or LPS-stimulated WT or Spata2-KO BMDMs. Bars and error bars represent the mean ± S.D. of triplicate experiments. Statistical analysis was performed using unpaired two-tailed Student's t-test.


ELISA of IL - 1β secretion by WT or Spata2 - KO BMDMs that were either untreated ( - ) or LPS - primed and then treated with the indicated NLRP3

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

ELISA of IL-1β secretion by WT or Spata2-KO BMDMs that were either untreated (-) or LPS-primed and then treated with the indicated NLRP3 inducers


ELISA of IL - 1β secretion by WT or Spata2 - KO BMDMs that were either untreated ( - ) or stimulated with the NLRC4 inducer ( Salmonella infection ) or AIM2 inducer ( Poly ( dA : dT ) transfection ) ( B ) .

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

ELISA of IL-1β secretion by WT or Spata2-KO BMDMs that were either untreated (-) or stimulated with the NLRC4 inducer (Salmonella infection) or AIM2 inducer ( Poly(dA:dT) transfection) (B).


Immunoblot analysis of the mature IL - 1β p17 and the active Caspase - 1 p20 in cell supernatants ( Sup ) and the indicated proteins in the cell extracts ( Cell ext ) of WT or Spata2 - KO BMDMs stimulated as in ( A ) .

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

Immunoblot analysis of the mature IL-1β p17 and the active Caspase-1 p20 in cell supernatants (Sup) and the indicated proteins in the cell extracts (Cell ext) of WT or Spata2-KO BMDMs stimulated as in (A).


WT and Spata2 CRISPR knock out ( KO ) iBMDMs stably expressing GFP - ASC were primed with LPS and stimulated with Nigericin for 1 h . GFP - ASC specks were imaged by confocal microscope , and percentage of ASC speck - positive cells was quantified . Scale bar , 10 µm .

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

WT and Spata2 CRISPR knock out (KO) iBMDMs stably expressing GFP-ASC were primed with LPS and stimulated with Nigericin for 1 h. GFP-ASC specks were imaged by confocal microscope, and percentage of ASC speck-positive cells was quantified. Scale bar, 10 µm.


BMDMs from Spata2 + / + ( WT ) and Spata2 - / - ( KO ) mice were pretreated with Caspase - 1 inhibitor , Z - YVAD - FMK , for 30 min , primed with LPS , and then stimulated with Nigericin for 1 h . ASC specks were assessed by Immunofluorescence with antibody against ASC and imaged by confocal microscope , and percentage of ASC speck - positive cells was quantified . Scale bar , 10 µm .

BMDMs from Spata2+/+ (WT) and Spata2-/- (KO) mice were pretreated with Caspase-1 inhibitor, Z-YVAD-FMK, for 30 min, primed with LPS, and then stimulated with Nigericin for 1 h. ASC specks were assessed by Immunofluorescence with antibody against ASC and imaged by confocal microscope, and percentage of ASC speck-positive cells was quantified. Scale bar, 10 µm.


Immunoblot analysis of ASC in soluble cell lysate and DSS cross - linked insoluble fraction of WT or Spata2 - KO BMDMs primed with LPS and stimulated without or with nigericin ( Nig ) for 1 h . Relative levels of ASC in cross - linked samples from LPS + Nig treated cells were quantitated and shown below .

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

Immunoblot analysis of ASC in soluble cell lysate and DSS cross-linked insoluble fraction of WT or Spata2-KO BMDMs primed with LPS and stimulated without or with nigericin (Nig) for 1 h. Relative levels of ASC in cross-linked samples from LPS+ Nig treated cells were quantitated and shown below.


Spata2 + / + ( WT ) and Spata2 - / - ( KO ) mice were injected intraperitoneally ( i . p . ) with alum ( 20 mg / kg bodyweight ) and sacrificed 6 h latter to collect peritoneal lavages . Absolute numbers of neutrophils recruited to the peritoneum were counted by FACS ( G )

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

Spata2+/+ (WT) and Spata2-/- (KO) mice were injected intraperitoneally (i.p.) with alum (20 mg/kg bodyweight) and sacrificed 6 h latter to collect peritoneal lavages. Absolute numbers of neutrophils recruited to the peritoneum were counted by FACS (G)


Spata2 + / + ( WT ) and Spata2 - / - ( KO ) mice were injected intraperitoneally ( i . p . ) with alum ( 20 mg / kg bodyweight ) and sacrificed 6 h latter to collect peritoneal lavages . levels of IL - 1β ( H ) and IL - 6 ( I ) in the peritoneal fluids were measured by ELISA .

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

Spata2+/+ (WT) and Spata2-/- (KO) mice were injected intraperitoneally (i.p.) with alum (20 mg/kg bodyweight) and sacrificed 6 h latter to collect peritoneal lavages. levels of IL-1β (H) and IL-6 (I) in the peritoneal fluids were measured by ELISA.


ELISA of IL - 1β secretion by WT or CYLD KO BMDMs that were either untreated ( - ) or LPS - primed and then treated with the indicated NLRP3 inducers .

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

ELISA of IL-1β secretion by WT or CYLD KO BMDMs that were either untreated (-) or LPS-primed and then treated with the indicated NLRP3 inducers.


Immunoblot analysis of IL - 1β and active caspase - 1 p20 in cell supernatants ( Sup ) and the indicated proteins in cell extracts ( Cell ext ) from WT and CYLD KO BMDMs , stimulated as in ( A ) .

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

Immunoblot analysis of IL-1β and active caspase-1 p20 in cell supernatants (Sup) and the indicated proteins in cell extracts (Cell ext) from WT and CYLD KO BMDMs, stimulated as in (A).


BMDMs from WT and CYLD KO mice were pretreated with Caspase - 1 inhibitor , Z - YVAD - FMK , for 30 min , primed with LPS , and then stimulated with Nigericin for 30 min . ASC specks were assessed by Immunofluorescence with antibody against ASC and the percentage of ASC speck - positive cells was quantified .

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

BMDMs from WT and CYLD KO mice were pretreated with Caspase-1 inhibitor, Z-YVAD-FMK, for 30 min, primed with LPS, and then stimulated with Nigericin for 30 min. ASC specks were assessed by Immunofluorescence with antibody against ASC and the percentage of ASC speck-positive cells was quantified.


LDH release from WT and Cyld KO BMDMs primed with LPS and stimulated as indicated .

LDH release from WT and Cyld KO BMDMs primed with LPS and stimulated as indicated.


Immunoblotting analysis of active caspase - 1 p20 and IL - 1β in cell supernatants ( Sup ) and the indicated proteins in cell extracts ( Cell ext ) of WT iBMDMs and Spata2 KO iBMDMs reconstituted with WT Spata2 or a Spata2 mutant ( F108A ) defective in CYLD binding that were primed with LPS and stimulated with Nigericin for 1 h .

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

Immunoblotting analysis of active caspase-1 p20 and IL-1β in cell supernatants (Sup) and the indicated proteins in cell extracts (Cell ext) of WT iBMDMs and Spata2 KO iBMDMs reconstituted with WT Spata2 or a Spata2 mutant (F108A) defective in CYLD binding that were primed with LPS and stimulated with Nigericin for 1 h.


Confocal immunofluorescence analysis of Spata2 centrosome localization in HEK293 cells stably expressing control shRNA or shRNA against Spata2 , stained with Spata2 and γ - tubulin antibodies and the nuclear dye DAPI . Scale bar , 10 µm .

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

Confocal immunofluorescence analysis of Spata2 centrosome localization in HEK293 cells stably expressing control shRNA or shRNA against Spata2, stained with Spata2 and γ-tubulin antibodies and the nuclear dye DAPI. Scale bar, 10 µm.


Confocal immunofluorescence analysis of Spata2 localization in RAW264 . 7 cells stably expressing GFP - Spata2 , stained with γ - tubulin antibody and DAPI . Scale bar , 10 µm .

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

Confocal immunofluorescence analysis of Spata2 localization in RAW264.7 cells stably expressing GFP-Spata2, stained with γ-tubulin antibody and DAPI. Scale bar, 10 µm.


Confocal microscopy of HEK293 cells stably expressing GFP - tagged Spata2 or Spata2 mutants ( ΔPUB orΔ51 - 81 ) that were stained with γ - tubulin antibody and DAPI .

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

Confocal microscopy of HEK293 cells stably expressing GFP-tagged Spata2 or Spata2 mutants (ΔPUB orΔ51-81) that were stained with γ-tubulin antibody and DAPI.


Confocal microscopy of HEK293 cells stably expressing GFP - tagged CYLD along with an empty vector or vectors encoding Flag - tagged Spata2 or Spata2 mutants ( ΔPUB orΔ51 - 81 ) stained as in ( C ) . Scale bar , 10 µm .

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

Confocal microscopy of HEK293 cells stably expressing GFP-tagged CYLD along with an empty vector or vectors encoding Flag-tagged Spata2 or Spata2 mutants (ΔPUB orΔ51-81) stained as in (C). Scale bar, 10 µm.


Co - immunoprecipitation analysis of CYLD physical interaction with Spata2 or Spata2 mutants ( ΔPUB orΔ51 - 81 ) in HEK293 cells transfected with the indicated expression vectors . CYLD and Spata2 proteins were detected by immunoblot using anti - HA and anti - Flag antibodies , respectively .

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

Co-immunoprecipitation analysis of CYLD physical interaction with Spata2 or Spata2 mutants (ΔPUB orΔ51-81) in HEK293 cells transfected with the indicated expression vectors. CYLD and Spata2 proteins were detected by immunoblot using anti-HA and anti-Flag antibodies, respectively.


Immunoblotting analysis of the active caspase - 1 p20 in cell supernatants ( Sup ) and pro - caspase - 1 and Flag - Spata2 proteins in cell extracts ( Cell ext ) from WT iBMDMs and Spata2 KO iBMDMs reconstituted with WT Spata2 or Spata2 mutant defective in centrosome localization ( ΔPUB orΔ51 - 81 ) . Cells were primed with LPS and stimulated with Nigericin for 1 h . Data information : For all Immunoblotting data , molecular weights in kDa are indicated to the left .

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

Immunoblotting analysis of the active caspase-1 p20 in cell supernatants (Sup) and pro-caspase-1 and Flag-Spata2 proteins in cell extracts (Cell ext) from WT iBMDMs and Spata2 KO iBMDMs reconstituted with WT Spata2 or Spata2 mutant defective in centrosome localization (ΔPUB orΔ51-81). Cells were primed with LPS and stimulated with Nigericin for 1 h. Data information: For all Immunoblotting data, molecular weights in kDa are indicated to the left.


WT and Spata2 KO iBMDMs stably expressing an empty vector or a vector encoding Flag - NEK7 were untreated ( NT ) , primed with LPS for 4 h , or primed with LPS for 4 h followed by stimulation with Nigericin for 30 min . Flag - NEK7 was immunoprecipitated and the association of endogenous NLRP3 was assessed by immunoblotting . Relative levels of NLRP3 in immunoprecipitates were normalized to Flag - NEK7 and shown below .

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

WT and Spata2 KO iBMDMs stably expressing an empty vector or a vector encoding Flag-NEK7 were untreated (NT), primed with LPS for 4 h, or primed with LPS for 4 h followed by stimulation with Nigericin for 30 min. Flag-NEK7 was immunoprecipitated and the association of endogenous NLRP3 was assessed by immunoblotting. Relative levels of NLRP3 in immunoprecipitates were normalized to Flag-NEK7 and shown below.


Co - immunoprecipitation analysis of PLK4 interaction with endogenous Spata2 and CYLD in parental HEK293 cells and HEK293 cells stably expressing GFP - PLK4 .

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

Co-immunoprecipitation analysis of PLK4 interaction with endogenous Spata2 and CYLD in parental HEK293 cells and HEK293 cells stably expressing GFP-PLK4.


iBMDMs stably expressing GFP - PLK4 and parental iBMDMs were unstimulated or stimulated with 0 . 5 ug / ml LPS for 4h . The PLK4 interaction with endogenous CYLD was analyzed by co - immunoprecipitation with anti - GFP antibody .

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

iBMDMs stably expressing GFP-PLK4 and parental iBMDMs were unstimulated or stimulated with 0.5 ug/ml LPS for 4h. The PLK4 interaction with endogenous CYLD was analyzed by co-immunoprecipitation with anti-GFP antibody.


Confocal microscopy analysis of ASC specks in iBMDMs stably expressing GFP - ASC , pretreated for 2 days with DMSO and two different PLK4 inhibitors , Centrinone ( 150 nM ) and Centrinone - B ( 500 nM ) , primed with LPS for 4 h , and stimulated with Nigericin for 30 min . GFP - ASC specks were imaged and quantified ( > 500 cells counted ) . Scale bar , 10 µm .

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

Confocal microscopy analysis of ASC specks in iBMDMs stably expressing GFP-ASC, pretreated for 2 days with DMSO and two different PLK4 inhibitors, Centrinone (150 nM) and Centrinone-B(500 nM), primed with LPS for 4 h, and stimulated with Nigericin for 30 min. GFP-ASC specks were imaged and quantified (>500 cells counted). Scale bar, 10 µm.


Immunoblot analysis of the indicated proteins in cell supernatants ( Sup ) and cell extracts ( Cell ext ) of LPS - primed and Nigericin - stimulated BMDMs that were pretreated for 2 days with PLK4 inhibitors , Centrinone ( 150 nM ) and Centrinone - B ( 500 nM ) ( E )

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

Immunoblot analysis of the indicated proteins in cell supernatants (Sup) and cell extracts (Cell ext) of LPS-primed and Nigericin-stimulated BMDMs that were pretreated for 2 days with PLK4 inhibitors, Centrinone (150 nM) and Centrinone-B(500 nM) (E)


LPS - primed and Nigericin - stimulated iBMDMs stably expressing a control shRNA or 3 different PLK4 - specific shRNAs ( F ) .

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

LPS-primed and Nigericin-stimulated iBMDMs stably expressing a control shRNA or 3 different PLK4-specific shRNAs (F).


Immunoblot analysis of active caspase - 1 ( p20 ) generation in HEK293 cells that were transfected with inflammasome components ( Flag - tagged NLRP3 , NEK7 , ASC , and Caspase - 1 ) along with EGFP , GFP - tagged PLK4 , PLK4 kinase dead mutant ( D154A ) , or PLK1 and stimulated with 10 µM Nigericin for 1h 24h post - transfection .

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

Immunoblot analysis of active caspase-1 (p20) generation in HEK293 cells that were transfected with inflammasome components (Flag-tagged NLRP3, NEK7, ASC, and Caspase-1) along with EGFP, GFP-tagged PLK4, PLK4 kinase dead mutant (D154A), or PLK1 and stimulated with 10 µM Nigericin for 1h 24h post-transfection.


WT and Spata2 CRISPR knock out ( KO ) iBMDMs stably expressing GFP - ASC were pretreated with DMSO or PLK4 inhibitor Centrinone at a concentration of 150nM for 2 days , primed with LPS , and stimulated with Nigericin for 1 h . GFP - ASC specks were imaged by confocal microscope , and percentage of ASC speck - positive cells was quantified .

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

WT and Spata2 CRISPR knock out (KO) iBMDMs stably expressing GFP-ASC were pretreated with DMSO or PLK4 inhibitor Centrinone at a concentration of 150nM for 2 days, primed with LPS, and stimulated with Nigericin for 1 h. GFP-ASC specks were imaged by confocal microscope, and percentage of ASC speck-positive cells was quantified.


Co - immunoprecipitation analysis of NEK7 / NLRP3 interaction in iBMDMs stably expressing Flag - NEK7 that were transduced with control or PLK4 - specific shRNA . The cells were untreated , primed with LPS , or primed with LPS and stimulated with Nigericin . Flag - NEK7 was immunoprecipitated and the association of endogenous NLRP3 was assessed by immunoblot analysis . Relative levels of NLRP3 in immunoprecipitates were normalized to Flag - NEK7 and shown below .

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

Co-immunoprecipitation analysis of NEK7/NLRP3 interaction in iBMDMs stably expressing Flag-NEK7 that were transduced with control or PLK4-specific shRNA. The cells were untreated, primed with LPS, or primed with LPS and stimulated with Nigericin. Flag-NEK7 was immunoprecipitated and the association of endogenous NLRP3 was assessed by immunoblot analysis. Relative levels of NLRP3 in immunoprecipitates were normalized to Flag-NEK7 and shown below.


Co - immunoprecipitation analysis of NEK7 / NLRP3 interaction in HEK293 cells transfected with HA - NEK7 and Flag - NLRP3 along with ( + ) or without ( - ) GFP - PLK4 . The cells were either treated ( + ) or not treated ( - ) with the PLK4 inhibitor Centrinone .

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

Co-immunoprecipitation analysis of NEK7/NLRP3 interaction in HEK293 cells transfected with HA-NEK7 and Flag-NLRP3 along with (+) or without (-) GFP-PLK4. The cells were either treated (+) or not treated (-) with the PLK4 inhibitor Centrinone.


iBMDMs stably expressing Flag - tagged NEK7 or NEK7 mutant S204A were treated with LPS . Anti - Flag immunoprecipitates were prepared for immunoblot analysis for the interaction between NEK7 and endogenous NLRP3 .

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

iBMDMs stably expressing Flag-tagged NEK7 or NEK7 mutant S204A were treated with LPS. Anti-Flag immunoprecipitates were prepared for immunoblot analysis for the interaction between NEK7 and endogenous NLRP3.


Immunoblot analysis of active caspase - 1 ( p20 ) in supernatants ( Sup ) and the indicated proteins in cell extracts ( Cell ext ) of iBMDMs stably expressing WT NEK7 or NEK7 mutant ( S204A ) , primed with LPS and stimulated with nigericin .

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

Immunoblot analysis of active caspase-1 (p20) in supernatants (Sup) and the indicated proteins in cell extracts (Cell ext) of iBMDMs stably expressing WT NEK7 or NEK7 mutant (S204A), primed with LPS and stimulated with nigericin.


iBMDMs stably expressing Flag - tagged WT NEK7 or NEK7 mutant S204A were left untreated or stimulated with LPS for indicated time . Anti - Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser 204 ( P - NEK7 ) .

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

iBMDMs stably expressing Flag-tagged WT NEK7 or NEK7 mutant S204A were left untreated or stimulated with LPS for indicated time. Anti-Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser 204 (P-NEK7).


iBMDMs stably expressing Flag - tagged WT NEK7 were left untreated or pretreated with PLK4 inhibitor Centrinone , and stimulated without or with LPS for 4h . Anti - Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser 204 .

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

iBMDMs stably expressing Flag-tagged WT NEK7 were left untreated or pretreated with PLK4 inhibitor Centrinone, and stimulated without or with LPS for 4h. Anti-Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser 204.


iBMDMs stably expressing GFP - tagged PLK4 and parental iBMDMs were left untreated or stimulated with LPS for 4h . Anti - GFP immunoprecipitates were prepared for immunoblot analysis for the interaction between GFP - PLK4 and endogenous NEK7 .

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

iBMDMs stably expressing GFP-tagged PLK4 and parental iBMDMs were left untreated or stimulated with LPS for 4h. Anti-GFP immunoprecipitates were prepared for immunoblot analysis for the interaction between GFP-PLK4 and endogenous NEK7.


Co - immunoprecipitation analysis of NEK7 / NLRP3 interaction in HEK293 cells transfected with the indicated expression vectors , treated with either DMSO or 150 nM PLK4 inhibitor Centrinone .

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

Co-immunoprecipitation analysis of NEK7/NLRP3 interaction in HEK293 cells transfected with the indicated expression vectors, treated with either DMSO or 150 nM PLK4 inhibitor Centrinone.


WT and Spata2 KO iBMDMs stably expressing Flag - tagged WT NEK7 were left untreated or pretreated with the PLK4 inhibitor Centrinone , and stimulated without or with LPS for 4h . Anti - Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser 204 .

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

WT and Spata2 KO iBMDMs stably expressing Flag-tagged WT NEK7 were left untreated or pretreated with the PLK4 inhibitor Centrinone, and stimulated without or with LPS for 4h. Anti-Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser 204.


HEK293 cells stably expressing GFP - PLK4Δ24 were transfected with a control siRNA or siRNAs targeting Spata2 or CYLD . GFP - PLK4Δ24 was immunoprecipitated under denaturing conditions and immunoblotted for K63 - , K48 - linked and linear ubiquitination . Relative levels of PLK4 ubiquitination were normalized to GFP - PLK4 and shown below .

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

HEK293 cells stably expressing GFP-PLK4Δ24 were transfected with a control siRNA or siRNAs targeting Spata2 or CYLD. GFP-PLK4Δ24 was immunoprecipitated under denaturing conditions and immunoblotted for K63-, K48-linked and linear ubiquitination. Relative levels of PLK4 ubiquitination were normalized to GFP-PLK4 and shown below.


Co - immunoprecipitation analysis of the PLK4 - NEK7 interaction in HEK293 cells co - transfected with Flag - NEK7 and HA - PLK4 in the presence ( + ) or absence ( - ) of GFP , GFP - Spata2 , GFP - CYLD , or GFP - CYLD C601S , a catalytically inactive mutant .

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

Co-immunoprecipitation analysis of the PLK4-NEK7 interaction in HEK293 cells co-transfected with Flag-NEK7 and HA-PLK4 in the presence (+) or absence (-) of GFP, GFP-Spata2, GFP-CYLD, or GFP-CYLD C601S, a catalytically inactive mutant.


( C ) NAD + / NADH ratio was measured in mBA cell lines and 17 . 5 days embryo liver . Data represented as mean ± SEMs , and obtained from three different cell line and embryo liver . Significance determined by Student ' s t test , two - tailed , * P < 0 . 05 , * * P < 0 . 01 .

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

(C) NAD+/NADH ratio was measured in mBA cell lines and 17.5 days embryo liver. Data represented as mean ±SEMs, and obtained from three different cell line and embryo liver. Significance determined by Student's t test, two-tailed, *P < 0.05, **P < 0.01.


( D ) Immunostaining of 17 . 5 days embryo tissues ( mid - brain , heart , liver , and skin ) with anti - PAR antibody . Insets show higher magnification . Scale bar indicates 100 μm ( insert ) .

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

(D) Immunostaining of 17.5 days embryo tissues (mid-brain, heart, liver, and skin) with anti-PAR antibody. Insets show higher magnification. Scale bar indicates 100 μm (insert).


( E ) Wild - type ( WT ) and Ews - / - embryos mid - brain at E17 . 5 days were subjected to TUNEL assay . Scale bar indicates 100 μm ( lnsert ) .

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

(E) Wild-type (WT) and Ews-/- embryos mid-brain at E17.5 days were subjected to TUNEL assay. Scale bar indicates 100 μm (lnsert).


( A ) Western blot analysis of Parp1 and DNA damage markers in Wild - type ( WT ) and Ews - / - mBA cells . Cells were treated with MMS ( 0 . 02 % ) in a time dependent manner . Proteins were fractionated into two groups , chromatin - bound ( Chro ) proteins and soluble ( Solu ) proteins . ( B ) After treatment with MMS ( 0 . 02 % , 1hour ) , the media was replaced to release the DNA damage . The proteins were fractionated and the specific protein kinetics at chromatin was analyzed by western blot .

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

(A) Western blot analysis of Parp1 and DNA damage markers in Wild-type (WT) and Ews-/- mBA cells. Cells were treated with MMS (0.02%) in a time dependent manner. Proteins were fractionated into two groups, chromatin-bound (Chro) proteins and soluble (Solu) proteins. (B) After treatment with MMS (0.02%, 1hour), the media was replaced to release the DNA damage. The proteins were fractionated and the specific protein kinetics at chromatin was analyzed by western blot.


( C ) GFP - PARP1 U2OS cells were transfected with siControl and siEWS . Local DNA damage was induced by micro - irradiation using a 405nm laser . Data represented as mean ± SEMs and more than six cells were analyzed from six independent experiments . The statistical significance determined by One - way ANOVA , * * * P < 0 . 001 . Scale bar indicates 5 μm .

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

(C) GFP-PARP1 U2OS cells were transfected with siControl and siEWS. Local DNA damage was induced by micro-irradiation using a 405nm laser. Data represented as mean ±SEMs and more than six cells were analyzed from six independent experiments. The statistical significance determined by One-way ANOVA, ***P< 0.001. Scale bar indicates 5 μm.


( D ) Fluorescence correlation spectroscopy ( FCS ) was measured after 15 minutes later following micro - irradiation ( Upper ) or under normal condition in GFP - PARP1 U2OS cells with either siControl or siEWS . Data represented as mean ± SEMs , more than 50 cells were analyzed , and significance determined by One - way ANOVA , * * * P < 0 . 001 . Scale bar indicate 5 μm .

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

(D) Fluorescence correlation spectroscopy (FCS) was measured after 15 minutes later following micro-irradiation (Upper) or under normal condition in GFP-PARP1 U2OS cells with either siControl or siEWS. Data represented as mean ±SEMs, more than 50 cells were analyzed, and significance determined by One-way ANOVA, ***P< 0.001. Scale bar indicate 5 μm.


( E ) AsiSI endonuclease - integrated U2OS cells were transfected with siControl and siEWS . Cells were incubated with doxycycline for 4 hours to induce DSBs . With or without changing the Dox - added media to fresh media for 2 hours ( for release samples ) , the amount of chromatin - associated PARP1 was measured using ChIP assay . ( N . T : Non - treat , T : AsiSI treat , Release : Damage released samples ) . Data represented as mean ± SEMs , and technical repeats ( n = 3 ) , significance determined by two - way ANOVA , * * * P < 0 . 001 .

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

(E) AsiSI endonuclease-integrated U2OS cells were transfected with siControl and siEWS. Cells were incubated with doxycycline for 4 hours to induce DSBs. With or without changing the Dox-added media to fresh media for 2 hours (for release samples), the amount of chromatin-associated PARP1 was measured using ChIP assay. (N.T: Non-treat, T: AsiSI treat, Release: Damage released samples). Data represented as mean ±SEMs, and technical repeats (n=3), significance determined by two-way ANOVA, ***P< 0.001.


( A ) Total levels of PAR in whole cell lysate were measured using western blot analysis . Wild - type ( WT ) and Ews - / - mBA cells were treated with H2O2 , ( 1 mM , 20 minutes ) with or without Olaparib ( 5 μM , 7 hours ) .

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

(A) Total levels of PAR in whole cell lysate were measured using western blot analysis. Wild-type (WT) and Ews-/- mBA cells were treated with H2O2, (1 mM, 20 minutes) with or without Olaparib (5 μM, 7 hours).


( B ) The kinetics of PAR accumulation was analyzed by western blotting . WT and Ews - / - cells were treated with H2O2 ( 1 mM , 20 minutes ) , followed by incubation in fresh media for the indicated times to allow release of DNA

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

(B) The kinetics of PAR accumulation was analyzed by western blotting. WT and Ews-/- cells were treated with H2O2 (1 mM, 20 minutes), followed by incubation in fresh media for the indicated times to allow release of DNA damage


( C ) Immunohistochemistry detection of PAR by ADP - ribose antibody in WT and Ews - / - cells after H2O2 treatment ( 1 mM , 20 minutes ) and recovery from DNA damage . Right graph displays mean of intensities measured from 500 cells . Data represented as mean ± SEMs , significance determined by One - way ANOVA , * * * P < 0 . 001 . Scale bar indicate 20 μm .

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

(C) Immunohistochemistry detection of PAR by ADP-ribose antibody in WT and Ews-/- cells after H2O2 treatment (1 mM, 20 minutes) and recovery from DNA damage. Right graph displays mean of intensities measured from 500 cells. Data represented as mean ±SEMs, significance determined by One-way ANOVA, ***P < 0.001. Scale bar indicate 20 μm.


( D , E ) Whole cell level of PAR was measured in WT and Ews - / - mBA cells upon treatment with ( D ) MMS ( 0 . 02 % , 60 minutes ) ( E ) with or without recovery from DNA damage .

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

(D, E) Whole cell level of PAR was measured in WT and Ews-/- mBA cells upon treatment with (D) MMS (0.02%, 60 minutes) (E) with or without recovery from DNA damage.


( F ) NAD + / NADH ratio was measured in mBA cell lines following treatment of H2O2 ( 1 mM , 10 minutes ) . Data represented as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Student ' s t test , two - tailed , * * P < 0 . 01 , * * * P < 0 . 001 .

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

(F) NAD+/NADH ratio was measured in mBA cell lines following treatment of H2O2 (1 mM, 10 minutes). Data represented as mean ±SEMs, and technical repeats (n=3). Significance determined by Student's t test, two-tailed, **P < 0.01, ***P< 0.001.


( A ) PARP1 and EWS interaction was visualized in cells using the CUPID system . After transfection of mRCD - EWS and GC3 - PARP1 ( EGFP - PARP1 ) plasmid , the cells were treated with either H2O2 ( 1 mM , 10 minutes ) or PMA . Scale bar indicates 5 μm .

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

(A) PARP1 and EWS interaction was visualized in cells using the CUPID system. After transfection of mRCD-EWS and GC3-PARP1 (EGFP-PARP1) plasmid, the cells were treated with either H2O2 (1 mM, 10 minutes) or PMA. Scale bar indicates 5 μm.


( B ) Endogenous interaction between EWS and PARP1 was measured using immunoprecipitation western blot analysis . Cells were treated with H2O2 ( 1 mM , 10 minutes ) with or without Olaparib ( 5 μM , 7 hours ) .

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

(B) Endogenous interaction between EWS and PARP1 was measured using immunoprecipitation western blot analysis. Cells were treated with H2O2 (1 mM, 10 minutes) with or without Olaparib (5 μM, 7 hours).


( C ) MMS - treated cells , with or without Olaparib , were immunoprecipitated using an anti - EWS antibody and immunoblotted by anti - PAR antibody ( upper ) . Pre - Olaparib group was treated with Olaparib for 3 hours before MMS treatment ( 0 . 02 % 30 minutes ) and after - Olaparib group treated Olaparib after 10 minutes following treatment of MMS . Lower blot indicates input of anti - EWS antibody .

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

(C) MMS-treated cells, with or without Olaparib, were immunoprecipitated using an anti-EWS antibody and immunoblotted by anti-PAR antibody (upper). Pre-Olaparib group was treated with Olaparib for 3 hours before MMS treatment (0.02% 30 minutes) and after-Olaparib group treated Olaparib after 10 minutes following treatment of MMS. Lower blot indicates input of anti-EWS antibody.


( D ) In vitro PAR binding assay . THRAP3 binding to PAR was used as a positive control and BSA binding to PAR was used as a negative control .

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

(D) In vitro PAR binding assay. THRAP3 binding to PAR was used as a positive control and BSA binding to PAR was used as a negative control.


( E ) Schematic map of nine EWS mutants ( left ) and IP - western blot analysis ( right ) . GFP - PARP1 and siPARG were transfected into HEK - 293 cells with each EWS mutants plasmid . After H2O2 treatment , the cells were conducted to IP with anti - Flag antibody .

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

(E) Schematic map of nine EWS mutants (left) and IP-western blot analysis (right). GFP-PARP1 and siPARG were transfected into HEK-293 cells with each EWS mutants plasmid. After H2O2 treatment, the cells were conducted to IP with anti-Flag antibody.


( F ) Western blot analysis of PARP1 accumulation on chromatin . EWS - WT and M4 mutants were transfected into EWS depleted HEK - 293 cells . After treatment of MMS ( 0 . 02 % , 1 hour ) , the proteins were fractionated to either chromatin - bound or soluble fraction . ( G ) Quantification of relative amount of PARP1 and γH2AX on chromatin , divided by chromatin H3 , from three independent western experiments are presented . Data represented as mean ± SEMs , were measured from 3 independent experiments . Significance determined by t - test . * P < 0 . 05 , * * P < 0 . 01 . n . s indicate non -

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

(F) Western blot analysis of PARP1 accumulation on chromatin. EWS-WT and M4 mutants were transfected into EWS depleted HEK-293 cells. After treatment of MMS (0.02%, 1 hour), the proteins were fractionated to either chromatin-bound or soluble fraction. (G) Quantification of relative amount of PARP1 and γH2AX on chromatin, divided by chromatin H3, from three independent western experiments are presented. Data represented as mean ±SEMs, were measured from 3 independent experiments. Significance determined by t-test. * P < 0.05, **P < 0.01. n.s indicate non-significance


( H ) WT and Ews - / - cells were transfected with EWS - WT and - M4 mutant and cell viability was measured 24 hours after MMS treatment ( indicated concentration ) . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * P < 0 . 01 . n . s indicate non - significance ,

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

(H) WT and Ews-/- cells were transfected with EWS-WT and -M4 mutant and cell viability was measured 24 hours after MMS treatment (indicated concentration). Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, **P < 0.01. n.s indicate non-significance,


( A ) Relative cell viability of cells transfected with siCon ( WT ) , siEWS ( EWS KD ) , PARP1 ( PARP - 1 KD ) , and double siRNA ( DKD ) after MMS treatment . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * * P < 0 . 001 . Analysis ( * * * ) indicates differences between DKD with EWS KD .

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

(A) Relative cell viability of cells transfected with siCon (WT), siEWS (EWS KD), PARP1 (PARP-1 KD), and double siRNA (DKD) after MMS treatment. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001. Analysis (***) indicates differences between DKD with EWS KD.


( B ) Additional Parp1 knockout in Ews knockout cells ( DKO ) were subjected to viability test after MMS treatment . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * * P < 0 . 001 . * * * indicated differences between DKO with EWS - KO .

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

(B) Additional Parp1 knockout in Ews knockout cells (DKO) were subjected to viability test after MMS treatment. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001. *** indicated differences between DKO with EWS-KO.


C ) After transfection of siCon , siEWS , PARP1 , and double siRNA , the effect of co - depletion of PARP1 and EWS on the γH2AX and phosphorylated CHK1 upon MMS treatment was analyzed by western blot .

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

C) After transfection of siCon, siEWS, PARP1, and double siRNA, the effect of co-depletion of PARP1 and EWS on the γH2AX and phosphorylated CHK1 upon MMS treatment was analyzed by western blot.


D ) After treatment of MMS , the effect of additional Parp1 knockout in Ews knockout cells ( Double KO ) to the γH2AX and phosphorylated CHK1 were analyzed using western blot .

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

D) After treatment of MMS, the effect of additional Parp1 knockout in Ews knockout cells (Double KO) to the γH2AX and phosphorylated CHK1 were analyzed using western blot.


E ) Whole cell expression of PAR in mBA ( WT : Ews - WT , KO : Ews - KO , DKO : double KO ) .

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

E) Whole cell expression of PAR in mBA (WT: Ews-WT, KO: Ews-KO, DKO: double KO).


F ) Relative NAD + / NADH ratios were measured in WT , Ews - / - and DKO cells . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * * P < 0 . 001 .

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

F) Relative NAD+/NADH ratios were measured in WT, Ews-/- and DKO cells. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001.


G ) PARGi ( 5 μM , 24 hours ) treated WT and Ews - / - cells were subjected to cellular viability assay . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA .

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

G) PARGi (5 μM, 24 hours) treated WT and Ews-/- cells were subjected to cellular viability assay. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA.


H ) Relative cellular viability was measured after NMN treatment , with or without MMS pretreatment , in WT and Ews - / - cells . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * * P < 0 . 001 .

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

H) Relative cellular viability was measured after NMN treatment, with or without MMS pretreatment, in WT and Ews-/- cells. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001.


I ) Chromatin - bound PARP1 was quantified by western blot in WT and Ews - / - cells treated with Olaparib ( 5 μM , 24 hours ) treatment .

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

I) Chromatin-bound PARP1 was quantified by western blot in WT and Ews-/- cells treated with Olaparib (5 μM, 24 hours) treatment.


J ) Relative viability was measured in WT and Ews - / - cells upon treatment of Olaparib for 24 hours . Error bars represent as mean ± SEMs , and technical repeats ( n = 3 ) . Significance determined by Two - way ANOVA , * * * P < 0 . 001 .

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

J) Relative viability was measured in WT and Ews-/- cells upon treatment of Olaparib for 24 hours. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001.


( A ) Western blot analysis of PARP1 in chromatin . Two Ewing Sarcoma cells ( CHP100 and A4573 ) were treated with MMS ( 0 . 02 % , 1 hour ) with or without release ( 1 hour ) from MMS ( N . T : non treat , T : treat , R : release sample ) .

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

(A) Western blot analysis of PARP1 in chromatin. Two Ewing Sarcoma cells (CHP100 and A4573) were treated with MMS (0.02%, 1 hour) with or without release (1 hour) from MMS (N.T: non treat, T: treat, R: release sample).


( B ) U2OS and two Ewing Sarcoma cells were treated MMS ( 0 . 02 % , 30 minutes ) and subjected to immunofluorescence by PARP1 antibody after chromatin bound fraction . Scale bar indicates 20 μm ( left ) . Red line indicates mean and more than 300 cells were analyzed . Significance determined by Two - way ANOVA , * * * P < 0 . 001 .

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

(B) U2OS and two Ewing Sarcoma cells were treated MMS (0.02%, 30 minutes) and subjected to immunofluorescence by PARP1 antibody after chromatin bound fraction. Scale bar indicates 20 μm (left). Red line indicates mean and more than 300 cells were analyzed. Significance determined by Two-way ANOVA, ***P < 0.001.


( C ) Human Ewing Sarcoma histology samples were analyzed by immuno - histochemistry using PAR antibody . Scale bar represents 100 mm .

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

(C) Human Ewing Sarcoma histology samples were analyzed by immuno-histochemistry using PAR antibody. Scale bar represents 100 mm.


( C ) Reaction progress was measured for the Twist COVID - 19 synthetic RNA ( MT007544 . 1 ) from 1 million molecules of virus ( 106 ) , then titrated down by log10 dilutions . The colorimetric findings of the LAMP assay are based on a yellow to pink gradient with higher copies of SARS - CoV - 2 RNA corresponding to a yellow color . The limit of detection ( LoD ) range is shown with a gradient after 30 minutes between 10 and 100 viral copies ( lower right ) .

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

(C) Reaction progress was measured for the Twist COVID-19 synthetic RNA (MT007544.1) from 1 million molecules of virus (106), then titrated down by log10 dilutions. The colorimetric findings of the LAMP assay are based on a yellow to pink gradient with higher copies of SARS-CoV-2 RNA corresponding to a yellow color. The limit of detection (LoD) range is shown with a gradient after 30 minutes between 10 and 100 viral copies (lower right).


( D ) Replicates of the titrated viral copies using LAMP , as measured by Quantifluor fluorescence .

(D) Replicates of the titrated viral copies using LAMP, as measured by Quantifluor fluorescence.


( E ) The sensitivity and specificity of the LAMP assay from 201 patients ( 132 negative and 69 positive for SARS - CoV - 2 , as measured by qRT - PCR ) . Thresholds are DNA quantified by the Quantifluor .

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

(E) The sensitivity and specificity of the LAMP assay from 201 patients (132 negative and 69 positive for SARS-CoV-2, as measured by qRT-PCR). Thresholds are DNA quantified by the Quantifluor.


LAMP . ( a ) Clinical samples tested by qRT - PCR ( Positive , dark red or Negative , light grey ) were sequenced and run through the LAMP assay . These results were compared to the buffer blanks ( Negative Control ) , dark grey , Synthetic RNAs or Vero 6 cell extracts with SARS - CoV - 2 infection ( Positive Controls , light red ) , and Subway Samples ( Environmental , blue ) . Read proportions are shown on the y - axis .

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

LAMP.(a) Clinical samples tested by qRT-PCR (Positive, dark red or Negative, light grey) were sequenced and run through the LAMP assay. These results were compared to the buffer blanks (Negative Control), dark grey, Synthetic RNAs or Vero 6 cell extracts with SARS-CoV-2 infection (Positive Controls, light red), and Subway Samples (Environmental, blue). Read proportions are shown on the y-axis.


( b ) SARS - CoV - 2 abundance , as measured with NGS and percentage of reads ( y - axis ) is compared to the Ct Threshold for qRT - PCR ( x - axis ) , with lower Ct values representing higher viral abundance , and the LAMP reaction output ( Fluorimeter values , black to yellow scale ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SARS-CoV-2 abundance, as measured with NGS and percentage of reads (y-axis) is compared to the Ct Threshold for qRT-PCR (x-axis), with lower Ct values representing higher viral abundance, and the LAMP reaction output (Fluorimeter values, black to yellow scale).


( c , d ) Read mapping to archaea ( red ) , bacteria ( green ) , fungi ( yellow ) , human ( blue ) , and SARS - CoV - 2 ( orange ) , and other viruses ( grey ) , across the clinical controls ( CN , CP ) , environmental samples , qRT - PCR negative , and qRT - PCR positive samples .

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

(c,d) Read mapping to archaea (red), bacteria (green), fungi (yellow), human (blue), and SARS-CoV-2 (orange), and other viruses (grey), across the clinical controls (CN, CP), environmental samples, qRT-PCR negative, and qRT-PCR positive samples.


coverage ( a ) The coverage plot across the SARS - CoV - 2 genomes ( viral coordinate on bottom , colored by sample ) from a representative set of clinical positive samples . Sample names with the suffixes CN and N are clinical negative ( buffer ) , P are qRT - PCR positive , and CP are Vero 6 cells with virus .

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

coverage(a) The coverage plot across the SARS-CoV-2 genomes (viral coordinate on bottom, colored by sample) from a representative set of clinical positive samples. Sample names with the suffixes CN and N are clinical negative (buffer), P are qRT-PCR positive, and CP are Vero 6 cells with virus.


The phylogenetic placement of these COVID samples is shown on the tree ( left ) and the global map of known COVID genomes ( right ) . Genetic variants called from the RNA - seq data show a range of variants that are distinct from the Wuhan reference strain , and the samples from this study , highlighted in blue , show enrichment for European and Asian alleles .

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

The phylogenetic placement of these COVID samples is shown on the tree (left) and the global map of known COVID genomes (right). Genetic variants called from the RNA-seq data show a range of variants that are distinct from the Wuhan reference strain, and the samples from this study, highlighted in blue, show enrichment for European and Asian alleles.


( b ) Proportion of the L ( green ) and S strain ( yellow ) are shown for the NYC viruses . Phylogeny of samples from this study on the left and total GISAID samples on the right , with a map of variants in this study ' s samples in the middle , colored by event type and sized by number of nucleotides impacted . Annotation track on the bottom shows frequency of alternate alleles in this study and in the GISAID database .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Proportion of the L (green) and S strain (yellow) are shown for the NYC viruses. Phylogeny of samples from this study on the left and total GISAID samples on the right, with a map of variants in this study's samples in the middle, colored by event type and sized by number of nucleotides impacted. Annotation track on the bottom shows frequency of alternate alleles in this study and in the GISAID database.


( c ) The 9 - bp deletion in ORRF1b ( NSP1 ) that was detected in samples from two patients was confirmed in the GISAID database ( below ) .

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

(c) The 9-bp deletion in ORRF1b (NSP1) that was detected in samples from two patients was confirmed in the GISAID database (below).


( d ) Comparison of the ORF1b of the SARS - CoV - 2 and SARS - CoV genomes with the 9 - bp deletion region shown enlarged .

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

(d) Comparison of the ORF1b of the SARS-CoV-2 and SARS-CoV genomes with the 9-bp deletion region shown enlarged.


( a ) Six variant alleles were found to be significantly enriched by population allelic fraction within this set of 93 WCM / NYP cases as compared with non - NYC strains of Nextstrain clade A2a ( 1059 : C > T P = 6 . 26e - 22 , 11916 : C > T P = 3 . 09e - 9 , 18998 : C > T P = 2 . 51e - 7 , 20755 : A > C P = 1 . 32e - 4 , 25563 : G > T P = 9 . 6e - 23 , 29540 : G > A P = 2 . 51e - 7 ) . These variants demonstrated similar PAF enrichment within all other NYC strains as compared to non - NYC clade A2a ( 1059 : C > T P = 4 . 35e - 84 , 11916 : C > T P = 2 . 16e - 43 , 18998 : C > T P = 1 . 34e - 25 , 20755 : A > C P = 1 . 67e - 4 , 25563 : G > T P = 2 . 60e - 83 , 29540 : G > A P = 1 . 34e - 46 ) .

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

(a) Six variant alleles were found to be significantly enriched by population allelic fraction within this set of 93 WCM/NYP cases as compared with non-NYC strains of Nextstrain clade A2a (1059:C>T P = 6.26e-22, 11916:C>T P = 3.09e-9, 18998:C>T P= 2.51e-7, 20755:A>C P = 1.32e-4, 25563:G>T P = 9.6e-23, 29540:G>A P = 2.51e-7). These variants demonstrated similar PAF enrichment within all other NYC strains as compared to non-NYC clade A2a (1059:C>T P = 4.35e-84, 11916:C>T P = 2.16e-43, 18998:C>T P= 1.34e-25, 20755:A>C P = 1.67e-4, 25563:G>T P = 2.60e-83, 29540:G>A P = 1.34e-46).


( b ) ( left ) Phylogenetic tree produced by the Nextstrain analysis with clade affiliations and nodes corresponding to WCM / NYP in red and other NYC cases in green . ( right ) occurrence of the six NYC - enriched alleles and the 9 nucleotide deletion across genomes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (left) Phylogenetic tree produced by the Nextstrain analysis with clade affiliations and nodes corresponding to WCM/NYP in red and other NYC cases in green. (right) occurrence of the six NYC-enriched alleles and the 9 nucleotide deletion across genomes.


Samples were quantified by a range of viral detection methods , including LAMP ( Quantifluor ) , RNA - seq ( log10 SARS - CoV - 2 % of reads ) , and qRT - PCR ( Ct values ) to create a three - tier range of viral load for the positive samples ( right ) compared to the clinically - annotated negative samples ( class , red or grey ) . ( bottom ) The differentially expressed genes of COVID + patients compared to COVID - patients showed up - regulated ( orange ) genes as well as down - regulated ( purple ) genes .

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

Samples were quantified by a range of viral detection methods, including LAMP (Quantifluor), RNA-seq (log10 SARS-CoV-2 % of reads), and qRT-PCR (Ct values) to create a three-tier range of viral load for the positive samples (right) compared to the clinically-annotated negative samples (class, red or grey). (bottom) The differentially expressed genes of COVID+ patients compared to COVID-patients showed up-regulated (orange) genes as well as down-regulated (purple) genes.