B ) Quantification graph of fold changes in the number of EB1 - GFP comets in the primary protrusion of qNSCs 6 h ALH from various genotypes compared with the control in ( A ) . n = 26 NSCs for control ; n = 18 NSCs for mspsP18 ; n = 21 NSCs for mspsP18 / P . * * * * p < 0 . 0001 . C ) Quantification graph of the percentage of retrograde EB1 - GFP comets in the primary protrusion of qNSCs from the control , mspsP18 with EB1 - GFP driven by grh - Gal4 at 6 h ALH . As EB1 - GFP comets were almost lost in mspsP18 , we were unable to capture adequate EB1 - GFP comets for each individual brain to calculate the standard deviation . 38 and 41 qNSCs were analyzed in control and mspsP18 , respectively . In control qNSCs , among total 383 EB1 - GFP comets , 353 were anterograde and 30 were retrograde movements . In mspsP18 qNSCs , we only found 12 EB1 - GFP comets with 6 anterograde and 6 retrograde movements

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

B) Quantification graph of fold changes in the number of EB1-GFP comets in the primary protrusion of qNSCs 6 h ALH from various genotypes compared with the control in (A). n=26 NSCs for control; n=18 NSCs for mspsP18; n=21 NSCs for mspsP18/P. ****p<0.0001. C) Quantification graph of the percentage of retrograde EB1-GFP comets in the primary protrusion of qNSCs from the control, mspsP18 with EB1-GFP driven by grh-Gal4 at 6 h ALH. As EB1-GFP comets were almost lost in mspsP18, we were unable to capture adequate EB1-GFP comets for each individual brain to calculate the standard deviation. 38 and 41 qNSCs were analyzed in control and mspsP18, respectively. In control qNSCs, among total 383 EB1-GFP comets, 353 were anterograde and 30 were retrograde movements. In mspsP18 qNSCs, we only found 12 EB1-GFP comets with 6 anterograde and 6 retrograde movements


D ) Larval brains at 24 h ALH from control ( grh - Gal4 UAS - CD8 - GFP + UAS - nod - lacZ ) and msps810 , UAS - nod - lacZ under the control of grh - Gal4 UAS - CD8 - GFP were labeled with β - Gal , Dpn , and GFP . Quiescent NSCs in the central brain were shown . Arrows point at the localization of Nod - lacZ in qNSCs . E ) Quantification graph of Nod - β - Gal localization in qNSCs from genotypes in ( D ) . n = 55 NSCs for control ; n = 45 NSCs for msps810 . * * * * p < 0 . 0001

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

D) Larval brains at 24 h ALH from control (grh-Gal4 UAS-CD8-GFP + UAS-nod-lacZ) and msps810,UAS-nod-lacZ under the control of grh-Gal4 UAS-CD8-GFP were labeled with β-Gal, Dpn, and GFP. Quiescent NSCs in the central brain were shown. Arrows point at the localization of Nod-lacZ in qNSCs. E) Quantification graph of Nod-β-Gal localization in qNSCs from genotypes in (D). n=55 NSCs for control; n=45 NSCs for msps810. ****p<0.0001


F ) Larval brains at 16 h ALH from control ( grh - Gal4 UAS - CD8 - GFP ; UAS - dicer2 + UAS - nod - lacZ ) and msps RNAi , UAS - nod - lacZ ( VDRC # 21982 ) with grh - Gal4 UAS - CD8 - GFP ; UAS - dicer2 were labeled with β - Gal , Dpn , and GFP . Quiescent NSCs in the central brain are shown . Arrows indicate the localization of Nod - lacZ in qNSCs . G ) Quantification graph of Nod - β - Gal localization in qNSCs from genotypes in ( F ) . n = 60 NSCs for control ; n = 68 NSCs for msps RNAi . * * * * p < 0 . 0001

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

F) Larval brains at 16 h ALH from control (grh-Gal4 UAS-CD8-GFP; UAS-dicer2 + UAS -nod-lacZ) and msps RNAi, UAS-nod-lacZ (VDRC#21982) with grh-Gal4 UAS-CD8-GFP; UAS-dicer2 were labeled with β-Gal, Dpn, and GFP. Quiescent NSCs in the central brain are shown. Arrows indicate the localization of Nod-lacZ in qNSCs. G) Quantification graph of Nod-β-Gal localization in qNSCs from genotypes in (F). n=60 NSCs for control; n=68 NSCs for msps RNAi. ****p<0.0001


H ) Larval brains at 24 h ALH from wild - type control ( grh - Gal4 > UAS - CD8 - GFP ) , msps924 , and mspsP18 expressing grh > CD8 - GFP were labeled with Dpn and GFP . The arrow indicates the second protrusion in msps924 . I ) Quantification graph of the thickness of the primary protrusion of qNSCs from wild - type , msps924 , and mspsP18 expressing grh > CD8 - GFP . The thickness was measured at the middle point of the primary protrusion . n = 18 NSCs for control ; n = 52 NSCs for msps924 ; n = 32 NSCs for mspsP18 . * * * * p < 0 . 0001

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

H) Larval brains at 24 h ALH from wild-type control (grh-Gal4>UAS-CD8-GFP), msps924, and mspsP18 expressing grh>CD8-GFP were labeled with Dpn and GFP. The arrow indicates the second protrusion in msps924. I) Quantification graph of the thickness of the primary protrusion of qNSCs from wild-type, msps924, and mspsP18 expressing grh>CD8-GFP. The thickness was measured at the middle point of the primary protrusion. n=18 NSCs for control; n=52 NSCs for msps924; n=32 NSCs for mspsP18. **** p<0.0001


L ) Larval brains at 24 h ALH from control ( grh - Gal4 UAS - CD8 - GFP + UAS - nod - lacZ ) and tacc74 , UAS - nod - lacZ under the control of grh - Gal4 UAS - CD8 - GFP ; tacc59 were labeled with β - Gal , Dpn , and GFP . Quiescent NSCs in the central brain are shown . M ) Quantification graph of Nod - β - Gal localization in qNSCs from genotypes in ( L ) . n = 51 NSCs for control ; n = 65 NSCs for tacc59 / 74 . * * p = 0 . 0051

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

L) Larval brains at 24 h ALH from control (grh-Gal4 UAS-CD8 -GFP + UAS-nod-lacZ) and tacc74,UAS-nod-lacZ under the control of grh-Gal4 UAS-CD8-GFP; tacc59 were labeled with β-Gal, Dpn, and GFP. Quiescent NSCs in the central brain are shown. M) Quantification graph of Nod-β-Gal localization in qNSCs from genotypes in (L). n=51 NSCs for control; n=65 NSCs for tacc59/74. **p=0.0051


A ) Larval VNCs at 24 h ALH from the control ( grh - Gal4 UAS - CD8 - GFP ) and msps810 with grh - Gal4 UAS - CD8 - GFP were labeled with E - cadherin , Dpn , and GFP . NSC - neuropil contact were marked by dashed lines , and NSC - neuropil contact points were circled . B ) Quantification of E - cadherin basal localization at NSC - neuropil contact sites in qNSCs from genotypes in ( A ) . " No E - cad " means absent or strongly reduced E - cad observed at the basal region of qNSCs . n = 32 NSCs for control ; n = 61 NSCs for msps810 . * * p = 0 . 0028 . C ) Quantification graph of the basal E - cad intensity at NSC - neuropil contact sites by normalizing against Dpn intensity in qNSCs from genotypes in ( A ) . n = 34 NSCs for control ; n = 40 NSCs for msps810 . * * * * p < 0 . 0001 .

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

A) Larval VNCs at 24 h ALH from the control (grh-Gal4 UAS-CD8-GFP) and msps810 with grh-Gal4 UAS-CD8-GFP were labeled with E-cadherin, Dpn, and GFP. NSC-neuropil contact were marked by dashed lines, and NSC-neuropil contact points were circled. B) Quantification of E-cadherin basal localization at NSC-neuropil contact sites in qNSCs from genotypes in (A). "No E-cad" means absent or strongly reduced E-cad observed at the basal region of qNSCs. n=32 NSCs for control; n=61 NSCs for msps810. **p=0.0028. C) Quantification graph of the basal E-cad intensity at NSC-neuropil contact sites by normalizing against Dpn intensity in qNSCs from genotypes in (A). n=34 NSCs for control; n=40 NSCs for msps810. ****p<0.0001.


D ) Larval VNCs at 24 h ALH from control ( grh - Gal4 UAS - CD8 - GFP ) and msps924 with grh - Gal4 UAS - CD8 - GFP were labeled with E - cadherin , Dpn , and GFP . NSC - neuropil contact were marked by dashed lines , and NSC - neuropil contact sites were circled . E ) Quantification of E - cadherin basal localization at NSC - neuropil contact sites in qNSCs from genotypes in ( D ) . " No E - cad " means absent or strongly reduced E - cad observed at the basal region of qNSCs . n = 77 NSCs for control ; n = 30 NSCs for msps924 . * * p = 0 . 0080 . F ) Quantification graph of the basal E - cad intensity at NSC - neuropil contact sites by normalizing against Dpn intensity in qNSCs from genotypes in ( D ) . n = 60 NSCs for control ; n = 37 NSCs for msps924 . * * * * p < 0 . 0001 .

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

D) Larval VNCs at 24 h ALH from control (grh-Gal4 UAS-CD8-GFP) and msps924 with grh-Gal4 UAS-CD8-GFP were labeled with E-cadherin, Dpn, and GFP. NSC-neuropil contact were marked by dashed lines, and NSC-neuropil contact sites were circled. E) Quantification of E-cadherin basal localization at NSC-neuropil contact sites in qNSCs from genotypes in (D). "No E-cad" means absent or strongly reduced E-cad observed at the basal region of qNSCs. n=77 NSCs for control; n=30 NSCs for msps924. **p=0.0080. F) Quantification graph of the basal E-cad intensity at NSC-neuropil contact sites by normalizing against Dpn intensity in qNSCs from genotypes in (D). n=60 NSCs for control; n=37 NSCs for msps924. ****p<0.0001.


H ) Larval brains at 16 h ALH from control t - GRASP under the control of single driver grh - Gal4 or nSyb - QF2 ( Q - system ) and t - GRASP under the control of driver pairs grh - Gal4 and nSyb - QF2 ( Q - system ) were analyzed for E - Cad , GFP ( reconstituted GFP ) , and Dpn . Arrows pointing at the cell body of qNSCs on the two different focal planes . NSC - neuropil contact sites were circled by dashed lines .

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

H) Larval brains at 16 h ALH from control t-GRASP under the control of single driver grh-Gal4 or nSyb-QF2 (Q-system) and t-GRASP under the control of driver pairs grh-Gal4 and nSyb-QF2 (Q-system) were analyzed for E-Cad, GFP (reconstituted GFP), and Dpn. Arrows pointing at the cell body of qNSCs on the two different focal planes. NSC-neuropil contact sites were circled by dashed lines.


A ) Larval brains at 16 h ALH from wild - type ( yw ) and E - cadR69 were analyzed for EdU incorporation , and larval brains were labeled with EdU , Dpn and Mira

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

A) Larval brains at 16 h ALH from wild-type (yw) and E-cadR69 were analyzed for EdU incorporation, and larval brains were labeled with EdU, Dpn and Mira


E ) Larval brains at 24 h ALH from the wild - type control ( yw ) and klp64Dk5h / Df , kap3V6 and kap3 V6 ; g - kap3 # 11 were stained with EdU , Dpn , and

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

E) Larval brains at 24 h ALH from the wild-type control (yw) and klp64Dk5h/Df, kap3V6 and kap3 V6; g-kap3#11 were stained with EdU, Dpn, and Mira


I ) Larval brains at 24h ALH from wild - type control ( yw ) , klp64Dk5 / Df , and kap3V5 were analyzed E - cad at the NSC - neuropil contact sites , and NSCs were labeled with Dpn and Msps

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

I) Larval brains at 24h ALH from wild-type control (yw), klp64Dk5/Df, and kap3V5 were analyzed E-cad at the NSC-neuropil contact sites, and NSCs were labeled with Dpn and Msps


A ) 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 .

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

A) 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 .

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

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 .

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

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 ) .

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

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).


LL - 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 .

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

LL-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 .

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

The 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 .

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

The 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 ) .

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

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 .

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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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.


Heatmap showing transcriptome profile correlation values of indicated lung tissue samples before and after PAO1 infection .

Heatmap 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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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.


Detection of LL - 37 expression in the engineered mDASCs by

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Detection of LL-37 expression in the engineered mDASCs by immunofluorescence


Detection of LL - 37 expression in the engineered mDASCs by real - time quantitative

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Detection of LL-37 expression in the engineered mDASCs by real-time quantitative PCR


Detection of LL - 37 expression in the engineered mDASCs by Western

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Detection of LL-37 expression in the engineered mDASCs by Western blot


Anti - Krt5 ( red ) and anti - P63 ( green ) immunostaining of WT - and LL - 37 - mDASC colonies . Scale bar , 70 μm .

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Anti-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 .

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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 .

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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 .

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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 .

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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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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.


Bright 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

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Bright 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 μm


Engraftment ratio of transplanted cells in mouse lungs on indicated days . n = 3 . Error bars , S . E . M . * P = 0 . 0474 .

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Engraftment 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 .

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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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Anti-Ki67 immunofluorescence of engrafted GFP-labeled WT- and LL-37-mDASCs in lung parenchyma 21 days after transplantation. Scale bar, 50 μm.


Distribution 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 .

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Distribution 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 .

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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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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.


H & 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 .

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H&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 .

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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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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.


Anti - Krt5 ( red ) and anti - P63 ( green ) immunofluorescence of isolated hDASCs . Scale bar , 90 μm .

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Anti-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 .

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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 .

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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 .

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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 )

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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 .

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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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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.


( 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

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(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

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(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

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(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 scored


F ) 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 ) .

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F) 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 .

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(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 .

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(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 .

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(J) Immunofluorescence analysis of MCAK levels in neonatal (HDF N) and elderly (HDF 87y) mitotic cells. Scale bar, 5µm.


( 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 .

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(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 .

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(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 .

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(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) 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.


( 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 ) .

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

(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 .

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

(F) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G, H) 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.


( 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 .

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

(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 .

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

(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 .

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

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 .

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

A 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 .

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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 .

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


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

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

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


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

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

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


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

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


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

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


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

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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 .

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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 .

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


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

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


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

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

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


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

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

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


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

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

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 .

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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 .

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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 .

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


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

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

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 .

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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 .

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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 .

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


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

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


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

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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

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


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

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


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

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


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

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


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

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


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

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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 .

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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 .

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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 .

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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.