Figure 7A ) Larval brains at 24 h ALH from wild - type control ( UAS - β - Gal RNAi ; UAS - β - Gal RNAi ) , msps RNAi control ( UAS - GFP + UAS - msps RNAi ( 21982 ) ) , and rescued animals ( UAS - klp64D + msps RNAi ( 21982 ) ) under the control of insc - Gal4 were analyzed for EdU incorporation , and larval brains were labeled with EdU and Dpn . B ) Quantification graph of EdU - negative NSCs per brain lobe for genotypes in ( A ) . n = 13 BL for contorl ; n = 13 BL for msps RNAi ; n = 20 BL for UAS - klp64D + msps RNAi . * * * * p < 0 . 0001 . D ) Larval brains at 24 h ALH from wild - type control ( UAS - β - Gal RNAi ) , RNAi control ( UAS - msps RNAi ( 21982 ) ) , and rescued animals ( g - kap3 + msps RNAi ( 21982 ) ) driven by insc - Gal4 were examined for EdU incorporation , and larval brains were stained with EdU and Dpn . E ) Quantification graph of NSCs negative for EdU incorporation per brain lobe for genotypes in ( D ) . n = 13 BL for control ; n = 15 BL for msps RNAi ; n = 19 BL for g - kap3 + msps RNAi . * * * * p < 0 . 0001 . G ) 24 h ALH Larval brains from wild - type control ( UAS - β - Gal RNAi ; UAS - β - Gal RNAi ) , RNAi control ( UAS - GFP + UAS - msps RNAi ( 21982 ) ) , and rescued animals ( UAS - E - cad7 + msps RNAi ( 21982 ) ) were assessed for EdU incorporation , and larval brains were stained with EdU and Dpn . H ) Quantification graph of NSCs negative for EdU incorporation per brain lobe for genotypes in ( G ) . n = 21 BL for control ; n = 21 BL for msps RNAi ; n = 22 BL for UAS - E - cad7 + msps RNAi . * * * * p < 0 . 0001 . J ) 24 h ALH Larval brains from wild - type control ( UAS - β - Gal RNAi ) , mutant control ( UAS - β - Gal RNAi + klp64Dk5h ) , and rescued animals ( UAS - E - cad7 + klp64Dk5h ) were processed for EdU incorporation , and larval brains were probed by with EdU , Dpn , and Mira . K ) Quantification graph of NSCs negative for EdU incorporation per brain lobe for genotypes in ( J ) . n = 10 BL for control ; n = 12 BL for klp64Dk5h ; n = 10 BL for UAS - E - cad7 + klp64Dk5h . * * * * p < 0 . 0001 ; p = 0 . 3006 ( ns ) .

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Figure 7A) Larval brains at 24 h ALH from wild-type control (UAS-β-Gal RNAi; UAS-β-Gal RNAi), msps RNAi control (UAS-GFP + UAS-msps RNAi (21982)), and rescued animals (UAS-klp64D+ msps RNAi (21982)) under the control of insc-Gal4 were analyzed for EdU incorporation, and larval brains were labeled with EdU and Dpn. B) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (A). n=13 BL for contorl; n=13 BL for msps RNAi; n=20 BL for UAS-klp64D + msps RNAi. ****p<0.0001.D) Larval brains at 24 h ALH from wild-type control (UAS-β-Gal RNAi), RNAi control (UAS-msps RNAi (21982)), and rescued animals (g-kap3 + msps RNAi (21982)) driven by insc-Gal4 were examined for EdU incorporation, and larval brains were stained with EdU and Dpn. E) Quantification graph of NSCs negative for EdU incorporation per brain lobe for genotypes in (D). n=13 BL for control; n=15 BL for msps RNAi; n=19 BL for g-kap3 + msps RNAi. ****p<0.0001. G) 24 h ALH Larval brains from wild-type control (UAS-β-Gal RNAi; UAS-β-Gal RNAi), RNAi control (UAS-GFP + UAS-msps RNAi (21982)), and rescued animals (UAS-E-cad7 + msps RNAi (21982)) were assessed for EdU incorporation, and larval brains were stained with EdU and Dpn. H) Quantification graph of NSCs negative for EdU incorporation per brain lobe for genotypes in (G). n=21 BL for control; n=21 BL for msps RNAi; n=22 BL for UAS-E-cad7 + msps RNAi. ****p<0.0001. J) 24 h ALH Larval brains from wild-type control (UAS-β-Gal RNAi), mutant control (UAS-β-Gal RNAi + klp64Dk5h), and rescued animals (UAS-E-cad7 + klp64Dk5h) were processed for EdU incorporation, and larval brains were probed by with EdU, Dpn, and Mira. K) Quantification graph of NSCs negative for EdU incorporation per brain lobe for genotypes in (J). n=10 BL for control; n=12 BL for klp64Dk5h; n=10 BL for UAS-E-cad7 + klp64Dk5h. ****p<0.0001; p=0.3006 (ns).


Figure 1LL - 37 ( 4 kD ) detection by Western blotting . Prior to loading , samples were centrifuged through 10 kD ultrafiltration membranes and the equal amount of ultrafiltrate ( 19μg / lane ) subject to immunoblotting . High - molecular - weight proteins ( GAPDH ) were not detected in ultrafiltrate . The CFU of PAO1 was measured by culturing in ultrafiltrate ( upper panel ) or retentate ( lower panel ) of mouse BALF samples from indicated mice . Initial additions of PAO1 were 1 × 103 CFU . Coculture duration , 6h . n = 3 . The bacterial CFU ( per gram ) in lungs of indicated mice with and without PAO1 infection ( 5 × 106 CFU ) for 6 , 24 and 48 hours . n = 3 . Error bars , S . E . M . Representative histological sections of indicated lungs with PAO1 infection ( 5 × 106 CFU ) for 6 hours . H & E staining . Scale bar , 1000 μm ( upper panel ) and 50 μm ( lower panel ) . Histopathological injury score of indicated mouse lungs with PAO1 infection ( 5 × 106 CFU ) for 6 h and 24 h based on blinded expert judgement . n = 3 . Error bars , S . E . M . Gene expression level of IL - 1β and IL - 6 of indicated mouse lung with PAO1 infection ( 5 × 106 CFU ) for 6 h and 24 h . n ≥ 3 . Error bars , S . E . M . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 .

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Figure 1LL-37(4 kD) detection by Western blotting. Prior to loading, samples were centrifuged through 10 kD ultrafiltration membranes and the equal amount of ultrafiltrate (19μg/lane) subject to immunoblotting. High-molecular-weight proteins (GAPDH) were not detected in ultrafiltrate.The CFU of PAO1 was measured by culturing in ultrafiltrate (upper panel) or retentate (lower panel) of mouse BALF samples from indicated mice. Initial additions of PAO1 were 1 × 103 CFU. Coculture duration, 6h. n=3.The bacterial CFU (per gram) in lungs of indicated mice with and without PAO1 infection (5 × 106 CFU) for 6, 24 and 48 hours. n=3. Error bars, S.E.M.Representative histological sections of indicated lungs with PAO1 infection (5 × 106 CFU) for 6 hours. H&E staining. Scale bar, 1000 μm (upper panel) and 50 μm (lower panel).Histopathological injury score of indicated mouse lungs with PAO1 infection (5 × 106 CFU) for 6 h and 24 h based on blinded expert judgement. n=3. Error bars, S.E.M.Gene expression level of IL-1β and IL-6 of indicated mouse lung with PAO1 infection (5 × 106 CFU) for 6 h and 24 h. n≥3. Error bars, S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.


Figure 2Heatmap showing transcriptome profile correlation values of indicated lung tissue samples before and after PAO1 infection . Histogram of selected differentially expressed genes of LL - 37 + / + mouse lung versus wild type FVB mouse lung prior to infection . Blue bars indicated genes up - regulated in wild type FVB mouse lungs , red bars indicated genes up - regulated in LL - 37 + / + mouse lungs .

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Figure 2Heatmap showing transcriptome profile correlation values of indicated lung tissue samples before and after PAO1 infection.Histogram of selected differentially expressed genes of LL-37+/+ mouse lung versus wild type FVB mouse lung prior to infection. Blue bars indicated genes up-regulated in wild type FVB mouse lungs, red bars indicated genes up-regulated in LL-37+/+ mouse lungs.


Figure 3Detection of LL - 37 expression in the engineered mDASCs by immunofluorescenceDetection of LL - 37 expression in the engineered mDASCs by real - time quantitative PCRDetection of LL - 37 expression in the engineered mDASCs by Western blotAnti - Krt5 ( red ) and anti - P63 ( green ) immunostaining of WT - and LL - 37 - mDASC colonies . Scale bar , 70 μm . Stem cell colony - forming efficiency of WT - and LL - 37 - mDASCs during 5 serial passages . n = 6 . Representative 3D organoid culture of mDASCs with expression of type I alveolar cell markers ( Aqp5 and Pdpn ) . Left panels , bright field imaging of 3D organoids . Right panels , immunofluorescence of organoid sections . Scale bar , 20 μm . Co - culture of bacteria with DASC shows the antimicrobial effects in dose - dependent manner . Initial additions of PAO1 were 0 . 1 × , 0 . 5 × and 1 × 104 CFU , respectively . Coculture duration , 6h . n = 4 . Error bars , S . E . M . MOI , Multiplicity of infection . Co - culture of bacteria with DASC shows antimicrobial effects in time - dependent manner . Initial additions of PAO1 was 1 × 104 CFU . MOI = 1 . n = 3 . Error bars , S . E . M . Preincubation of cells with anti - LL - 37 antibody , but not IgG control , significantly reduced anti - PAO1 and anti - E . coli ( J ) effects of LL - 37 - mDASCs . Initial dose of bacteria was 103 CFU .

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Figure 3Detection of LL-37 expression in the engineered mDASCs by immunofluorescenceDetection of LL-37 expression in the engineered mDASCs by real-time quantitative PCRDetection of LL-37 expression in the engineered mDASCs by Western blotAnti-Krt5 (red) and anti-P63 (green) immunostaining of WT- and LL-37-mDASC colonies. Scale bar, 70 μm.Stem cell colony-forming efficiency of WT- and LL-37-mDASCs during 5 serial passages. n=6.Representative 3D organoid culture of mDASCs with expression of type I alveolar cell markers (Aqp5 and Pdpn). Left panels, bright field imaging of 3D organoids. Right panels, immunofluorescence of organoid sections. Scale bar, 20 μm.Co-culture of bacteria with DASC shows the antimicrobial effects in dose-dependent manner. Initial additions of PAO1 were 0.1×, 0.5× and 1 × 104 CFU, respectively. Coculture duration, 6h. n=4. Error bars, S.E.M. MOI, Multiplicity of infection.Co-culture of bacteria with DASC shows antimicrobial effects in time-dependent manner. Initial additions of PAO1 was 1 × 104 CFU. MOI=1. n=3. Error bars, S.E.M.Preincubation of cells with anti-LL-37 antibody, but not IgG control, significantly reduced anti-PAO1 and anti-E.coli (J) effects of LL-37-mDASCs. Initial dose of bacteria was 103 CFU.


Figure 4Bright field and direct fluorescence images of mouse lungs following transplantation of 1 × 106 GFP - labeled WT - mDASCs ( WT - Lung ) or LL - 37 - mDASCs ( LL - 37 - Lung ) on indicated days . Scale bar , 1000 μmEngraftment ratio of transplanted cells in mouse lungs on indicated days . n = 3 . Error bars , S . E . M . * P = 0 . 0474 . Morphology of engrafted GFP - labeled cells in lung parenchyma by direct fluorescence . Blue color indicates nucleus DAPI staining . Scale bar , 200 μm . Anti - Ki67 immunofluorescence of engrafted GFP - labeled WT - and LL - 37 - mDASCs in lung parenchyma 21 days after transplantation . Scale bar , 50 μm .

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Figure 4Bright field and direct fluorescence images of mouse lungs following transplantation of 1 × 106 GFP-labeled WT-mDASCs (WT-Lung) or LL-37-mDASCs (LL-37-Lung) on indicated days. Scale bar, 1000 μmEngraftment ratio of transplanted cells in mouse lungs on indicated days. n=3. Error bars, S.E.M. *P=0.0474.Morphology of engrafted GFP-labeled cells in lung parenchyma by direct fluorescence. Blue color indicates nucleus DAPI staining. Scale bar, 200 μm.Anti-Ki67 immunofluorescence of engrafted GFP-labeled WT- and LL-37-mDASCs in lung parenchyma 21 days after transplantation. Scale bar, 50 μm.


Figure 5Distribution of engrafted GFP - labeled cells in lung parenchyma by immunofluorescence 7 days after transplantation . WT - Lung : WT - mDASCs engrafted ; LL - 37 - Lung : LL - 37 - mDASCs engrafted . Scale bar , 200 μm . Arrows showed the representative cells with overlapping fluorescence of GFP and LL - 37 . Intratracheal instillation of equal amount of PAO1 ( 5 × 106 CFU per mouse ) into WT - Lung ( WT - mDASCs engrafted ) and LL - 37 - Lung ( LL - 37 - mDASCs engrafted ) followed by bacterial CFU analysis in whole lung homogenates 6 , 24 and 48 hours after infection . n = 3 . Error bars , S . E . M . Intratracheal instillation of equal amount of PAO1 into WT - Lung and LL - 37 - Lung followed by bacterial CFU analysis in BAL fluid 6 , 24 and 48 hours after infection . n = 3 . Error bars , S . E . M . Arterial blood gas analysis of mice with WT - Lung and LL - 37 - Lung following PAO1 infection . n = 6 . Error bars , S . E . M . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 .

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Figure 5Distribution of engrafted GFP-labeled cells in lung parenchyma by immunofluorescence 7 days after transplantation. WT-Lung: WT-mDASCs engrafted; LL-37-Lung: LL-37-mDASCs engrafted. Scale bar, 200 μm. Arrows showed the representative cells with overlapping fluorescence of GFP and LL-37.Intratracheal instillation of equal amount of PAO1(5 ×106 CFU per mouse) into WT-Lung (WT-mDASCs engrafted) and LL-37-Lung (LL-37-mDASCs engrafted) followed by bacterial CFU analysis in whole lung homogenates 6, 24 and 48 hours after infection. n=3. Error bars, S.E.M. Intratracheal instillation of equal amount of PAO1 into WT-Lung and LL-37-Lung followed by bacterial CFU analysis in BAL fluid 6, 24 and 48 hours after infection. n=3. Error bars, S.E.M.Arterial blood gas analysis of mice with WT-Lung and LL-37-Lung following PAO1 infection. n=6. Error bars, S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.


Figure 6H & E staining showing histology of injured lung infected by PAO1 with WT - mDASCs or LL - 37 - mDASCs transplantation . Scale bar , 100 μm . Histopathological examination according to the lung injury scoring system based on blinded expert judgement . n = 5 mice per group . Error bars , S . E . M . CD68 immunochemistry ( brown ) in infected lung with WT - mDASCs or LL - 37 - mDASCs transplantation . Scale bar , 50 μm . Quantification of brown - stained ( CD68 + ) area by Image J software . n = 5 . Error bars , S . E . M . Gene expression level of indicated pro - inflammatory cytokines of lung infected by PAO1 with WT - mDASCs or LL - 37 - mDASCs transplantation . n = 3 . Error bars , S . E . M . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 .

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Figure 6H&E staining showing histology of injured lung infected by PAO1 with WT-mDASCs or LL-37-mDASCs transplantation. Scale bar, 100 μm. Histopathological examination according to the lung injury scoring system based on blinded expert judgement. n=5 mice per group. Error bars, S.E.M.CD68 immunochemistry (brown) in infected lung with WT-mDASCs or LL-37-mDASCs transplantation. Scale bar, 50 μm. Quantification of brown-stained (CD68+) area by Image J software. n=5. Error bars, S.E.M.Gene expression level of indicated pro-inflammatory cytokines of lung infected by PAO1 with WT-mDASCs or LL-37-mDASCs transplantation. n=3. Error bars, S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.


Figure 7Anti - Krt5 ( red ) and anti - P63 ( green ) immunofluorescence of isolated hDASCs . Scale bar , 90 μm . The mRNA expression levels of LL - 37 was measured by qPCR for WT - and LL - 37 - hDASCs . n = 5 . Error bars , S . E . M . H & E staining of native rat lung tissue and decellularized scaffold showed the absence of nuclei following decellularization . Scale bar , 40 μm . Ex vivo biomimetic culture of LL - 37 - hDASCs recellularized lungs with constant media perfusion . Direct fluorescence image showing GFP - labeled LL - 37 - hDASCs before ( left ) and 7 days after ( right ) being engrafted onto the decellularized scaffold . Scale bar , 100 μm ( right panel ) and 1000 μm ( right panel ) Immunofluorescence of recellularized lung with indicated antibodies . Blue color indicates nucleus DAPI staining . Scale bar , 50 μm . The recellularized lung by LL - 37 - hDASCs displayed growth inhibitory effect on PAO1 and E . coli . Initial dose of bacteria was 2 × 104 CFU . Culture duration , 24h . n = 6 . Error bars , S . E . M . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 .

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Figure 7Anti-Krt5 (red) and anti-P63 (green) immunofluorescence of isolated hDASCs. Scale bar, 90 μm.The mRNA expression levels of LL-37 was measured by qPCR for WT- and LL-37-hDASCs. n=5. Error bars, S.E.M.H&E staining of native rat lung tissue and decellularized scaffold showed the absence of nuclei following decellularization. Scale bar, 40 μm.Ex vivo biomimetic culture of LL-37-hDASCs recellularized lungs with constant media perfusion.Direct fluorescence image showing GFP-labeled LL-37-hDASCs before (left) and 7 days after (right) being engrafted onto the decellularized scaffold. Scale bar, 100 μm (right panel) and 1000 μm (right panel)Immunofluorescence of recellularized lung with indicated antibodies. Blue color indicates nucleus DAPI staining. Scale bar, 50 μm.The recellularized lung by LL-37-hDASCs displayed growth inhibitory effect on PAO1 and E.coli. Initial dose of bacteria was 2× 104 CFU. Culture duration, 24h. n=6. Error bars, S.E.M. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.


Figure 1 ( A , B ) Representative images ( A ) and quantification ( B ) of calcium - stable k - fiber intensity levels by immunofluorescence analysis of n ≥ 41 tubulin - stained mitotic cells of neonatal ( N ) and elderly ( 87y ) human dermal fibroblasts ( HDF ) at prometaphase and metaphase stages . Intensity levels were normalized to neonatal samples . Scale bar , 5µm . Data information : All values shown are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by two tailed Mann - Whitney test ( C ) Intra - kinetochore distance ( between Hec1 and ACA immunostainings of a kinetochore ) in n = 50 kinetochore pairs scored from 10 elderly vs . neonatal cells in prometaphase and metaphase . ( D ) Inter - kinetochore distance ( between Hec1 staining of sister kinetochores ) in n = 50 kinetochore pairs scored from 10 elderly vs . neonatal cells in prometaphase and metaphase . Data information : All values shown are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by two tailed Mann - Whitney test ( E , Live cell imaging of neonatal ( N ) and elderly ( 87y ) fibroblasts expressing H2B - GFP / α - Tubulin - mCherry treated with kinesin - 5 inhibitor ( STLC ) to induce monopolar spindles , followed by washout ( WO ) into medium with DMSO ( - ) or ZM447439 ( + ; 500nM ) . ( E ) Quantification of anaphases with lagging chromosomes in n = cells scoredF ) Live cell imaging of neonatal ( N ) and elderly ( 87y ) fibroblasts expressing H2B - GFP / α - Tubulin - mCherry treated with kinesin - 5 inhibitor ( STLC ) to induce monopolar spindles , followed by washout ( WO ) into medium with DMSO ( - ) or ZM447439 ( + ; 500nM ) . ( F ) representative movie frame series of a young and an elderly dividing cell that underwent correct ( Normal ) and incorrect ( Lagging ) chromosome segregation , respectively . Time , min : sec . Scale bars : 5µm ( images ) or 2µm ( insets ) . ( G ) Representative images of anaphases without ( top ) and with ( bottom ) mis - segregation ( MS ) , FISH - stained for three chromosome pairs ( 7 , 12 and 18 ) . Arrowheads indicate micronuclei containing centromeric signal for chromosome 18 . Scale bar , 10µm . ( H ) Percentage of anaphases with MS in neonatal ( N ) vs . elderly ( 87y ) n = cells scored by FISH analysis . Data information : All values shown are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by ( H ) chi - square test . ( I ) Western blot analysis of total MCAK protein levels in neonatal ( HDF N ) and elderly ( HDF 87y ) fibroblasts . GAPDH is shown as loading control . ( J ) Immunofluorescence analysis of MCAK levels in neonatal ( HDF N ) and elderly ( HDF 87y ) mitotic cells . Scale bar , 5µm .

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

Figure 1(A, B) Representative images (A) and quantification (B) of calcium-stable k-fiber intensity levels by immunofluorescence analysis of n≥41 tubulin-stained mitotic cells of neonatal (N) and elderly (87y) human dermal fibroblasts (HDF) at prometaphase and metaphase stages. Intensity levels were normalized to neonatal samples. Scale bar, 5µm. Data information: All values shown are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, **** p<0.0001 by two tailed Mann-Whitney test(C) Intra-kinetochore distance (between Hec1 and ACA immunostainings of a kinetochore) in n=50 kinetochore pairs scored from 10 elderly vs. neonatal cells in prometaphase and metaphase. (D) Inter-kinetochore distance (between Hec1 staining of sister kinetochores) in n=50 kinetochore pairs scored from 10 elderly vs. neonatal cells in prometaphase and metaphase. Data information: All values shown are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, **** p<0.0001 by two tailed Mann-Whitney test(E, Live cell imaging of neonatal (N) and elderly (87y) fibroblasts expressing H2B-GFP/α-Tubulin-mCherry treated with kinesin-5 inhibitor (STLC) to induce monopolar spindles, followed by washout (WO) into medium with DMSO (-) or ZM447439 (+; 500nM). (E) Quantification of anaphases with lagging chromosomes in n=cells scoredF) Live cell imaging of neonatal (N) and elderly (87y) fibroblasts expressing H2B-GFP/α-Tubulin-mCherry treated with kinesin-5 inhibitor (STLC) to induce monopolar spindles, followed by washout (WO) into medium with DMSO (-) or ZM447439 (+; 500nM). (F) representative movie frame series of a young and an elderly dividing cell that underwent correct (Normal) and incorrect (Lagging) chromosome segregation, respectively. Time, min:sec. Scale bars: 5µm (images) or 2µm (insets).(G) Representative images of anaphases without (top) and with (bottom) mis-segregation (MS), FISH-stained for three chromosome pairs (7, 12 and 18). Arrowheads indicate micronuclei containing centromeric signal for chromosome 18. Scale bar, 10µm. (H) Percentage of anaphases with MS in neonatal (N) vs. elderly (87y) n=cells scored by FISH analysis. Data information: All values shown are mean ± s.d. of at least two independent experiments. ns p>0.05, * p<0.05, ** p<0.01, **** p<0.0001 by (H) chi-square test.(I) Western blot analysis of total MCAK protein levels in neonatal (HDF N) and elderly (HDF 87y) fibroblasts. GAPDH is shown as loading control.(J) Immunofluorescence analysis of MCAK levels in neonatal (HDF N) and elderly (HDF 87y) mitotic cells. Scale bar, 5µm.


Figure 2 ( A ) GFP protein levels in FACS - sorted neonatal ( N ) and elderly ( 87y ) fibroblasts transduced with empty ( control , - ) , GFP - MCAK ( + ) and GFP - Kif2b ( + ) lentiviral plasmids . Tubulin is shown as loading control . ( B ) Mitotic duration scored by time - lapse phase - contrast microscopy of n ≥ 50 cells per condition measured from nuclear envelope breakdown ( NEB ) to anaphase onset . 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 . 0001 by two tailed ( B Mann - Whitney test . ( C , D ) Representative images ( C ) and quantification ( D ) of calcium - stable k - fiber intensity levels by immunofluorescence analysis of n ≥ 26 tubulin - stained metaphase cells transduced as indicated . Levels were normalized to the control neonatal sample . 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 . 01 , * * * * p < 0 . 0001 by two tailed D ) Mann - Whitney test . ( E ) Representative images of cytochalasin D - induced binucleated ( BN ) cells without ( top ) and with ( bottom ) mis - segregation ( MS ) , FISH - stained for three chromosome pairs ( 7 , 12 and 18 ) . Scale bar , 10µm . ( F ) Percentage of BN cells with MS in n = cells scored . ( G ) Percentage of micronuclei in n = cells analyzed . 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 . 0001 by ( F , G ) chi - square test .

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

Figure 2(A) GFP protein levels in FACS-sorted neonatal (N) and elderly (87y) fibroblasts transduced with empty (control, -), GFP-MCAK (+) and GFP-Kif2b (+) lentiviral plasmids. Tubulin is shown as loading control.(B) Mitotic duration scored by time-lapse phase-contrast microscopy of n≥50 cells per condition measured from nuclear envelope breakdown (NEB) to anaphase onset. 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<0.0001 by two tailed (B Mann-Whitney test.(C, D) Representative images (C) and quantification (D) of calcium-stable k-fiber intensity levels by immunofluorescence analysis of n≥26 tubulin-stained metaphase cells transduced as indicated. Levels were normalized to the control neonatal sample. 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<0.01, **** p<0.0001 by two tailed D) Mann-Whitney test.(E) Representative images of cytochalasin D-induced binucleated (BN) cells without (top) and with (bottom) mis-segregation (MS), FISH-stained for three chromosome pairs (7, 12 and 18). Scale bar, 10µm. (F) Percentage of BN cells with MS in n=cells scored. (G) Percentage of micronuclei in n=cells analyzed. 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<0.0001 by (F,G) chi-square test.


Figure 3 ( A ) Representative images of elderly cells scored as negative ( - ) or positive ( + ) for Cdkn1a / p21 ( cell cycle inhibitor ) and 53BP1 ( ≥ 1 foci ; DNA damage ) senescence biomarkers . Scale bar 20µm . ( B ) Percentage of n = cells staining double - positive for Cdkn1a / p21 and 53BP1 in neonatal ( N / N ) and elderly ( 75 / 87y ) human dermal fibroblasts ( HDF ) transduced with empty , GFP - MCAK or GFP - Kif2b lentiviral plasmids . ( C ) Representative images of elderly cells scored as negative ( - ) or positive ( + ) for SA - β - galactosidase ( SA - β - gal ) activity . Scale bar 20µm . ( D ) Percentage of n = cells positive for SA - β - gal in neonatal and elderly HDFs transduced with empty , GFP - MCAK or GFP - Kif2b lentiviral plasmids . Data Information : Values are mean ± s . d . of at least two independent experiments . ns p > 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed chi - square test ( B , D ) . ( F ) Total number of genes altered ( up or downregulated , as indicated ) for the comparisons shown between control and kinesin - 13 - overexpressing young ( HDF N ) and / or aged ( HDF 87y ) cells . ( G , H ) Heatmaps of differentially expressed ( G ) SASP and senescence genes , and ( H ) cGAS / STING / NF - κB - related genes of neonatal and 87y HDFs . Down and Up refer to the expected changes as reported for senescent cells Gene symbols highlighted in grey indicate genes that were not modulated by overexpression of Kinesin - 13 proteins . Z - score row color intensities indicate higher ( red ) to lower ( blue ) expression .

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

Figure 3(A) Representative images of elderly cells scored as negative (-) or positive (+) for Cdkn1a/p21 (cell cycle inhibitor) and 53BP1 (≥1 foci; DNA damage) senescence biomarkers. Scale bar 20µm. (B) Percentage of n=cells staining double-positive for Cdkn1a/p21 and 53BP1 in neonatal (N/N) and elderly (75/87y) human dermal fibroblasts (HDF) transduced with empty, GFP-MCAK or GFP-Kif2b lentiviral plasmids. (C) Representative images of elderly cells scored as negative (-) or positive (+) for SA-β-galactosidase (SA-β-gal) activity. Scale bar 20µm. (D) Percentage of n=cells positive for SA-β-gal in neonatal and elderly HDFs transduced with empty, GFP-MCAK or GFP-Kif2b lentiviral plasmids. Data Information: Values are mean ± s.d. of at least two independent experiments. ns p>0.05, *** p<0.001, **** p<0.0001 by two tailed chi-square test (B,D).(F) Total number of genes altered (up or downregulated, as indicated) for the comparisons shown between control and kinesin-13-overexpressing young (HDF N) and/or aged (HDF 87y) cells.(G, H) Heatmaps of differentially expressed (G) SASP and senescence genes, and (H) cGAS/STING/NF-κB-related genes of neonatal and 87y HDFs. Down and Up refer to the expected changes as reported for senescent cells Gene symbols highlighted in grey indicate genes that were not modulated by overexpression of Kinesin-13 proteins. Z-score row color intensities indicate higher (red) to lower (blue) expression.


Figure 4 ( A ) Mitotic duration of neonatal ( N ) and elderly ( 87y ) human dermal fibroblasts ( HDF ) treated for 24 hrs with different concentrations of UMK57 ( MCAK agonist ) . n ≥ 58 cells were analyzed per condition . For all subsequent experiments UMK57 was used at 1µM for 24 hrs . 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 ( A Mann - Whitney tests . ( B , Representative images ( B ) of calcium - stable k - fiber intensity levels in metaphase , scored by immunofluorescence analysis of n ≥ 34 tubulin - stained mitotic cells of neonatal and elderly samples treated with DMSO ( - ) and UMK57 ( + ) . Levels were normalized to neonatal DMSO - treated condition . Scale bar , 5µm . C ) quantification ( C ) of calcium - stable k - fiber intensity levels in metaphase , of neonatal and elderly samples treated with DMSO ( - ) and UMK57 ( + ) . Levels were normalized to neonatal DMSO - treated condition . 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 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by two tailed C ) Mann - Whitney tests . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Mitotic duration of neonatal (N) and elderly (87y) human dermal fibroblasts (HDF) treated for 24 hrs with different concentrations of UMK57 (MCAK agonist). n≥58 cells were analyzed per condition. For all subsequent experiments UMK57 was used at 1µM for 24 hrs. 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 (A Mann-Whitney tests.(B, Representative images (B) of calcium-stable k-fiber intensity levels in metaphase, scored by immunofluorescence analysis of n≥34 tubulin-stained mitotic cells of neonatal and elderly samples treated with DMSO (-) and UMK57 (+). Levels were normalized to neonatal DMSO-treated condition. Scale bar, 5µm.C) quantification (C) of calcium-stable k-fiber intensity levels in metaphase, of neonatal and elderly samples treated with DMSO (-) and UMK57 (+). Levels were normalized to neonatal DMSO-treated condition. 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<0.01, *** p<0.001, **** p<0.0001 by two tailed C) Mann-Whitney tests.(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.(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.


Figure 5 ( 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 . ( 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 . ( 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.(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.(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.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.


Figure 1A 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 µmD 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A 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 µmD 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


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

Figure 2A 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.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.


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

Figure 3A 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).


Figure 4A 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A 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.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.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.


Figure 5A 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 . 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 . 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 . 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 . 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 .

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

Figure 5A 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.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.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.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.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.


Figure 6Expression 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 . 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 cordE , 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 cordG , 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 cordI Tau expression in worm lysates , as detected using the human tau - specific antibody HT7 .

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

Figure 6Expression 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.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 cordE, 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 cordG, 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 cordI Tau expression in worm lysates, as detected using the human tau-specific antibody HT7.


Figure 1Survival 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Survival 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.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.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.


Figure 2ELISA of IL - 1β secretion by WT or Spata2 - KO BMDMs that were either untreated ( - ) or LPS - primed and then treated with the indicated NLRP3 inducersELISA 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 ) . 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 . 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 ) 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2ELISA of IL-1β secretion by WT or Spata2-KO BMDMs that were either untreated (-) or LPS-primed and then treated with the indicated NLRP3 inducersELISA 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).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.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)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.


Figure 3ELISA 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 ) . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3ELISA 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).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.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.


Figure 4Confocal 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 . 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 . 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 .

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Figure 4Confocal 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.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.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.


Figure 5WT 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 . 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 . 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 ) . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5WT 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.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.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).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.