( a ) HCT116 were treated with serum starvation for 24 h ( upper panels ) or 0 . 5 mM H2O2 for 12 h ( lower panels ) ; were extracted for co - immunoprecipitation with anti - FoxO1 probed with anti - acetylated lysine ( Ace - lyn ) . Both bottom panels display FoxO1 . IP , immunoprecipitation .

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(a) HCT116 cells were treated with serum starvation for 24 h (upper panels) or 0.5 mM H2O2 for 12 h (lower panels); cytosolic proteins were extracted for co-immunoprecipitation with anti-FoxO1 and probed with anti-acetylated lysine (Ace-lyn). Both bottom panels display cytosolic FoxO1. IP, immunoprecipitation.


( b ) HCT116 were serum - starved for 24 h were extracted for co - immunoprecipitation with anti - FoxO1 or anti - SIRT2 , followed by probing with anti - SIRT2 or anti - FoxO1 , respectively ( top middle panels ) . FoxO1 ( WT ) was also transfected into H1299 was extracted , followed by co - immunoprecipitation with anti - FoxO1 probing with anti - SIRT2 ( bottom panels ) .

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(b) HCT116 cells were serum-starved for 24 h and the cytosolic proteins were extracted for co-immunoprecipitation with anti-FoxO1 or anti-SIRT2, followed by probing with anti-SIRT2 or anti-FoxO1, respectively (top and middle panels). FoxO1(WT) was also transfected into H1299 cells and the cytosolic protein was extracted, followed by co-immunoprecipitation with anti-FoxO1 and probing with anti-SIRT2 (bottom panels).


( c ) HCT116 were transfected with SIRT2 RNAi plasmid or an empty control plasmid . At 48 h after transfection , lysate was extracted for probing with SIRT2 or FoxO1 ( upper panels ) , or for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti - acetylated lysine ( lower panels ) . IB , immunoblot .

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(c) HCT116 cells were transfected with SIRT2 RNAi plasmid or an empty control plasmid. At 48 h after transfection, the cytosolic lysate was extracted for probing with SIRT2 or FoxO1 (upper panels), or for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-acetylated lysine (lower panels). IB, immunoblot.


( d ) HCT116 were treated with AGK2 for 24 h at different doses , as indicated . lysate was extracted for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti - acetylated lysine .

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(d) HCT116 cells were treated with AGK2 for 24 h at different doses, as indicated. The cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-acetylated lysine.


( a , b ) H1299 were transfected with an empty control plasmid or with Flag - FoxO1 ( WT ) plasmid . At 24 h after transfection , lysate was extracted for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti - mTOR , Beclin 1 , Vps34 or Atg1 ( a ) with Atg3 , Atg4 , Atg10 , Atg7 or LC3 ( b ) .

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(a, b) H1299 cells were transfected with an empty control plasmid or with the Flag-FoxO1(WT) plasmid. At 24 h after transfection, the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-mTOR, Beclin 1, Vps34 or Atg1 (a) and with Atg3, Atg4, Atg10, Atg7 or LC3 (b).


( c ) H1299 were co - transfected with Myc - Atg7 , with Flag - FoxO1 ( WT ) or with an empty control plasmid . At 24 h after transfection , lysate was extracted for Flag with anti - Flag , followed by probing with anti - Myc .

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(c) H1299 cells were co-transfected with Myc-Atg7, with Flag-FoxO1(WT) or with an empty control plasmid. At 24 h after transfection, the cytosolic lysate was extracted for Flag with anti-Flag, followed by probing with anti-Myc.


HCT116 were serum - starved for 24 h ( d ) lysate was extracted for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti -

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HCT116 cells were serum-starved for 24 h (d)and the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-Atg7


( e ) Different Myc - tagged FoxO1 fragments Flag - tagged Atg7 were co - transfected into H1299 , lysate was extracted for co - immunoprecipitation with anti - Myc , followed by probing with anti - Flag .

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(e) Different Myc-tagged FoxO1 fragments and Flag-tagged Atg7 were co-transfected into H1299 cells, and the cytosolic lysate was extracted for co-immunoprecipitation with anti-Myc, followed by probing with anti-Flag.


( f ) HCT116 were transfected with an empty control plasmid , an SIRT2 ( WT ) - expressing plasmid or an SIRT2 ( 168A ) - expressing plasmid . At 24 h after transfection , were serum - starved for 24 h , lysate was extracted for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti - Atg7 .

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(f) HCT116 cells were transfected with an empty control plasmid, an SIRT2(WT)-expressing plasmid or an SIRT2(168A)-expressing plasmid. At 24 h after transfection, cells were serum-starved for 24 h, and the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-Atg7.


HCT116 weretreated with AGK2 for 24 h ( g ) , lysate was extracted for co - immunoprecipitation with anti - FoxO1 , followed by probing with anti -

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HCT116 cells weretreated with AGK2 for 24 h (g), and the cytosolic lysate was extracted for co-immunoprecipitation with anti-FoxO1, followed by probing with anti-Atg7


( a ) HCT116 or stable FoxO1 - RNAi HCT116 were treated with a non - specific RNAi control or SIRT2 - RNAi , LC3 - II accumulation or p62 degradation was detected in presence or absence of E64 pepstatin A .

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(a) HCT116 cells or stable FoxO1-RNAi HCT116 cells were treated with a non-specific RNAi control or SIRT2-RNAi, and LC3-II accumulation or p62 degradation was detected in the presence or absence of E64 and pepstatin A.


( b ) HCT116 or stable FoxO1 - RNAi HCT116 were treated with 10 μM AGK2 for 24 h , in presence or absence of E64 pepstatin A . Changes in FoxO1 , p62 LC3 - II were determined by western blotting ( upper panels ) , or formation of LC3 punctate signals was observed ( lower panels ) . HCT116 were treated with 10 μM AGK2 for 24 h formation of LC3 punctate signals was observed . of of LC3 punctate to overall HCT116 is shown in lower right panel . Data are means ± s . d . ( n = 3 ) .

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(b) HCT116 cells or stable FoxO1-RNAi HCT116 cells were treated with 10 μM AGK2 for 24 h, in the presence or absence of E64 and pepstatin A. Changes in FoxO1, p62 and LC3-II were determined by western blotting (upper panels), or the formation of LC3 punctate signals was observed (lower panels). HCT116 cells were treated with 10 μM AGK2 for 24 h and the formation of LC3 punctate signals was observed. Quantification of the ratio of LC3 punctate cells to overall HCT116 cells is shown in the lower right panel. Data are means ± s.d. (n = 3).


( c ) H1299 were transfected with Flag - FoxO1 ( WT ) , SIRT2 ( WT ) or SIRT2 ( 168A ) . At 24 h after transfection , lysate was extracted immunoblotted with anti - Flag , anti - SIRT2 , anti - LC3 , anti - p62 or anti - β - actin . .

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(c) H1299 cells were transfected with Flag-FoxO1(WT), SIRT2(WT) or SIRT2(168A). At 24 h after transfection, the cell lysate was extracted and immunoblotted with anti-Flag, anti-SIRT2, anti-LC3, anti-p62 or anti-β-actin..


( d ) HCT116 were transfected with a control plasmid , a SIRT2 ( WT ) - expressing plasmid or a SIRT2 ( 168A ) - expressing plasmid . At 24 h after transfection , were subjected to serum starvation for 24 h . lysate was extracted immunoblotted with anti - SIRT2 , anti - LC3 , anti - p62 or anti - β - actin .

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(d) HCT116 cells were transfected with a control plasmid, a SIRT2(WT)-expressing plasmid or a SIRT2(168A)-expressing plasmid. At 24 h after transfection, the cells were subjected to serum starvation for 24 h. The cell lysate was extracted and immunoblotted with anti-SIRT2, anti-LC3, anti-p62 or anti-β-actin.


( e ) H1299 were transfected with Myc - Atg7 , Flag - FoxO1 ( WT ) or mutated Flag - FoxO1 ( KR ) . At 24 h after transfection , lysate was extracted for co - immunoprecipitation with anti - Myc , followed by probing with anti - Flag .

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(e) H1299 cells were transfected with Myc-Atg7, Flag-FoxO1(WT) or mutated Flag-FoxO1(KR). At 24 h after transfection, the cytosolic lysate was extracted for co-immunoprecipitation with anti-Myc, followed by probing with anti-Flag.


( f ) H1299 were transfected with a control plasmid , Flag - FoxO1 ( WT ) , Flag - FoxO1 ( K262R ) , Flag - FoxO1 ( K265R ) , Flag - FoxO1 ( K274R ) or Flag - FoxO1 ( KR ) . At 24h after transfection in presence or absence of E64 pepstatin A immunoblotted , lysate was extracted immunoblotted with anti - Flag , anti - LC3 , anti - p62 or anti - β - actin .

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

(f) H1299 cells were transfected with a control plasmid, Flag-FoxO1(WT), Flag-FoxO1(K262R), Flag-FoxO1(K265R), Flag-FoxO1(K274R) or Flag-FoxO1(KR). At 24h after transfection in the presence or absence of E64 and pepstatin A and immunoblotted, the cell lysate was extracted and immunoblotted with anti-Flag, anti-LC3, anti-p62 or anti-β-actin.


( g ) Left : H1299 were co - transfected with GFP - LC3 , Flag - FoxO1 ( WT ) or Flag - FoxO1 ( KR ) . formation of GFP - LC3 - labelled punctate signals was observed 24 h after transfection . Right : of of GFP - LC3 - labelled punctate to overall survived H1299 . Data in g are means ± s . d . ( n = 3 ) . Scale bars , 25 μm ( b ) , 10 μm ( g ) .

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(g) Left: H1299 cells were co-transfected with GFP-LC3, Flag-FoxO1(WT) or Flag-FoxO1(KR). The formation of GFP-LC3-labelled punctate signals was observed 24 h after transfection. Right: quantification of the ratio of GFP-LC3-labelled punctate cells to overall survived H1299 cells. Data in g are means ± s.d. (n = 3). Scale bars, 25 μm (b), 10 μm (g).


( a ) H1299 were transfected with a control plasmid , Flag - FoxO1 ( WT ) , Flag - FoxO1 ( 3A ) , Flag - FoxO1 ( ΔDB ) or Flag - FoxO1 ( ΔDB3A ) , in presence or absence of 10 mM 3 - MA ( upper panel ) or in presence or absence of 50 μM Z - VAD - fmk ( lower panel ) ; were stained with propidium iodide ( PI ) 48 h after transfection . PI - stained ( dead ) were counted by flow cytometry loss was calculated .

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(a) H1299 cells were transfected with a control plasmid, Flag-FoxO1(WT), Flag-FoxO1(3A), Flag-FoxO1(ΔDB) or Flag-FoxO1(ΔDB3A), in the presence or absence of 10 mM 3-MA (upper panel) or in the presence or absence of 50 μM Z-VAD-fmk (lower panel); cells were stained with propidium iodide (PI) 48 h after transfection. PI-stained (dead) cells were counted by flow cytometry and the percentage cell loss was calculated.


( b ) FoxO1 ( WT ) or Flag - FoxO1 ( ΔDB ) was transfected into H1299 ; 48 h later autophagic vesicles were observed under an electron microscope . Enlarged ( right ) show clearer autophagic vesicles indicated by arrows . Scale bars , 2 μm .

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(b) FoxO1(WT) or Flag-FoxO1(ΔDB) was transfected into H1299 cells; 48 h later the autophagic vesicles were observed under an electron microscope. Enlarged images (right) show clearer autophagic vesicles indicated by arrows. Scale bars, 2 μm.


( c ) 48 h with or without Atg5 RNAi were transfected with FoxO1 ( WT ) , was determined by with PI at 48 h after transfection . Data in c are means ± s . d . ( n = 3 ) .

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(c) 48 h cells with or without Atg5 RNAi were transfected with FoxO1(WT), and cell death was determined by staining with PI at 48 h after transfection. Data in c are means ± s.d. (n = 3).


( d ) stable Atg5 knockdown H1299 was further transfected with a control plasmid , Flag - FoxO1 ( WT ) , or Flag - FoxO1 ( ΔDB ) , loss was quantified by flow cytometry at 48 h after transfection . Data in d are means ± s . d . ( n = 3 ) .

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(d) The stable Atg5 knockdown H1299 cells was further transfected with a control plasmid, Flag-FoxO1(WT), or Flag-FoxO1(ΔDB), and the percentage cell loss was quantified by flow cytometry at 48 h after transfection. Data in d are means ± s.d. (n = 3).


( a ) Stable H1299 with or without Atg7 knockdown were transfected with a control plasmid ( group 1 ) , Flag - FoxO1 ( ΔDB ) ( group 2 ) or Flag - FoxO1 ( ΔDB - 3A ) ( group 3 ) ; 107 were then injected into 18 nude mice , with 6 mice per group . Seven later mice were killed tumours were weighed analysed ( bottom graph ) . Group 1 : tumour weights were 1 . 22 ± 0 . 78 g ( without Atg7 RNAi ) 1 . 31 ± 0 . 43 g ( with Atg7 RNAi ) , P = 0 . 426 . Group 2 : tumour weights were 0 . 49 ± 0 . 18 g ( without Atg7 RNAi ) 0 . 80 ± 0 . 20 g ( with Atg7 RNAi ) , P = 0 . 017 . Group 3 : tumour weights were 1 . 00 ± 0 . 40 g ( without Atg7 RNAi ) 1 . 10 ± 0 . 36 g ( with Atg7 RNAi ) , P = 0 . 500 . Data are means ± s . d . ( n = 6 ) .

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(a) Stable H1299 cells with or without Atg7 knockdown were transfected with a control plasmid (group 1), Flag-FoxO1(ΔDB) (group 2) or Flag-FoxO1(ΔDB-3A) (group 3); 107 cells were then injected into 18 nude mice, with 6 mice per group. Seven weeks later the mice were killed and the tumours were weighed and analysed (bottom graph). Group 1: tumour weights were 1.22 ± 0.78 g (without Atg7 RNAi) and 1.31 ± 0.43 g (with Atg7 RNAi), P = 0.426. Group 2: tumour weights were 0.49 ± 0.18 g (without Atg7 RNAi) and 0.80 ± 0.20 g (with Atg7 RNAi), P = 0.017. Group 3: tumour weights were 1.00 ± 0.40 g (without Atg7 RNAi) and 1.10 ± 0.36 g (with Atg7 RNAi), P = 0.500. Data are means ± s.d. (n = 6).


( b ) Expression of FoxO1 Atg7 conjugate in mice tumours were determined by western blotting .

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(b) Expression levels of FoxO1 and Atg7 conjugate in mice tumours were determined by western blotting.


( c ) HCT116 ( 107 ) or stable FoxO1 - knockdown HCT116 ( 107 ) were injected into left or right hind legs of three nude mice . Six later mice were killed tumours were weighed analysed ( bottom graph ) . Tumour weights were 0 . 08 ± 0 . 10 g ( without FoxO1 RNAi ) 0 . 48 ± 0 . 06 g ( with FoxO1 RNAi ) , P = 0 . 020 . Data are means ± sd . n = 3 .

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(c) HCT116 cells (107) or stable FoxO1-knockdown HCT116 cells (107) were injected into the left or right hind legs of three nude mice. Six weeks later the mice were killed and the tumours were weighed and analysed (bottom graph). Tumour weights were 0.08 ± 0.10 g (without FoxO1 RNAi) and 0.48 ± 0.06 g (with FoxO1 RNAi), P = 0.020. Data are means ± sd. n = 3.


( d ) was extracted from one paired colon tumour ( T ) or adjacent normal tissue ( N ) from one , western blotting was performed to detect changes in FoxO1 or p62 .

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(d) Protein was extracted from one paired colon tumour (T) or adjacent normal tissue (N) from one patient, and western blotting was performed to detect changes in FoxO1 or p62.


( e ) were extracted from 16 paired samples from for western blotting with anti - FoxO1 or with anti - p62 antibody . bands were scanned with a phosphorimager relative band was normalized to β - actin band . FoxO1 of normal tissue , 0 . 37 ; FoxO1 of tumour tissue , 0 . 10 ; P = 0 . 001 , two - sided t - . p62 of normal tissue , 0 . 32 ; p62 of tumour tissue , 1 . 00 ; P = 0 . 009 , two - sided t - . Data in e are means ± s . d . ( n = 16 ) .

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(e) Proteins were extracted from 16 paired samples from patients for western blotting with anti-FoxO1 or with anti-p62 antibody. Protein bands were scanned with a phosphorimager and relative band intensity was normalized to the β-actin band. FoxO1 of normal tissue, 0.37; FoxO1 of tumour tissue, 0.10; P = 0.001, two-sided t-test. p62 of normal tissue, 0.32; p62 of tumour tissue, 1.00; P = 0.009, two-sided t-test. Data in e are means ± s.d. (n = 16).


( B ) Time course of SUMO - 1 modification of His - tagged HTTex1p ( 25Q ) - purified ( WT , K6R , K9R , K6 , 9R , K6 , 9 , 15R , S13 , 16D ) was performed in vitro . SUMOylation was visualized using anti - His antibody . WT HTTex1p is SUMOylated within 16 min , K6R or K9R mutations delay SUMOylation , combined mutations ( K6 , 9R or K6 , 9 , 15R ) greatly reduce SUMOylation . Mutations that mimic phosphorylation ( S13 , 16D ) alter kinetics of SUMO - 1 modification with SUMO modification observed beginning by 4 min .

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(B) Time course of SUMO-1 modification of His-tagged HTTex1p (25Q)-purified proteins (WT, K6R, K9R, K6,9R, K6,9,15R, and S13,16D) was performed in vitro. SUMOylation was visualized using anti-His antibody. WT HTTex1p is SUMOylated within 16 min, K6R or K9R mutations delay SUMOylation, and combined mutations (K6,9R or K6,9,15R) greatly reduce SUMOylation. Mutations that mimic phosphorylation (S13,16D) alter kinetics of SUMO-1 modification with SUMO modification observed beginning by 4 min.


( A ) qRT - PCR of SUMO - modifying enzymes in cortex striata of WT mice at 12 . Relative expression for SUMO enzymes is normalized to mouse β - actin .

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(A) qRT-PCR analysis of SUMO-modifying proteins and enzymes in cortex and striata of WT mice at 12 weeks. Relative expression for all the SUMO enzymes is normalized to mouse β-actin.


( B C ) qRT - PCR of SUMO mRNAs from 12 - week - old WT R6 / 2 mouse cortex ( B ) striatum ( C ) . SUMO enzyme mRNAs are differentially expressed in R6 / 2 versus control with statistically significant increases in SENP1 ( p = 0 . 01 ) , SENP3 ( p = 0 . 02 ) , SUMO - 1 ( p = 0 . 003 ) in cortex SENP1 ( p = 0 . 02 ) , SENP6 ( p = 0 . 04 ) , PIAS3 ( p = 0 . 007 ) , SUMO - 1 ( p = 0 . 02 ) , SUMO - 2 ( p = 0 . 02 ) in striatum . Samples were analyzed in quadruplicate normalized to mouse β - actin . Data are shown as R6 / 2 expression relative to WT set at 1 for each enzyme with ± SD ( n = 4 ) . * p < 0 . 05 . n . s . , not significant .

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(B and C) qRT-PCR of SUMO mRNAs from 12-week-old WT and R6/2 mouse cortex (B) and striatum (C). SUMO enzyme mRNAs are differentially expressed in R6/2 versus control with statistically significant increases in SENP1 (p = 0.01), SENP3 (p = 0.02), and SUMO-1 (p = 0.003) in cortex and SENP1 (p = 0.02), SENP6 (p = 0.04), PIAS3 (p = 0.007), SUMO-1 (p = 0.02), and SUMO-2 (p = 0.02) in striatum. Samples were analyzed in quadruplicate and normalized to mouse β-actin. Data are shown as R6/2 expression relative to WT levels set at 1 for each enzyme with ± SD (n = 4). *p < 0.05. n.s., not significant.


( D E ) Western blot of SUMO - 2 in 14 - week R6 / 2 cortex ( D ) striatum ( E ) versus aged - matched controls . SUMO - 2 is upregulated in R6 / 2 striatum versus control ( p = 0 . 026 ; n = 4 ) . is normalized to α - tubulin quantitated using ImageJ .

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(D and E) Western blot analysis of SUMO-2 in 14-week R6/2 cortex (D) and striatum (E) versus aged-matched controls. SUMO-2 is upregulated in R6/2 striatum versus control (p = 0.026; n = 4). Protein is normalized to α-tubulin and quantitated using ImageJ.


( A ) HeLa transfected with His - tagged HTTex1p ( 46Q ) or 46QP - K6 , 9 , 15R ( 3R ) along with GFP - SUMO - 1 or GFP - SUMO - 2 , lysed under denaturing conditions , nickel purified ( Ni - NTA ) . Unmodified HTT - 46Q is indicated by arrow SUMO - modified HTT by boxed region . lysine mutant ( 3R ) serves as a negative control . Ni - NTA represents nickel - purified His - tagged HTT , WC TCA represents 10 % of whole - lysate expression of HTT myc - actin ( transfection control ) . HTT is modified by SUMO - 1 ( left ) SUMO - 2 ( right ) .

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(A) HeLa cells transfected with His-tagged HTTex1p (46Q) or 46QP-K6,9,15R (3R) along with GFP-SUMO-1 or GFP-SUMO-2, lysed under denaturing conditions, and nickel purified (Ni-NTA). Unmodified HTT-46Q is indicated by the arrow and SUMO-modified HTT by the boxed region. The lysine mutant (3R) serves as a negative control. Ni-NTA represents nickel-purified His-tagged HTT, and WC TCA represents 10% of the whole-cell lysate expression of HTT and myc-actin (transfection control). HTT is modified by SUMO-1 (left) and SUMO-2 (right).


( B ) SUMO isopeptidases ( SENP1 , SENP2 , SENP3 , SENP5 , SENP6 ) modulate SUMO - modified HTT when overexpressed together with HTT ( 46QP - H4 or 3R ) SUMO - 1 ( GFP - SUMO - 1 ) . SENP1 , SENP2 , SENP6 decrease HTT SUMOylation . Graph depicts quantitation of western blot using Odyssey Infrared Imaging System ( LI - COR ) to calculate of HTT purified versus HTT modified by SUMO multiplied by 100 .

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(B) SUMO isopeptidases (SENP1, SENP2, SENP3, SENP5, and SENP6) modulate SUMO-modified HTT when overexpressed together with HTT (46QP-H4 or 3R) and SUMO-1 (GFP-SUMO-1). SENP1, SENP2, and SENP6 decrease HTT SUMOylation. Graph depicts quantitation of western blot using the Odyssey Infrared Imaging System (LI-COR) to calculate the ratio of HTT purified versus the HTT modified by SUMO multiplied by 100.


( C ) Titration of SUMO - 1 . Denaturing nickel purification of HTTex1p ( 46QP - H4 ) following transfection with decreasing amounts of SUMO - 1 reduces amount of SUMO - modified HTT to undetectable . Ni - NTA blot displayed in gray scale shows purified HTTex1 SUMO - modified HTTex1 using HTT antibody . Note that 0 . 5 μg of SUMO - 1 ( ¼ amount of SUMO - 1 cDNA ) was used for identifying SUMO - 1 E3 ligase for HTTex1p . Graph depicts quantitation of western blot using Odyssey Infrared Imaging System to calculate of HTT purified versus HTT modified by SUMO multiplied by 100 . Note that experiments were performed in triplicate , a representative figure is shown .

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(C) Titration of SUMO-1. Denaturing nickel purification of HTTex1p (46QP-H4) following transfection with decreasing amounts of SUMO-1 reduces the amount of SUMO-modified HTT to undetectable levels. The Ni-NTA blot displayed in the gray scale shows purified HTTex1 and SUMO-modified HTTex1 using HTT antibody. Note that 0.5 μg of SUMO-1 (¼ the amount of SUMO-1 cDNA ) was used for identifying the SUMO-1 E3 ligase for HTTex1p. Graph depicts quantitation of western blot using the Odyssey Infrared Imaging System to calculate the ratio of HTT purified versus the HTT modified by SUMO multiplied by 100. Note that all experiments were performed in triplicate, and a representative figure is shown.


( A ) Under limiting SUMO conditions ( 1 / 4 SUMO - 1 , lanes 3 - 9 ) , PIAS1 increases HTT - SUMO modification above 1 / 4 SUMO alone . Purified HTT ( arrow ) SUMO - HTT ( boxed region ) were detected using anti - HTT . Graph depicts quantitation of Ni - NTA western blot using Odyssey Infrared Imaging Software ( LI - COR ) to calculate of HTT purified versus HTT modified by SUMO multiplied by 100 .

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(A) Under limiting SUMO conditions (1/4 SUMO-1, lanes 3-9), PIAS1 increases HTT-SUMO modification above 1/4 SUMO alone. Purified HTT (arrow) and SUMO-HTT (boxed region) were detected using anti-HTT. Graph depicts quantitation of the Ni-NTA western blot using the Odyssey Infrared Imaging Software (LI-COR) to calculate the ratio of HTT purified versus the HTT modified by SUMO multiplied by 100.


( B ) Western of overexpression of HTTex1 ( 46QP - H4 or 3R ) , SUMO - 2 ( GFP - SUMO - 2 ) , PIAS ( PIAS1 , PIASxα , PIASxβ , PIAS3 , PIASy ) . Under nonlimiting SUMO - 2 conditions , PIAS1 enhances SUMO modification of HTT . WC TCA shows overall myc - actin ( transfection control ) HTT . Graph quantitating of HTT purified versus HTT modified using Odyssey ( LICOR ) .

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(B) Western analysis of overexpression of HTTex1 (46QP-H4 or 3R), SUMO-2 (GFP-SUMO-2), and all the PIAS proteins (PIAS1, PIASxα, PIASxβ, PIAS3, and PIASy). Under nonlimiting SUMO-2 conditions, PIAS1 enhances SUMO modification of HTT. WC TCA shows overall myc-actin (transfection control) and HTT levels. Graph quantitating the ratio of HTT purified versus the HTT modified using the Odyssey (LICOR).


( C ) Left panel is autoradiography results of a GST pull - down showing that radiolabeled human PIAS1 interacts with HTTex1p . Right panel is a phosphorimager of GST pull - downs , performed in triplicate , showing of 35S - labeled PIAS that bound GST : GST alone , unexpanded HTT with without proline - rich region ( 20QP 20Q ) , expanded HTT with without proline - rich region ( 51QP 51Q ) , proline - rich region alone ( Pro ) . Error bars were calculated as an estimate of SE = STDEV ( n ) / SQRT ( n - 1 ) .

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(C) Left panel is the autoradiography results of a GST pull-down assay showing that radiolabeled human PIAS1 interacts with HTTex1p. Right panel is a phosphorimager analysis of GST pull-downs, performed in triplicate, showing the percentage of 35S-labeled PIAS protein that bound the GST proteins: GST alone, unexpanded HTT with and without the proline-rich region (20QP and 20Q), expanded HTT with and without the proline-rich region (51QP and 51Q), and the proline-rich region alone (Pro). Error bars were calculated as an estimate of SE = STDEV(n)/SQRT(n-1).


( B ) Longer HTT polypeptides are modified by SUMO - 1 . Western blot overexpressing HTTex1p ( 46QP - H4 or 3R ) , unexpanded HTT - 586 fragment ( 25Q - 586 or 25Q - 3R - 586 aa ) , expanded HTT - 586 fragment ( 137Q - 586 or 137Q - 3R - 586 aa ) with SUMO - 1 ( GFP - SUMO - 1 ) . lysates were subjected to HTT immunoprecipitation ( IP ) using HTT antibody . Western performed with Odyssey ( LI - COR ) allows detection of HTT ( data not shown ) SUMO simultaneously shows that forms of HTT are SUMO - 1 modified using anti - GFP antibody . HTTex1p is covalently SUMO - 1 modified ( lane 3 ) , modification disappears when Lys are mutated to Arg ( 3R ) ( lane 4 ) . Both unexpanded ( lane 4 5 ) expanded HTT - 586 fragments ( lane 6 7 ) are covalently SUMO - 1 modified in both presence absence of three Lys in N - terminal region of HTT ( 3R ) . Free SUMO - 1 is indicated with arrow , SUMO - modified HTT is indicated by boxes . Inset on right , from a replicate experiment , shows comigration of expanded HTT ( anti - HTT ) SUMO - 1 ( anti - GFP ) displayed in gray scale in color when two antibodies are merged ( HTT in red , SUMO - 1 in green , yellow when colocalizing ) .

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(B) Longer HTT polypeptides are modified by SUMO-1. Western blot overexpressing HTTex1p (46QP-H4 or 3R), unexpanded HTT-586 fragment (25Q-586 or 25Q-3R-586 aa), and expanded HTT-586 fragment (137Q-586 or 137Q-3R-586 aa) with SUMO-1 (GFP-SUMO-1). Cell lysates were subjected to HTT immunoprecipitation (IP) using HTT antibody. Western analysis performed with the Odyssey (LI-COR) allows detection of HTT (data not shown) and SUMO simultaneously and shows that all forms of HTT are SUMO-1 modified using the anti-GFP antibody. HTTex1p is covalently SUMO-1 modified (lane 3), and the modification disappears when Lys are mutated to Arg (3R) (lane 4). Both unexpanded (lane 4 and 5) and expanded HTT-586 fragments (lane 6 and 7) are covalently SUMO-1 modified in both the presence and absence of the three Lys in the N-terminal region of HTT (3R). Free SUMO-1 is indicated with the arrow, and SUMO-modified HTT is indicated by the boxes. Inset on the right, from a replicate experiment, shows comigration of expanded HTT (anti-HTT) and SUMO-1 (anti-GFP) displayed in the gray scale and in color when the two antibodies are merged (HTT in red, SUMO-1 in green, and yellow when colocalizing).


( C ) Bait plasmids ( HTT - 586 - 25Q or HTT - 586 - 73Q aa ) were transformed into L40ccua MATa yeast . Yeast clones encoding bait were individually mated against a matrix of MATα yeast clones encoding 16 , 888 prey ( with Gal4 activation domain fusions ) using pipetting spotting robots . Diploid yeasts were spotted onto SDIV ( - Leu - Trp - Ura - His ) agar plates for selection of PPIs as well as nylon membranes placed on SDIV agar plates for β - galactosidase assays . After 5 - 6 days of incubation at 30ºC , digitized of agar plates nylon membranes were assessed for growth β - galactosidase activity using software Visual Grid ( GPC Biotech ) .

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(C) Bait plasmids (HTT-586-25Q or HTT-586-73Q aa) were transformed into the L40ccua MATa yeast strain. Yeast clones encoding bait proteins were individually mated against a matrix of MATα yeast clones encoding 16,888 prey proteins (with Gal4 activation domain fusions) using pipetting and spotting robots. Diploid yeasts were spotted onto SDIV (-Leu-Trp-Ura-His) agar plates for selection of PPIs as well as nylon membranes placed on SDIV agar plates for β-galactosidase assays. After 5-6 days of incubation at 30ºC, digitized images of the agar plates and nylon membranes were assessed for growth and β-galactosidase activity using the software Visual Grid (GPC Biotech).


( D ) Overexpression of PIAS1 alone , with unexpanded HTT constructs ( 25Q - 586 aa ) plus SUMO - 1 ( GFP - SUMO - 1 ) , shows that PIAS1 binds both full - HTT HTT - 586 fragment ( 25Q - 586 aa ) . WT HTT was used in these experiments to preclude confounding aggregation effects . Western detection was performed using Odyssey is displayed in gray scale but is shown in color on merge ( HTT is red , PIAS1 is green ) . WB , western blot .

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(D) Overexpression of PIAS1 alone, with unexpanded HTT constructs (25Q-586 aa) plus SUMO-1 (GFP-SUMO-1), shows that PIAS1 binds both full-length HTT and HTT-586 fragment (25Q-586 aa). WT HTT was used in these experiments to preclude confounding aggregation effects. Western analysis detection was performed using Odyssey and is displayed in the gray scale but is shown in color on the merge (HTT is red, and PIAS1 is green). WB, western blot.


( E ) HeLa overexpressing either expanded 586 aa - HTT or phosphomimetic ( S13 , 16D = DD ) with SUMO - 2 plus minus PIAS1 . HTT was purified by IP using hydrazide beads ( Bioclone ) crosslinked to HTT ( Enzo ) antibody subjected to western using anti - HTT ( MAB5490 ) . Arrows indicate SUMO - 2 - modified HTT .

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(E) HeLa cells overexpressing either expanded 586 aa-HTT or the phosphomimetic (S13,16D = DD) with SUMO-2 plus and minus PIAS1. HTT was purified by IP using hydrazide beads (Bioclone) crosslinked to HTT (Enzo) antibody and subjected to western analysis using anti-HTT (MAB5490). Arrows indicate SUMO-2-modified HTT.


( F ) IP of HTT with hydrazide - linked beads shows that both unexpanded expanded HTT 586 aa phosphomimetics ( S13 , 16D - 586 aa ) are modified by SUMO - 2 . Arrows indicate SUMO - conjugated HTT . Note that experiments including Y2H were done in triplicate ; representative experiments are shown . Arrows indicate SUMO - 2 - modified HTT .

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(F) IP of HTT with hydrazide-linked beads shows that both unexpanded and expanded HTT 586 aa phosphomimetics (S13, 16D-586 aa) are modified by SUMO-2. Arrows indicate SUMO-conjugated HTT. Note that all experiments including the Y2H assay were done in triplicate; representative experiments are shown. Arrows indicate SUMO-2-modified HTT.


( A ) Western of whole - lysates from HeLa transfected with His - SUMO - 1 or SUMO - 2 / or 97Q - HTT exon 1 treated with 5 μM MG132 for 18 hr . Lysates were separated using differential centrifugation into a detergent - soluble fraction ( SOLUBLE ) with 1 % Triton X - 100 a detergent - insoluble fraction ( INSOLUBLE ) with 4 % SDS . Western blot probed with anti - HTT shows full - endogenous HTT in SOLUBLE fraction ( upper arrow ) 97Q - HTTex1 ( lower arrow ) ( left panel ) . In INSOLUBLE fraction , HTT HMW species are indicated by bracket asterisks ( right panel ) .

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(A) Western analysis of whole-cell lysates from HeLa cells transfected with His-SUMO-1 or SUMO-2 and/or 97Q-HTT exon 1 and treated with 5 μM MG132 for 18 hr. Lysates were separated using differential centrifugation into a detergent-soluble fraction (SOLUBLE) with 1% Triton X-100 and a detergent-insoluble fraction (INSOLUBLE) with 4% SDS. Western blot probed with anti-HTT shows full-length endogenous HTT in the SOLUBLE fraction (upper arrow) and 97Q-HTTex1 (lower arrow) (left panel). In the INSOLUBLE fraction, HTT HMW species are indicated by the bracket and asterisks (right panel).


( B ) MG132 SUMO - 2 cause mutant HTT to accumulate as HMW species ( bracke asterisk ) . Western blot showing IP with HTT antibody crosslinked beads from detergent - insoluble fraction probed with anti - HTT antibody .

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(B) MG132 and SUMO-2 cause mutant HTT to accumulate as HMW species (bracke and asterisk). Western blot showing IP with HTT antibody crosslinked beads from the detergent-insoluble fraction probed with the anti-HTT antibody.


( C ) Mutant HTT ( 97Q - Httex1 ) fibrils are detected with anti - HTT in insoluble fraction with treatment of MG132 or addition of exogenous SUMO - 2 .

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(C) Mutant HTT (97Q-Httex1) fibrils are detected with anti-HTT in the insoluble fraction with treatment of MG132 or addition of exogenous SUMO-2.


( D ) Western blot with increasing concentrations of SUMO - 2 detected with anti - His antibody ( left panel ) . Middle panel is same western blot probed with anti - HTT showing soluble forms of HTT . Right panel presents western blot from detergent - insoluble fraction with monomeric HTT ( 97Q ) at 55 kDa , asterisk ( * ) indicates HMW species . Note that experiments were performed in triplicate ; representative figures are shown .

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(D) Western blot with increasing concentrations of SUMO-2 detected with anti-His antibody (left panel). Middle panel is the same western blot probed with anti-HTT showing soluble forms of HTT. Right panel presents western blot from detergent-insoluble fraction with monomeric HTT (97Q) at 55 kDa, and the asterisk (*) indicates the HMW species. Note that all experiments were performed in triplicate; representative figures are shown.


( A ) Western blot of HeLa over - expressing exogenous PIAS1 in presence of mutant HTT ( 97Q ) when separated into detergent - soluble detergent - insoluble fractions . No difference is detected in monomeric HTT ( top panel , Soluble ) , but HMW HTT increase with PIAS1 overexpression . Anti - PIAS1 antibody ( Invitrogen ) was used to detect PIAS1 .

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(A) Western blot analysis of HeLa cells over-expressing exogenous PIAS1 in the presence of mutant HTT (97Q) when separated into detergent-soluble and detergent-insoluble fractions. No difference is detected in monomeric HTT (top panel, Soluble), but HMW HTT levels increase with PIAS1 overexpression. Anti-PIAS1 antibody (Invitrogen) was used to detect PIAS1.


( B ) Acute knockdown of PIAS1 decreases HMW HTT species in detergent - insoluble fraction . PIAS1 knockdown is detected in detergent - soluble - insoluble fractions using anti - PIAS1 antibody .

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(B) Acute knockdown of PIAS1 decreases HMW HTT species in the detergent-insoluble fraction. PIAS1 knockdown is detected in detergent-soluble and -insoluble fractions using anti-PIAS1 antibody.


( C ) Drosophila melanogaster expressing mutant HTTex1p ( 93Q ) in a reduced Su ( var ) 2 - 10 / dPIAS genetic background exhibits statistically significantly reduced photoreceptor neuron degeneration ( left panel , p = 0 . 033 ) when comparing dPIAS / + to + / + flies increased overall survival ( right panel , p = 0 . 047 ) when comparing dPIAS1 / + to + / + flies . Significance was measured by Student ' s t .

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

(C) Drosophila melanogaster expressing mutant HTTex1p (93Q) in a reduced Su(var)2-10/dPIAS genetic background exhibits statistically significantly reduced photoreceptor neuron degeneration (left panel, p = 0.033) when comparing dPIAS/+ to +/+ flies and increased overall survival (right panel, p = 0.047) when comparing dPIAS1/+ to +/+ flies. Significance was measured by Student's t test.


( D ) HMW SUMO - 2 accumulates in postmortem HD striata . Western blot of insoluble fraction from three control three HD postmortem striata as described ( Experimental Procedures ) .

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 O O O O

(D) HMW SUMO-2 accumulates in postmortem HD striata. Western blot analysis of the insoluble fraction from three control and three HD postmortem striata as described (Experimental Procedures).


B ) Larval brains at 6 h ALH from grh - Gal4 ; UAS - CD8 - GFP were labeled with Sas - 4 , Dpn , GFP .

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B) Larval brains at 6 h ALH from grh-Gal4; UAS-CD8-GFP were labeled with Sas-4, Dpn, and GFP.


C ) Larval brains at 0 h ALH from grh - Gal4 ; UAS - CD8 - GFP were labeled with γ - tubulin ( γ - tub ) , Asl , GFP imaged under super - resolution microscopy . D ) Larval brains at 0 h ALH from grh - Gal4 ; UAS - CD8 - GFP were labeled with Centrosomin ( CNN ) , Asl GFP imaged under spinning disc super - resolution microscopy .

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

C) Larval brains at 0 h ALH from grh-Gal4; UAS-CD8-GFP were labeled with γ-tubulin (γ-tub), Asl, and GFP and imaged under super-resolution microscopy. D) Larval brains at 0 h ALH from grh-Gal4; UAS-CD8-GFP were labeled with Centrosomin (CNN), Asl and GFP and imaged under spinning disc super-resolution microscopy.


E ) Larval brains at 0 h ALH from Jupiter - GFP ( G147 ) were labeled with GFP , Msps , Dpn . F ) Larval brains at 24 h ALH from Jupiter - GFP were raised on normal food ( fed ) food depleted of ( nutritional restriction / NR ) labelled with GFP Dpn .

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E) Larval brains at 0 h ALH from Jupiter-GFP (G147) were labeled with GFP, Msps, and Dpn. F) Larval brains at 24 h ALH from Jupiter-GFP were raised on normal food (fed) and food depleted of amino acids (nutritional restriction/NR) and labelled with GFP and Dpn.


A ) Larval brains at 16 h ALH , in which kin - lacZ was expressed under control of insc - Gal4 , tub - Gal80ts , were labelled with β - Gal , Msps , Dpn . protrusion of qNSCs was marked by Msps . Quiescent NSCs at ventral nerve cord ( VNC ) are shown . Arrows indicate localization of Kin - lacZ in qNSCs . B ) Larval brains at 6 h ALH from insc - Gal4 ; UAS - nod - lacZ were labeled with β - Gal , Msps , Dpn . Quiescent NSCs at both CB VNC are shown . Arrows indicate localization of Nod - lacZ in qNSCs

O O O O O O O O O O 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 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 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

A) Larval brains at 16 h ALH, in which kin-lacZ was expressed under the control of insc-Gal4, tub-Gal80ts, were labelled with β-Gal, Msps, and Dpn. The primary protrusion of qNSCs was marked by Msps. Quiescent NSCs at the ventral nerve cord (VNC) are shown. Arrows indicate the localization of Kin-lacZ in qNSCs. B) Larval brains at 6 h ALH from insc-Gal4; UAS-nod-lacZ were labeled with β-Gal, Msps, and Dpn. Quiescent NSCs at both CB and VNC are shown. Arrows indicate the localization of Nod-lacZ in qNSCs


A ) Larval brains at 24 h ALH from control ( insc - Gal4 ; UAS - dicer2 / UAS - β - Gal RNAi ) msps RNAi ( VDRC # 21982 ) controlled under insc - Gal4 were analyzed for EdU incorporation . NSCs were marked by Dpn Mira . B ) graph of EdU - negative NSCs per brain lobe for genotypes in ( A ) . n = 13 BL for both control 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 B-CONTROLLED_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 O O 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

A) Larval brains at 24 h ALH from control (insc-Gal4; UAS-dicer2/UAS-β-Gal RNAi) and msps RNAi (VDRC#21982) controlled under insc-Gal4 were analyzed for EdU incorporation. NSCs were marked by Dpn and Mira. B) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (A). n=13 BL for both control and msps RNAi. ****p< 0.0001


C ) Larval brains at 24 h ALH from wild - type various msps loss - of - function alleles msps810 with a genomic rescue construct ( g - msps ) were analyzed for EdU incorporation . NSCs were marked by Dpn Mira . D ) graph of EdU - negative NSCs per brain lobe for genotypes in ( C ) . n = 13 BL for control ; n = 13 BL for msps810 ; n = 17 BL for msps924 ; n = 14 BL for mspsP18 ; n = 6 BL for mspsP ; n = 12 BL for g - msps ; msps924 . * * * * p < 0 . 0001 ; p = 0 . 7502 ( ns ) . E ) graph of diameter of in NSCs at 24 h ALH from various genotypes . n = 238 NSCs for control ; n = 219 NSC for msps924 ; n = 170 NSCs for mspsP . * * * * p < 0 . 0001 . F ) graph of of qNSCs with a protrusion in wild - type , msps924 , mspsP18 larval brains . protrusion was labelled by grh > CD8 - GFP . n = 5 BL for control ; n = 6 BL for msps924 ; n = 4 BL for mspsP . * * * * p < 0 . 0001

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

C) Larval brains at 24 h ALH from wild-type and various msps loss-of-function alleles and msps810 with a genomic rescue construct (g-msps) were analyzed for EdU incorporation. NSCs were marked by Dpn and Mira. D) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (C). n=13 BL for control; n=13 BL for msps810; n=17 BL for msps924; n=14 BL for mspsP18; n=6 BL for mspsP; n=12 BL for g-msps; msps924. ****p< 0.0001; p=0.7502 (ns). E) Quantification graph of the diameter of the cell body in NSCs at 24 h ALH from various genotypes. n=238 NSCs for control; n=219 NSC for msps924; n=170 NSCs for mspsP. ****p< 0.0001. F) Quantification graph of the percentage of qNSCs with a primary protrusion in wild-type, msps924, and mspsP18 larval brains. The protrusion was labelled by grh>CD8-GFP. n=5 BL for control; n=6 BL for msps924; n=4 BL for mspsP. ****p< 0.0001


G ) Larval brains at 24 h ALH from wild - type tacc loss - of - function tacc74 were examined for EdU incorporation . NSCs were marked by Dpn Mira . H ) graph of EdU - negative NSCs per brain lobe for genotypes in ( G ) . n = 17 BL for control ; n = 15 BL for tacc74 . * * * * p < 0 . 0001 . I ) graph of qNSCs retaining protrusion per brain lobe for genotypes in ( G ) . n = 15 BL for both control tacc74 . * * * * p < 0 . 0001 . J ) graph of NSCs are positive for mitotic marker PH3 per brain lobe for genotypes in ( G ) . n = 15 BL for both control tacc74 . * * * * p < 0 . 0001

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

G) Larval brains at 24 h ALH from wild-type and tacc loss-of-function tacc74 were examined for EdU incorporation. NSCs were marked by Dpn and Mira. H) Quantification graph of EdU-negative NSCs per brain lobe for genotypes in (G). n=17 BL for control; n=15 BL for tacc74. ****p< 0.0001. I) Quantification graph of qNSCs retaining primary protrusion per brain lobe for genotypes in (G). n=15 BL for both control and tacc74. ****p< 0.0001. J) Quantification graph of NSCs are positive for mitotic marker PH3 per brain lobe for genotypes in (G). n=15 BL for both control and tacc74. ****p< 0.0001


B ) graph of fold changes in number of EB1 - GFP comets in protrusion of qNSCs 6 h ALH from various genotypes compared with control in ( A ) . n = 26 NSCs for control ; n = 18 NSCs for mspsP18 ; n = 21 NSCs for mspsP18 / P . * * * * p < 0 . 0001 . C ) graph of of retrograde EB1 - GFP comets in protrusion of qNSCs from 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 brain to calculate standard deviation . 38 41 qNSCs were analyzed in control mspsP18 , respectively . In control qNSCs , among total 383 EB1 - GFP comets , 353 were anterograde 30 were retrograde movements . In mspsP18 qNSCs , we only found 12 EB1 - GFP comets with 6 anterograde 6 retrograde movements

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 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 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 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 B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR 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 ) msps810 , UAS - nod - lacZ under control of grh - Gal4 UAS - CD8 - GFP were labeled with β - Gal , Dpn , GFP . Quiescent NSCs in central brain were shown . Arrows point at localization of Nod - lacZ in qNSCs . E ) 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

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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 ) msps RNAi , UAS - nod - lacZ ( VDRC # 21982 ) with grh - Gal4 UAS - CD8 - GFP ; UAS - dicer2 were labeled with β - Gal , Dpn , GFP . Quiescent NSCs in central brain are shown . Arrows indicate localization of Nod - lacZ in qNSCs . G ) 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

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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 , mspsP18 expressing grh > CD8 - GFP were labeled with Dpn GFP . arrow indicates second protrusion in msps924 . I ) graph of thickness of protrusion of qNSCs from wild - type , msps924 , mspsP18 expressing grh > CD8 - GFP . thickness was measured at middle point of protrusion . n = 18 NSCs for control ; n = 52 NSCs for msps924 ; n = 32 NSCs for mspsP18 . * * * * p < 0 . 0001

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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 ) tacc74 , UAS - nod - lacZ under control of grh - Gal4 UAS - CD8 - GFP ; tacc59 were labeled with β - Gal , Dpn , GFP . Quiescent NSCs in central brain are shown . M ) 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 B-CONTROLLED_VAR 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 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 control ( grh - Gal4 UAS - CD8 - GFP ) msps810 with grh - Gal4 UAS - CD8 - GFP were labeled with E - cadherin , Dpn , GFP . NSC - neuropil contact were marked by dashed lines , NSC - neuropil contact points were circled . B ) 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 basal region of qNSCs . n = 32 NSCs for control ; n = 61 NSCs for msps810 . * * p = 0 . 0028 . C ) graph of basal E - cad at NSC - neuropil contact sites by normalizing against Dpn 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 B-MEASURED_VAR 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 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 B-CONTROLLED_VAR 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 B-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

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 ) msps924 with grh - Gal4 UAS - CD8 - GFP were labeled with E - cadherin , Dpn , GFP . NSC - neuropil contact were marked by dashed lines , NSC - neuropil contact sites were circled . E ) 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 basal region of qNSCs . n = 77 NSCs for control ; n = 30 NSCs for msps924 . * * p = 0 . 0080 . F ) graph of basal E - cad at NSC - neuropil contact sites by normalizing against Dpn in qNSCs from genotypes in ( D ) . n = 60 NSCs for control ; n = 37 NSCs for msps924 . * * * * 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.


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

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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 ) E - cadR69 were analyzed for EdU incorporation , larval brains were labeled with EdU , Dpn Mira

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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 wild - type control ( yw ) klp64Dk5h / Df , kap3V6 kap3 V6 ; g - kap3 # 11 were stained with EdU , Dpn ,

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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 , kap3V5 were analyzed E - cad at NSC - neuropil contact sites , NSCs were labeled with Dpn Msps

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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 ) ) , rescued ( UAS - klp64D + msps RNAi ( 21982 ) ) under control of insc - Gal4 were analyzed for EdU incorporation , larval brains were labeled with EdU Dpn . B ) 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 .

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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 ) ) , rescued ( g - kap3 + msps RNAi ( 21982 ) ) driven by insc - Gal4 were examined for EdU incorporation , larval brains were stained with EdU Dpn . E ) 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 .

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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 ) ) , rescued ( UAS - E - cad7 + msps RNAi ( 21982 ) ) were assessed for EdU incorporation , larval brains were stained with EdU Dpn . H ) 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 .

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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 ) , rescued ( UAS - E - cad7 + klp64Dk5h ) were processed for EdU incorporation , larval brains were probed by with EdU , Dpn , Mira . K ) graph of NSCs negative for EdU incorporation per brain lobe for genotypes in ( J ) . n = 10 BL for control ; n = 12 BL for klp64Dk5h ; n = 10 BL for UAS - E - cad7 + klp64Dk5h . * * * * p < 0 . 0001 ; p = 0 . 3006 ( ns ) .

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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 equal amount of ultrafiltrate ( 19μg / lane ) subject to immunoblotting . High - molecular - ( GAPDH ) were not detected in ultrafiltrate .

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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.


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 .

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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.


CFU ( per gram ) in lungs of indicated mice with without PAO1 infection ( 5 × 106 CFU ) for 6 , 24 48 hours . n = 3 . Error bars , S . E . M .

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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 . Scale bar , 1000 μm ( upper panel ) 50 μm ( lower panel ) .

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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 24 h 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 IL - 6 of indicated mouse lung with PAO1 infection ( 5 × 106 CFU ) for 6 h 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.


showing transcriptome profile correlation values of indicated lung tissue samples before 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 engineered mDASCs by

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


Detection of LL - 37 expression in 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 engineered mDASCs by Western

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


Anti - Krt5 ( red ) anti - P63 ( green ) immunostaining of WT - 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.


colony - forming efficiency of WT - 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 markers ( Aqp5 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 with DASC shows antimicrobial effects in dose - dependent manner . Initial additions of PAO1 were 0 . 1 × , 0 . 5 × 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 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 with anti - LL - 37 antibody , but not IgG control , significantly reduced anti - PAO1 anti - E . coli ( J ) effects of LL - 37 - mDASCs . Initial dose of 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 direct 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 of transplanted 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.


of engrafted GFP - labeled in lung parenchyma by direct . Blue color indicates nucleus DAPI . 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 - 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 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 representative with overlapping of GFP 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 ) LL - 37 - Lung ( LL - 37 - mDASCs engrafted ) followed by CFU in whole lung homogenates 6 , 24 48 hours after infection . n = 3 . Error bars , S . E . M . Intratracheal instillation of equal amount of PAO1 into WT - Lung LL - 37 - Lung followed by CFU in BAL fluid 6 , 24 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 of mice with WT - Lung 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 showing histology of injured lung infected by PAO1 with WT - mDASCs or LL - 37 - mDASCs transplantation . Scale bar , 100 μm . Histopathological examination according to lung injury scoring system 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 . of brown - stained ( CD68 + ) 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 ) 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.


mRNA expression of LL - 37 was measured by qPCR for WT - 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 of native rat lung tissue decellularized scaffold showed absence of nuclei following decellularization . Scale bar , 40 μm .

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 image showing GFP - labeled LL - 37 - hDASCs before ( left ) 7 days after ( right ) being engrafted onto decellularized scaffold . Scale bar , 100 μm ( right panel ) 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 . Scale bar , 50 μm .

Immunofluorescence of recellularized lung with indicated antibodies. Blue color indicates nucleus DAPI staining. Scale bar, 50 μm.


recellularized lung by LL - 37 - hDASCs displayed growth inhibitory effect on PAO1 E . coli . Initial dose of 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 ( A ) ( B ) of calcium - stable k - fiber by immunofluorescence of n ≥ 41 tubulin - stained mitotic of neonatal ( N ) elderly ( 87y ) human dermal fibroblasts ( HDF ) at prometaphase metaphase stages . were normalized to neonatal samples . Scale bar , 5µm . Data information : 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 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 ( between Hec1 ACA immunostainings of a kinetochore ) in n = 50 kinetochore pairs scored from 10 elderly vs . neonatal in prometaphase metaphase . ( D ) Inter - kinetochore ( between Hec1 of sister kinetochores ) in n = 50 kinetochore pairs scored from 10 elderly vs . neonatal in prometaphase metaphase . Data information : 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 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 imaging of neonatal ( N ) 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 ) of anaphases with lagging chromosomes in n =

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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 imaging of neonatal ( N ) 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 an elderly dividing that underwent correct ( Normal ) incorrect ( Lagging ) chromosome segregation , respectively . Time , min : sec . Scale bars : 5µm ( ) 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 of anaphases without ( top ) with ( bottom ) mis - segregation ( MS ) , FISH - stained for three chromosome pairs ( 7 , 12 18 ) . Arrowheads indicate micronuclei containing centromeric signal for chromosome 18 . Scale bar , 10µm . ( H ) of anaphases with MS in neonatal ( N ) vs . elderly ( 87y ) n = scored by FISH . Data information : 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 .

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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 of total MCAK in neonatal ( HDF N ) 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 of MCAK in neonatal ( HDF N ) elderly ( HDF 87y ) mitotic . 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 in FACS - sorted neonatal ( N ) elderly ( 87y ) fibroblasts transduced with empty ( control , - ) , GFP - MCAK ( + ) 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 per condition measured from envelope breakdown ( NEB ) to anaphase onset . Data Information : 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 ( B Mann - Whitney .

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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.