( C ) The increase in p62 protein expression induced by β ‐ catenin siRNA was attenuated in HT29 cells treated with 10 μg / μl cycloheximide and starvation for 8 and 24 h compared to vehicle control . A complementary experiment using the transcription inhibitor actinomycin D is shown in Supplementary Figure S1C .

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(C) The increase in p62 protein expression induced by β‐catenin siRNA was attenuated in HT29 cells treated with 10 μg/μl cycloheximide and starvation for 8 and 24 h compared to vehicle control. A complementary experiment using the transcription inhibitor actinomycin D is shown in Supplementary Figure S1C.


( D ) Overexpression of Fzd6 ( green , left panel ) increased p62 ( red , right panel ) protein expression in HEK293T cells . As an internal control , cells not overexpressing Fzd6 are shown in the field of view .

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(D) Overexpression of Fzd6 (green, left panel) increased p62 (red, right panel) protein expression in HEK293T cells. As an internal control, cells not overexpressing Fzd6 are shown in the field of view.


( E ) Doxycycline induction ( 1 μg / ml ) of DNTCF4 increased p62 and LC3 ‐ II protein expression in doxycycline ‐ inducible DNTCF4 LS174T ‐ L8 cells . LGR5 downregulation by DNTCF4 confirmed inhibition of β ‐ catenin / TCF4 signalling .

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(E) Doxycycline induction (1 μg/ml) of DNTCF4 increased p62 and LC3‐II protein expression in doxycycline‐inducible DNTCF4 LS174T‐L8 cells. LGR5 downregulation by DNTCF4 confirmed inhibition of β‐catenin/TCF4 signalling.


( F ) Relative p62 mRNA levels ( 48 h ) by qRT - PCR increased following doxycycline induction of DNTCF4 in LS174T ‐ L8 cells ( mean ± s . e . m . , three independent experiments performed in triplicate , * * * P0 . 001 ) .

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(F) Relative p62 mRNA levels (48 h) by qRT-PCR increased following doxycycline induction of DNTCF4 in LS174T‐L8 cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001).


( G ) Upper panel : Wnt3a ( 200 ng / ml ) treatment of HCT116 β ‐ cateninWT / − cells ( 24 h ) decreased LC3 ‐ II and p62 protein expression by western blotting . Lower panel : quantification by densitometry of the LC3 ‐ II / β ‐ actin ratio ( mean ± s . e . m . of three independent experiments ) . Source data for this figure is available on the online supplementary information page .

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(G) Upper panel: Wnt3a (200 ng/ml) treatment of HCT116 β‐cateninWT/− cells (24 h) decreased LC3‐II and p62 protein expression by western blotting. Lower panel: quantification by densitometry of the LC3‐II/β‐actin ratio (mean±s.e.m. of three independent experiments).Source data for this figure is available on the online supplementary information page.


( A ) Chromatin immunoprecipitation ( ChIP ) demonstrates β ‐ catenin and TCF4 binding to the p62 promoter . HT29 cells were grown in normal or starvation conditions for 2 h and subjected to ChIP analysis with the indicated antibodies ( IgG was used as a negative control ) . Binding of RNA Pol II , β ‐ catenin and TCF4 to the p62 promoter region was measured by quantitative PCR and expressed as percent enrichment relative to the input chromatin . During nutrient deprivation , binding of RNA Pol II to the p62 promoter increased ; binding of β ‐ catenin to the p62 promoter decreased ; TCF4 binding did not change ( data are from one representative experiment of at least three independent experiments performed in triplicate ) . PCR products subjected to agarose gel electrophoresis are shown in Supplementary Figure S2A .

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(A) Chromatin immunoprecipitation (ChIP) demonstrates β‐catenin and TCF4 binding to the p62 promoter. HT29 cells were grown in normal or starvation conditions for 2 h and subjected to ChIP analysis with the indicated antibodies (IgG was used as a negative control). Binding of RNA Pol II, β‐catenin and TCF4 to the p62 promoter region was measured by quantitative PCR and expressed as percent enrichment relative to the input chromatin. During nutrient deprivation, binding of RNA Pol II to the p62 promoter increased; binding of β‐catenin to the p62 promoter decreased; TCF4 binding did not change (data are from one representative experiment of at least three independent experiments performed in triplicate). PCR products subjected to agarose gel electrophoresis are shown in Supplementary Figure S2A.


( B ) Relative p62 mRNA expression by qRT - PCR increases ∼ 14 ‐ fold after 24 h starvation in HT29 cells ( mean ± s . e . m . , three independent experiments performed in triplicate , * * * P0 . 001 ) .

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(B) Relative p62 mRNA expression by qRT-PCR increases ∼14‐fold after 24 h starvation in HT29 cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001).


( C ) Binding of acetyl ‐ Histone H3 to the p62 promoter increased under starvation conditions , suggesting p62 gene derepression ( data are from one representative experiment of at least three independent experiments performed in triplicate ) .

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(C) Binding of acetyl‐Histone H3 to the p62 promoter increased under starvation conditions, suggesting p62 gene derepression (data are from one representative experiment of at least three independent experiments performed in triplicate).


( D ) Binding of β ‐ catenin to the p62 promoter ( relative to β ‐ catenin binding to a non ‐ target gene promoter , GAPDH ) significantly decreased under starvation conditions ( mean ± s . e . m . , three independent experiments performed in triplicate , * * P0 . 01 ) .

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(D) Binding of β‐catenin to the p62 promoter (relative to β‐catenin binding to a non‐target gene promoter, GAPDH) significantly decreased under starvation conditions (mean±s.e.m., three independent experiments performed in triplicate, **P0.01).


( A ) Western blotting showing that autophagy was induced after 2 h starvation , as evidenced by decreased p62 protein and increased LC3 ‐ II protein expression . β ‐ Catenin protein expression decreased over a 24 ‐ h period of starvation in HT29 cells .

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(A) Western blotting showing that autophagy was induced after 2 h starvation, as evidenced by decreased p62 protein and increased LC3‐II protein expression. β‐Catenin protein expression decreased over a 24‐h period of starvation in HT29 cells.


( B ) Autophagy decreased following Atg7 knockdown as shown by increased p62 and decreased LC3 ‐ II protein expression . β ‐ Catenin protein expression increased following Atg7 siRNA . Reduction of LC3 puncta with Atg7 siRNA was confirmed by immunofluorescence and is shown in Supplementary Figure S2B .

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(B) Autophagy decreased following Atg7 knockdown as shown by increased p62 and decreased LC3‐II protein expression. β‐Catenin protein expression increased following Atg7 siRNA. Reduction of LC3 puncta with Atg7 siRNA was confirmed by immunofluorescence and is shown in Supplementary Figure S2B.


( C , D ) Reduction of TopFlash activity in HT29 cells after 24 h of autophagy induction using ( C ) starvation or ( D ) 100 nM mTOR inhibitor PP242 ( C and D , mean ± s . e . m . , three independent experiments performed in triplicate , * * * P0 . 001 ) . Autophagy induction was confirmed by western blotting and is shown in Supplementary Figures S2C and S2D ) .

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(C, D) Reduction of TopFlash activity in HT29 cells after 24 h of autophagy induction using (C) starvation or (D) 100 nM mTOR inhibitor PP242 (C and D, mean±s.e.m., three independent experiments performed in triplicate, ***P0.001). Autophagy induction was confirmed by western blotting and is shown in Supplementary Figures S2C and S2D).


( E , F ) qRT - PCR shows reduction of Wnt ‐ target gene expression ( E ) Axin2 ( * P = 0 . 017 ) and ( F ) Cyclin D1 ( * * P = 0 . 0036 ) after 8 h starvation ( mean ± s . e . m . , three ( Axin2 ) or six ( Cyclin D1 ) independent experiments performed in triplicate ) .

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(E, F) qRT-PCR shows reduction of Wnt‐target gene expression (E) Axin2 (*P=0.017) and (F) Cyclin D1 (**P=0.0036) after 8 h starvation (mean±s.e.m., three (Axin2) or six (Cyclin D1) independent experiments performed in triplicate).


( G - I ) Inhibition of Wnt ‐ induced TopFlash activity by autophagy induction in ( G ) HCT116 β ‐ cateninWT / − cells ( mean ± s . e . m . , three independent experiments performed in triplicate , * * P0 . 01 ) and ( H ) RKO cells ( mean ± s . e . m . , three independent experiments performed in triplicate , * P0 . 05 ) . ( I ) Reduction of Wnt3a ‐ induced Cyclin D1 gene expression RKO cells after 12 h treatment with autophagy induction using starvation or PP242 ( mean ± s . e . m . , two independent experiments ) .

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(G-I) Inhibition of Wnt‐induced TopFlash activity by autophagy induction in (G) HCT116 β‐cateninWT/− cells (mean±s.e.m., three independent experiments performed in triplicate, **P0.01) and (H) RKO cells (mean±s.e.m., three independent experiments performed in triplicate, *P0.05). (I) Reduction of Wnt3a‐induced Cyclin D1 gene expression RKO cells after 12 h treatment with autophagy induction using starvation or PP242 (mean±s.e.m., two independent experiments).


( J , K ) TopFlash activity following Atg7 knockdown in ( J ) HT29 cells ( mean ± s . e . m . , four independent experiments performed in triplicate , * P = 0 . 0488 ) and ( K ) RKO cells with 24 hWnt3a treatment ( mean ± s . e . m . , three independent experiments performed in triplicate , * P = 0 . 0282 ) .

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(J, K) TopFlash activity following Atg7 knockdown in (J) HT29 cells (mean±s.e.m., four independent experiments performed in triplicate, *P=0.0488) and (K) RKO cells with 24 hWnt3a treatment (mean±s.e.m., three independent experiments performed in triplicate, *P=0.0282).


( L ) A representative western blot of RKO cells following Wnt3a treatment and Atg7 siRNA .

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(L) A representative western blot of RKO cells following Wnt3a treatment and Atg7 siRNA.


( M ) Relative β ‐ catenin mRNA levels did not change after 8 h starvation by qRT - PCR ( mean ± s . e . m . , three independent experiments performed in triplicate ) .

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(M) Relative β‐catenin mRNA levels did not change after 8 h starvation by qRT-PCR (mean±s.e.m., three independent experiments performed in triplicate).


( N ) Western blot showing prevented β ‐ catenin protein degradation during starvation in the presence of lysosomal autophagy inhibitors chloroquine ( 10 μM ) and bafilomycin A1 ( 100 nM ) compared to starvation alone .

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(N) Western blot showing prevented β‐catenin protein degradation during starvation in the presence of lysosomal autophagy inhibitors chloroquine (10 μM) and bafilomycin A1 (100 nM) compared to starvation alone.


( O ) Inhibition of autophagy using wortmannin ( 50 nM ) prevented β ‐ catenin protein degradation during starvation .

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(O) Inhibition of autophagy using wortmannin (50 nM) prevented β‐catenin protein degradation during starvation.


( P ) Western blot analysis of RKO cells expressing a myc - tagged β ‐ catenin mutant ( S33A , S37A , T41A , S45A ) resistant to proteasomal degradation ( myc - β ‐ cateninAAAA ) . RKO cells were subject to nutrient starvation for 2 , 8 and 24 h , and autophagy induction was confirmed by increased LC3 ‐ II and decreased p62 protein expression . Both endogenous β ‐ catenin and myc - β ‐ cateninAAAA protein expression decreased during starvation .

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(P) Western blot analysis of RKO cells expressing a myc-tagged β‐catenin mutant (S33A, S37A, T41A, S45A) resistant to proteasomal degradation (myc-β‐cateninAAAA). RKO cells were subject to nutrient starvation for 2, 8 and 24 h, and autophagy induction was confirmed by increased LC3‐II and decreased p62 protein expression. Both endogenous β‐catenin and myc-β‐cateninAAAA protein expression decreased during starvation.


( Q ) Proteasome inhibition with MG132 ( 10 μM ) did not prevent starvation ‐ induced decrease in β ‐ catenin protein expression . Source data for this figure is available on the online supplementary information page .

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(Q) Proteasome inhibition with MG132 (10 μM) did not prevent starvation‐induced decrease in β‐catenin protein expression.Source data for this figure is available on the online supplementary information page.


LC3 and β ‐ catenin co ‐ localise in the mouseintestinal epithelium . ( A and B ) Immunofluorescence of LC3 ( red ) and β ‐ catenin ( green ) expression in the intestinal epithelium following 2 daystamoxifen treatment in control ( A : β ‐ catenin + / lox ‐ villin ‐ creERT2 ) and β ‐ catenin deleted ( B : β ‐ catenin − / lox ‐ villin ‐ creERT2 ) mice . ( C and D ) Magnified areas from B revealing co ‐ localisation of β ‐ catenin ( red ) and β ‐ catenin ( green ) . Arrowheads indicate co ‐ localisation of LC3 and β ‐ catenin . ( E ) Linescan analyses from C and D showing staining intensity of indicated co ‐ localised puncta . Red and green channel levels were adjusted post acquisition ( equal changes applied across the entire figure ) and the blue channel was removed for clarity .

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LC3 and β‐catenin co‐localise in the mouseintestinal epithelium. (A and B) Immunofluorescence of LC3 (red) and β‐catenin (green) expression in the intestinal epithelium following 2 daystamoxifen treatment in control (A: β‐catenin+/lox‐villin‐creERT2) and β‐catenin deleted (B: β‐catenin−/lox‐villin‐creERT2) mice. (C and D) Magnified areas from B revealing co‐localisation of β‐catenin (red) and β‐catenin (green). Arrowheads indicate co‐localisation of LC3 and β‐catenin. (E) Linescan analyses from C and D showing staining intensity of indicated co‐localised puncta. Red and green channel levels were adjusted post acquisition (equal changes applied across the entire figure) and the blue channel was removed for clarity.


( A ) Co ‐ immunoprecipitation of YFP - LC3 or negative control YFP in HT29 cells . Binding of endogenous β ‐ catenin to YFP - LC3 was detected after 8 h of autophagy induction by starvation and starvation in the presence of lysosomal autophagy flux inhibitor chloroquine ( 10 μM ) . Input and immunodepleted lysates are shown .

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(A) Co‐immunoprecipitation of YFP-LC3 or negative control YFP in HT29 cells. Binding of endogenous β‐catenin to YFP-LC3 was detected after 8 h of autophagy induction by starvation and starvation in the presence of lysosomal autophagy flux inhibitor chloroquine (10 μM). Input and immunodepleted lysates are shown.


( B ) Immunoprecipitation of endogenous LC3 in HT29 cells . IgG was used as a negative control .

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(B) Immunoprecipitation of endogenous LC3 in HT29 cells. IgG was used as a negative control.


( D ) Pulldown assays using recombinant GST or GST - LC3B and lysates from HEK293 cells expressing HA - tagged wild ‐ type β ‐ catenin ( HA - β ‐ cateninWT ) or W504A / I507A β ‐ catenin ( HA - β ‐ cateninW504A / I507A ) . HA - β ‐ cateninW504A / I507A exhibited reduced GST - LC3B binding compared to HA - β ‐ cateninWT .

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(D) Pulldown assays using recombinant GST or GST-LC3B and lysates from HEK293 cells expressing HA-tagged wild‐type β‐catenin (HA-β‐cateninWT) or W504A/I507A β‐catenin (HA-β‐cateninW504A/I507A). HA-β‐cateninW504A/I507A exhibited reduced GST-LC3B binding compared to HA-β‐cateninWT.


( E ) Recombinant myc - tagged His - LC3 ( His - LC3 - myc ) interacted with recombinant GST - β ‐ cateninWTin vitro , but binding to GST - β ‐ cateninW504A / I507A was reduced ( upper panel ) . Input recombinant proteins visualised with Coomassie blue are shown ( lower panels ) .

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(E) Recombinant myc-tagged His-LC3 (His-LC3-myc) interacted with recombinant GST-β‐cateninWTin vitro, but binding to GST-β‐cateninW504A/I507A was reduced (upper panel). Input recombinant proteins visualised with Coomassie blue are shown (lower panels).


( F ) Co ‐ immunoprecipitation experiments using lysates from HEK293 cells transiently expressing YFP - LC3 with β ‐ cateninWT or β ‐ cateninW504A / I507A starved for 8 h with chloroquine ( 10 μM ) . YFP - LC3 immunoprecipitated β ‐ cateninWT but not β ‐ cateninW504A / I507A . Input lysates are shown and immunoprecipitated p62 and YFP - LC3 served as positive controls .

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(F) Co‐immunoprecipitation experiments using lysates from HEK293 cells transiently expressing YFP-LC3 with β‐cateninWT or β‐cateninW504A/I507A starved for 8 h with chloroquine (10 μM). YFP-LC3 immunoprecipitated β‐cateninWT but not β‐cateninW504A/I507A. Input lysates are shown and immunoprecipitated p62 and YFP-LC3 served as positive controls.


( G ) HA - β ‐ cateninS33A / S37A / T41A / S45A / W504A / I507A ( HA - β ‐ catenin6A ) was more resistant to the starvation ‐ induced reduction in β ‐ catenin protein expression than HA - β ‐ cateninS33Y .

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(G) HA-β‐cateninS33A/S37A/T41A/S45A/W504A/I507A (HA-β‐catenin6A) was more resistant to the starvation‐induced reduction in β‐catenin protein expression than HA-β‐cateninS33Y.


( H ) Relative cell death in HT29 cells subject to nutrient starvation for 24 h . Atg7 knockdown increased cell survival . The increase in cell survival following Atg7 knockdown was significantly reversed by simultaneously depleting β ‐ catenin ( double knockdown of Atg7 and β ‐ catenin ) . Data are the mean ± s . e . m . of three independent experiments performed in triplicate , * * * P0 . 001 ; * * P0 . 01 . Source data for this figure is available on the online supplementary information page .

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(H) Relative cell death in HT29 cells subject to nutrient starvation for 24 h. Atg7 knockdown increased cell survival. The increase in cell survival following Atg7 knockdown was significantly reversed by simultaneously depleting β‐catenin (double knockdown of Atg7 and β‐catenin). Data are the mean±s.e.m. of three independent experiments performed in triplicate, ***P0.001; **P0.01.Source data for this figure is available on the online supplementary information page.


Identification of Rtt105 - TAP associated proteins . Purified proteins were resolved on a 4 - 12 % gel , revealed by silver staining , and identified by mass spectrometry ( Table EV1 ) . * Non - specific band also in control .

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Identification of Rtt105-TAP associated proteins. Purified proteins were resolved on a 4-12% gel, revealed by silver staining, and identified by mass spectrometry (Table EV1). *Non-specific band also in control.


Cells containing Rfa1 - TAP were synchronized at G1 , S , and G2 / M phase and used for the TAP purification . Rfa1 - TAP associated protein complexes were analyzed by Western blot using CBP , Rfa2 , and Rtt105 antibodies ( left panel ) . DNA content was monitored by flow cytometry ( right panel ) .

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Cells containing Rfa1-TAP were synchronized at G1, S, and G2/M phase and used for the TAP purification. Rfa1-TAP associated protein complexes were analyzed by Western blot using CBP, Rfa2, and Rtt105 antibodies (left panel). DNA content was monitored by flow cytometry (right panel).


MBP - Rtt105 pulled down recombinant RPA , resolved on SDS - PAGE gels and visualized by Coomassie Brilliant Blue ( CBB ) staining . MBP was used as a negative control .

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MBP-Rtt105 pulled down recombinant RPA, resolved on SDS-PAGE gels and visualized by Coomassie Brilliant Blue (CBB) staining. MBP was used as a negative control.


GST - tagged full length ( FL ) and Rtt105 deletion mutants ( Schematic of the Rtt105 truncations is shown in the upper panel ) were purified and used to pull down Rfa1 protein . GST proteins were used as a negative control . Isolated protein complexes were resolved on 15 % SDS - PAGE gels and visualized by CBB staining .

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GST-tagged full length (FL) and Rtt105 deletion mutants (Schematic of the Rtt105 truncations is shown in the upper panel) were purified and used to pull down Rfa1 protein. GST proteins were used as a negative control. Isolated protein complexes were resolved on 15% SDS-PAGE gels and visualized by CBB staining.


Rfa1 - TAP purification was performed using Rfa1 - TAP rtt105 ∆ strains expressing WT and indicated Rtt105 mutant forms . pRS313 serves as vector control ( Vc ) .

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Rfa1-TAP purification was performed using Rfa1-TAP rtt105∆ strains expressing WT and indicated Rtt105 mutant forms. pRS313 serves as vector control (Vc).


Snapshots of Rfa1 ChIP - Seq peak at ARS305 from WT and rtt105 ∆ cells released into HU - medium . The average Rfa1 ChIP - Seq read density from cells released into HU - medium around ACS sites . ACS : ARS consensus sequence .

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Snapshots of Rfa1 ChIP-Seq peak at ARS305 from WT and rtt105∆ cells released into HU-medium. The average Rfa1 ChIP-Seq read density from cells released into HU-medium around ACS sites. ACS: ARS consensus sequence.


Snapshots of Rfa2 ChIP - Seq peaks at ARS305 from cells released at 16 ° C for 72 min . The average Rfa2 ChIP - Seq read density surrounding ACS sites from cells released at 16 ° C for 72 min .

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Snapshots of Rfa2 ChIP-Seq peaks at ARS305 from cells released at 16°C for 72 min. The average Rfa2 ChIP-Seq read density surrounding ACS sites from cells released at 16°C for 72 min.


Rfa1 occupancy analysis of cells progression through S phase . G1 - arrested cells were released into fresh YPD medium to allow cell progression through S phase at standard growth temperature ( 25 ° C ) . Equal amounts of cells were collected at each time point . Rfa1 ChIP were performed using IgG beads for TAP - tag . ARS305 and ARS607 , early fired replication origins ; ARS607 + 14kb , a corresponding 14kb away region of ARS607 . The mean and standard deviation ( SD ) of three biological replicates were shown .

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Rfa1 occupancy analysis of cells progression through S phase. G1-arrested cells were released into fresh YPD medium to allow cell progression through S phase at standard growth temperature (25°C). Equal amounts of cells were collected at each time point. Rfa1 ChIP were performed using IgG beads for TAP-tag. ARS305 and ARS607, early fired replication origins; ARS607+14kb, a corresponding 14kb away region of ARS607. The mean and standard deviation (SD) of three biological replicates were shown.


The nuclear localization of Rfa1 proteins is altered in rtt105 ∆ mutant cells . Rfa1 tagged with GFP at its C terminus was used to analyze the localization of Rfa1 - GFP fusion protein ( Green ) and the nuclear envelop is visualized with the Nup49 - mCherry fusion protein ( Red ) using fluorescence microscopy . DIC : differential interference contrast . Scale bar : 5μm .

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The nuclear localization of Rfa1 proteins is altered in rtt105∆ mutant cells. Rfa1 tagged with GFP at its C terminus was used to analyze the localization of Rfa1-GFP fusion protein (Green) and the nuclear envelop is visualized with the Nup49-mCherry fusion protein (Red) using fluorescence microscopy. DIC: differential interference contrast. Scale bar: 5μm.


NLS - RFA1 - GFP rescues the nuclear localization of Rfa1 in rtt105 ∆ mutant cells . The wild type RFA1 gene was fused with an SV40 large T Antigen nuclear localization sequence ( NLS ) at its 5 ' - end and a GFP gene at its 3 ' end to obtain a construct , driven by the RFA1 promoter to express NLS - RFA1 - GFP fusion protein . The engineered construct was then transformed into WT or rtt105 ∆ mutant yeast cells to replace its endogenous RFA1 expression . The resulting yeast cells were then visualized under microscope , and GFP signals enriched in the nuclei were scored . RFA1 - GFP lacking the NLS sequence was transformed into rtt105 ∆ rfa1 ∆ mutant cells as a control . DAPI staining indicates nuclear DNA .

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NLS-RFA1-GFP rescues the nuclear localization of Rfa1 in rtt105∆ mutant cells. The wild type RFA1 gene was fused with an SV40 large T Antigen nuclear localization sequence (NLS) at its 5'-end and a GFP gene at its 3' end to obtain a construct, driven by the RFA1 promoter to express NLS-RFA1-GFP fusion protein. The engineered construct was then transformed into WT or rtt105∆ mutant yeast cells to replace its endogenous RFA1 expression. The resulting yeast cells were then visualized under microscope, and GFP signals enriched in the nuclei were scored. RFA1-GFP lacking the NLS sequence was transformed into rtt105∆ rfa1∆ mutant cells as a control. DAPI staining indicates nuclear DNA.


An increase in nuclear localization of Rfa1 could not rescue the RPA binding defects at the replication regions in rtt105 ∆ cells . The percentage of Rfa2 ChIP - DNA over the total input DNA was calculated . The mean and standard error ( SE ) of three biological replicates were shown . Statistical significance was evaluated based on Student ' s t - tests ( * : 0 . 01 ≤ P value < 0 . 05 ) .

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An increase in nuclear localization of Rfa1 could not rescue the RPA binding defects at the replication regions in rtt105∆ cells. The percentage of Rfa2 ChIP-DNA over the total input DNA was calculated. The mean and standard error (SE) of three biological replicates were shown. Statistical significance was evaluated based on Student's t-tests (*: 0.01≤P value<0.05).


Purified RPA and Rtt105 - RPA complex were resolved on 15 % SDS - PAGE gels and analyzed by silver staining .

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Purified RPA and Rtt105-RPA complex were resolved on 15% SDS-PAGE gels and analyzed by silver staining.


EMSA assay was performed to analyze ssDNA binding ability of RPA or Rtt105 - RPA complex . Cy3 - labeled 30nt ssDNA was used as the substrate , and reaction mixtures were resolved on native PAGE gels . Quantification of the RPA - bound ssDNA is shown in ( C ) .

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EMSA assay was performed to analyze ssDNA binding ability of RPA or Rtt105-RPA complex. Cy3-labeled 30nt ssDNA was used as the substrate, and reaction mixtures were resolved on native PAGE gels. Quantification of the RPA-bound ssDNA is shown in (C).


Recombinant Rtt105 - RPA complex was mixed with a 30nt - ssDNA in a 2 : 1 ratio , and the reaction product were subjected to gel filtration chromatography . Equal volume of fraction 5 - 10 were resolved on SDS - PAGE for detecting proteins by Ruby staining ( upper panel ) or were resolved by native agarose gel for detection of Cy3 - ssDNA signal ( lower panel ) .

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Recombinant Rtt105-RPA complex was mixed with a 30nt-ssDNA in a 2:1 ratio, and the reaction product were subjected to gel filtration chromatography. Equal volume of fraction 5-10 were resolved on SDS-PAGE for detecting proteins by Ruby staining (upper panel) or were resolved by native agarose gel for detection of Cy3-ssDNA signal (lower panel).


GST - RPA pull down Rtt105 with increased amount of 30nt ssDNA , and the bound proteins were resolved on SDS - PAGE and visualized by silver staining .

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GST-RPA pull down Rtt105 with increased amount of 30nt ssDNA, and the bound proteins were resolved on SDS-PAGE and visualized by silver staining.


Kymograms showing most ssDNA molecules two distinct stretching patterns of RPA - ssDNA molecules . Pattern 1 , slow stretching ; Pattern 2 , fast stretching . The counts of the two patterns in each reaction were plotted in ( C ) . Please note a fraction of individual ssDNA shows both patterns . Rtt105 increases the rate of ssDNA stretching upon RPA binding . Stretching rate was defined as the length change of ssDNA upon binding to RPA - mCherry in unit time as shown in ( B , white arrow ) . Rtt105 increases the extent of ssDNA stretching upon RPA binding . Length increment = DNA length at 8 minutes / DNA length at 3 minutes .

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Kymograms showing most ssDNA molecules two distinct stretching patterns of RPA-ssDNA molecules. Pattern 1, slow stretching; Pattern 2, fast stretching. The counts of the two patterns in each reaction were plotted in (C). Please note a fraction of individual ssDNA shows both patterns. Rtt105 increases the rate of ssDNA stretching upon RPA binding. Stretching rate was defined as the length change of ssDNA upon binding to RPA-mCherry in unit time as shown in (B, white arrow). Rtt105 increases the extent of ssDNA stretching upon RPA binding. Length increment = DNA length at 8 minutes / DNA length at 3 minutes.


Snapshots of BrdU IP - Seq peaks at ARS305 from cells released into YPD medium containing HU for 45 min . The average BrdU IP - Seq read density around fired ACS from cells released into YPD medium containing HU for 45 min sites . Snapshots of BrdU IP - Seq peaks at ARS305 from cells released into YPD medium at 16 ° C for 72 min . The average BrdU IP - Seq read density around ACS sites from cells released into 16 ° C for 72 min .

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Snapshots of BrdU IP-Seq peaks at ARS305 from cells released into YPD medium containing HU for 45 min. The average BrdU IP-Seq read density around fired ACS from cells released into YPD medium containing HU for 45 min sites. Snapshots of BrdU IP-Seq peaks at ARS305 from cells released into YPD medium at 16°C for 72 min. The average BrdU IP-Seq read density around ACS sites from cells released into 16°C for 72 min.


Rad52 - GFP foci were counted in WT and rtt105 ∆ cells , the percentage of cells with Rad52 - foci is reported . Error bars indicate one standard deviation from three independent experiments with at least 150 cells counted in each replicate .

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Rad52-GFP foci were counted in WT and rtt105∆ cells, the percentage of cells with Rad52-foci is reported. Error bars indicate one standard deviation from three independent experiments with at least 150 cells counted in each replicate.


Rad53 phosphorylation was analyzed in WT and rtt105 ∆ cells by immunoblotting of protein extracts with anti - Rad53 antibodies . Ponceau S ( Pon S ) staining was applied as a loading control .

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Rad53 phosphorylation was analyzed in WT and rtt105∆ cells by immunoblotting of protein extracts with anti-Rad53 antibodies. Ponceau S (Pon S) staining was applied as a loading control.


Deletion of RTT105 leads to an increased rate of gross chromosomal rearrangements ( GCRs ) . Wild - type ( WT ) and rtt105 ∆ mutant strains integrated with the yWSS439 - 5ori ∆ yeast artificial chromosomes ( YACs ) . GCRs rates in the evaluation of telomere marker loss were

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Deletion of RTT105 leads to an increased rate of gross chromosomal rearrangements (GCRs). Wild-type (WT) and rtt105∆ mutant strains integrated with the yWSS439-5ori∆ yeast artificial chromosomes (YACs). GCRs rates in the evaluation of telomere marker loss were assayed


Ten - fold serial dilutions WT or rtt105 ∆ cells in two different backgrounds were assayed on normal growth media ( YPD ) and on media containing the indicated DNA damaging agents , methyl - methane sulfonate ( MMS ) , camptothecin ( CPT ) , bleomycin ( Bleo ) , and hydroxyurea ( HU ) .

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Ten-fold serial dilutions WT or rtt105∆ cells in two different backgrounds were assayed on normal growth media (YPD) and on media containing the indicated DNA damaging agents, methyl-methane sulfonate (MMS), camptothecin (CPT), bleomycin (Bleo), and hydroxyurea (HU).


( A ) HOIP , HOIL - 1L , and SHARPIN co - localize with Htt - Q97 aggregates . SH - SY5Y cells co - expressing Htt - Q25 - GFP or Htt - Q97 - GFP ( green ) and HOIP , HOIL - 1L , SHARPIN , or HHARI ( red ) ; DAPI ( blue ) . Scale bar , 10 µm .

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(A) HOIP, HOIL-1L, and SHARPIN co-localize with Htt-Q97 aggregates. SH-SY5Y cells co-expressing Htt-Q25-GFP or Htt-Q97-GFP (green) and HOIP, HOIL-1L, SHARPIN, or HHARI (red); DAPI (blue). Scale bar, 10 µm.


HOIP co - localizes with Htt inclusion bodies in human HD frontal cortex . Perinuclear Htt aggregates visualized by immunofluorescence ( B ) in human HD frontal cortex . Scale bar , 10 µm ( B )

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HOIP co-localizes with Htt inclusion bodies in human HD frontal cortex. Perinuclear Htt aggregates visualized by immunofluorescence (B) in human HD frontal cortex. Scale bar, 10 µm (B)


HOIP co - localizes with Htt inclusion bodies in human HD frontal cortex . Perinuclear Htt aggregates visualized by DAB immunohistochemistry ( C ) in human HD frontal cortex . Scale bar , 15 µm ( C ) .

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HOIP co-localizes with Htt inclusion bodies in human HD frontal cortex. Perinuclear Htt aggregates visualized by DAB immunohistochemistry (C) in human HD frontal cortex. Scale bar, 15 µm (C).


M1 - linked ubiquitin co - localizes with Htt - polyQ aggregates in cultured SH - SY5Y cells ( A ) Autofluorescent lipofuscin appears in the green channel in human brain . Scale bar , 20 µm ( A )

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M1-linked ubiquitin co-localizes with Htt-polyQ aggregates in cultured SH-SY5Y cells (A) Autofluorescent lipofuscin appears in the green channel in human brain. Scale bar, 20 µm (A)


M1 - linked ubiquitin co - localizes with Htt - polyQ aggregates in cultured R6 / 2 mouse cortex and striatum ( B ) Scale bar 10

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M1-linked ubiquitin co-localizes with Htt-polyQ aggregates in cultured R6/2 mouse cortex and striatum (B) Scale bar 10 µm


M1 - linked ubiquitin co - localizes with Htt - polyQ aggregates in cultured human HD frontal cortex ( C ) . Autofluorescent lipofuscin appears in the green channel in human brain . Scale bar , 10

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M1-linked ubiquitin co-localizes with Htt-polyQ aggregates in cultured human HD frontal cortex (C). Autofluorescent lipofuscin appears in the green channel in human brain. Scale bar, 10 µm


( D ) Htt - Q25 - or Htt - Q97 - expressing HEK293T were analyzed by filter retardation assays . As indicated , the M1 ubiquitin antibody was preincubated with recombinant tetra - ubiquitin linked via M1 ( 4 x M1 ubiquitin ) or K63 ( 4 x K63 ubiquitin ) .

O O O B-GENEPROD O O O O B-GENEPROD O O O B-EXP_ASSAY B-CELL_LINE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O

(D) Htt-Q25- or Htt-Q97-expressing HEK293T were analyzed by filter retardation assays. As indicated, the M1 ubiquitin antibody was preincubated with recombinant tetra-ubiquitin linked via M1 (4 x M1 ubiquitin) or K63 (4 x K63 ubiquitin).


( B ) Quantification of the co - localization of WT HOIP or HOIP mutants and Htt - Q97 aggregates . Data are displayed as mean ± SD and were analyzed by Mann - Whitney U - test , n = 3 . Data information : * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the co-localization of WT HOIP or HOIP mutants and Htt-Q97 aggregates. Data are displayed as mean ± SD and were analyzed by Mann-Whitney U-test, n = 3. Data information: *p ≤ 0.05, ***p ≤ 0.001.


( C ) Htt - Q97 - GFP ( green ) was co - expressed with HA - tagged WT or HOIP mutants ( red ) in SH - SY5Y cells and analyzed by immunocytochemistry using an antibody against HA . DAPI ( blue ) . Scale bar , 10 µm .

O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O

(C) Htt-Q97-GFP (green) was co-expressed with HA-tagged WT or HOIP mutants (red) in SH-SY5Y cells and analyzed by immunocytochemistry using an antibody against HA. DAPI (blue). Scale bar, 10 µm.


( D ) Recruitment of HOIP to Htt - Q97 is decreased in p97 / VCP - deficient cells . p97 / VCP - deficient SH - SY5Y cells co - expressing Htt - Q97 and HA - HOIP were reconstituted with either WT VCP or VCP ∆ PIM and analyzed by immunocytochemistry . Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Tukey ' s Multiple Comparison Test , n = 9 . Data information : * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O 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) Recruitment of HOIP to Htt-Q97 is decreased in p97/VCP-deficient cells. p97/VCP-deficient SH-SY5Y cells co-expressing Htt-Q97 and HA-HOIP were reconstituted with either WT VCP or VCP∆PIM and analyzed by immunocytochemistry. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9. Data information: *p ≤ 0.05, ***p ≤ 0.001.


( E ) Recruitment of p97 / VCP to Htt - Q97 is decreased in HOIP KO cells . WT HAP1 cells ( control ) or HOIP KO HAP1 cells reconstituted with either WT HOIP or HOIP ∆ PUB were analyzed by immunocytochemistry . Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Bonferroni Multiple Comparison Test , n = 5 . Data information : * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O 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) Recruitment of p97/VCP to Htt-Q97 is decreased in HOIP KO cells. WT HAP1 cells (control) or HOIP KO HAP1 cells reconstituted with either WT HOIP or HOIP∆PUB were analyzed by immunocytochemistry. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Bonferroni Multiple Comparison Test, n = 5. Data information: *p ≤ 0.05, ***p ≤ 0.001.


( F ) The interaction of soluble Htt - Q60 with p97 / VCP is dependent on HOIP . WT HAP1 cells or HOIP KO HAP1 cells were transfected with Htt - Q60 - HA . 48 h after transfection cells were lysed with 1 % Triton X - 100 , followed by an immunoprecipitation of Htt - Q60 via the HA tag . Immunopurified proteins were detected by Western blotting using an anti - VCP and anti - HOIP antibody .

O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O

(F) The interaction of soluble Htt-Q60 with p97/VCP is dependent on HOIP. WT HAP1 cells or HOIP KO HAP1 cells were transfected with Htt-Q60-HA. 48 h after transfection cells were lysed with 1% Triton X-100, followed by an immunoprecipitation of Htt-Q60 via the HA tag. Immunopurified proteins were detected by Western blotting using an anti-VCP and anti-HOIP antibody.


( A ) Increased expression of HOIP decreases nuclear phospho - c - Jun in primary neurons . Primary mouse hippocampal neurons expressing Htt - Q25 or Htt - Q97 + / - HOIP were analyzed by immunocytochemistry using an antibody against phospho - c - Jun . Data are displayed as mean ± SD and were analyzed by 2 independent Mann - Whitney U - tests , n = 3 . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

O O O O O O B-GENEPROD O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O 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) Increased expression of HOIP decreases nuclear phospho-c-Jun in primary neurons. Primary mouse hippocampal neurons expressing Htt-Q25 or Htt-Q97 +/- HOIP were analyzed by immunocytochemistry using an antibody against phospho-c-Jun. Data are displayed as mean ± SD and were analyzed by 2 independent Mann-Whitney U-tests, n = 3. *p ≤ 0.05, ***p ≤ 0.001.


( B ) Htt - Q97 - induced toxicity is increased in cells silenced for HOIP . SH - SY5Y cells transiently transfected with Htt - Q25 or Htt - Q97 and either control siRNA ( CO ) or HOIP siRNA were analyzed by immunocytochemistry using an antibody against phospho - c - Jun . For rescue experiments , HOIP was co - expressed 24 h after silencing . Quantification of Htt - expressing cells with nuclear phospho - c - Jun is based on Student ' s t test from 3 independent experiments . Data are displayed as mean ± SD unless otherwise stated . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O 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) Htt-Q97-induced toxicity is increased in cells silenced for HOIP. SH-SY5Y cells transiently transfected with Htt-Q25 or Htt-Q97 and either control siRNA (CO) or HOIP siRNA were analyzed by immunocytochemistry using an antibody against phospho-c-Jun. For rescue experiments, HOIP was co-expressed 24 h after silencing. Quantification of Htt-expressing cells with nuclear phospho-c-Jun is based on Student's t test from 3 independent experiments. Data are displayed as mean ± SD unless otherwise stated. *p ≤ 0.05, ***p ≤ 0.001.


OTULIN silencing decreases the toxicity of Htt - Q97 . SH - SY5Y cells transiently transfected with either control siRNA ( CO ) or OTULIN siRNA and Htt - Q25 or Htt - Q97 were analyzed by immunocytochemistry using antibodies against phospho - c - Jun ( C ) Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Tukey ' s Multiple Comparison Test , n = 9 - 11 . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

B-GENEPROD O O O O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

OTULIN silencing decreases the toxicity of Htt-Q97. SH-SY5Y cells transiently transfected with either control siRNA (CO) or OTULIN siRNA and Htt-Q25 or Htt-Q97 were analyzed by immunocytochemistry using antibodies against phospho-c-Jun (C) Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9-11. *p ≤ 0.05, ***p ≤ 0.001.


OTULIN silencing decreases the toxicity of Htt - Q97 . SH - SY5Y cells transiently transfected with either control siRNA ( CO ) or OTULIN siRNA and Htt - Q25 or Htt - Q97 were analyzed by immunocytochemistry using antibodies against cleaved caspase - 3 ( D ) . Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Tukey ' s Multiple Comparison Test , n = 9 - 11 . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

B-GENEPROD O O O O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

OTULIN silencing decreases the toxicity of Htt-Q97. SH-SY5Y cells transiently transfected with either control siRNA (CO) or OTULIN siRNA and Htt-Q25 or Htt-Q97 were analyzed by immunocytochemistry using antibodies against cleaved caspase-3 (D). Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9-11. *p ≤ 0.05, ***p ≤ 0.001.


( E ) The protective effect of HOIP is independent of NF - κB signaling . SH - SY5Y cells transiently transfected with Htt - Q97 - GFP and either control vector ( CO ) or HOIP ± the NF - κB super - repressor IκB - 2S / A were analyzed by immunocytochemistry using an antibody against phospho - c - Jun . Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Tukey ' s Multiple Comparison Test , n = 9 . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

O O O O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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) The protective effect of HOIP is independent of NF-κB signaling. SH-SY5Y cells transiently transfected with Htt-Q97-GFP and either control vector (CO) or HOIP ± the NF-κB super-repressor IκB-2S/A were analyzed by immunocytochemistry using an antibody against phospho-c-Jun. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9. *p ≤ 0.05, ***p ≤ 0.001.


( F ) SDS - soluble Htt - polyQ species are modified by linear ubiquitin chains . HEK293T cells were co - transfected with Htt - Q60 - GFP ( as a control for specific immunoprecipitation of HA - tagged Htt - Q60 ) , Htt - Q60 - HA or Htt - Q60 - 3R ( 3 lysine residues replaced by arginines ) and the plasmids indicated . Cells were lysed under denaturing conditions ( 1 % SDS ) , followed by immunoprecipitation of Htt via the HA tag ( 1 % Triton X - 100 , 0 . 1 % SDS ) . Immunopurified proteins were detected by Western blotting using the M1 ubiquitin - specific antibody 1E3 and anti - HA .

O O O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_LINE O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD O

(F) SDS-soluble Htt-polyQ species are modified by linear ubiquitin chains. HEK293T cells were co-transfected with Htt-Q60-GFP (as a control for specific immunoprecipitation of HA-tagged Htt-Q60), Htt-Q60-HA or Htt-Q60-3R (3 lysine residues replaced by arginines) and the plasmids indicated. Cells were lysed under denaturing conditions (1% SDS), followed by immunoprecipitation of Htt via the HA tag (1% Triton X-100, 0.1% SDS). Immunopurified proteins were detected by Western blotting using the M1 ubiquitin-specific antibody 1E3 and anti-HA.


( G ) Silencing of OTULIN increases linear ubiquitination of Htt - Q97 . A linear ubiquitination assay was performed as described in ( F ) with HEK293T cells transfected with Htt - Q97 - HA and control siRNA or OTULIN siRNA .

O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O B-GENEPROD O O

(G) Silencing of OTULIN increases linear ubiquitination of Htt-Q97. A linear ubiquitination assay was performed as described in (F) with HEK293T cells transfected with Htt-Q97-HA and control siRNA or OTULIN siRNA.


( H ) SDS - insoluble Htt - polyQ species are modified by linear ubiquitin chains . HEK293T cells expressing Htt - Q97 - HA or Htt - Q97 - GFP were lysed under denaturing conditions in 1 . 5 % SDS . After centrifugation , the pellets containing the SDS - insoluble aggregates ( SDS - insoluble fraction ) were dissolved in formic acid . Formic acid - dissolved aggregates were analyzed by immunoblotting using the M1 ubiquitin - specific 1F11 / 3F5 / Y102L antibody .

O O O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_LINE O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O

(H) SDS-insoluble Htt-polyQ species are modified by linear ubiquitin chains. HEK293T cells expressing Htt-Q97-HA or Htt-Q97-GFP were lysed under denaturing conditions in 1.5% SDS. After centrifugation, the pellets containing the SDS-insoluble aggregates (SDS-insoluble fraction) were dissolved in formic acid. Formic acid-dissolved aggregates were analyzed by immunoblotting using the M1 ubiquitin-specific 1F11/3F5/Y102L antibody.


( I ) Endogenous NEMO co - localizes with Htt - Q97 aggregates . NEMO ( red ) and Htt - Q97 - GFP ( green ) were analyzed by immunocytochemistry in SH - SY5Y cells . DAPI ( blue ) . Scale bar , 10 µm . ( J ) Endogenous Optineurin co - localizes with Htt - Q97 aggregates . Optineurin ( red ) and Htt - Q97 - GFP ( green ) were analyzed by immunocytochemistry in SH - SY5Y cells . DAPI ( blue ) . Scale bar , 10 µm .

O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O O O

(I) Endogenous NEMO co-localizes with Htt-Q97 aggregates. NEMO (red) and Htt-Q97-GFP (green) were analyzed by immunocytochemistry in SH-SY5Y cells. DAPI (blue). Scale bar, 10 µm. (J) Endogenous Optineurin co-localizes with Htt-Q97 aggregates. Optineurin (red) and Htt-Q97-GFP (green) were analyzed by immunocytochemistry in SH-SY5Y cells. DAPI (blue). Scale bar, 10 µm.


( A ) The interaction between Htt - Q97 and Sp1 is decreased by linear ubiquitination . HEK293T cells were co - transfected with Htt - Q97 - HA and the siRNAs indicated . Htt - Q97 - GFP was transfected as a control for specific immunoprecipitation of HA - Htt - Q97 . Nuclear ( left panel ) and cytosolic fractions ( middle panel ) were subjected to an anti - HA immunoprecipitation followed by Western blotting using an anti - Sp1 antibody . Right panel : Input controls ( whole cell lysates ) .

O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O

(A) The interaction between Htt-Q97 and Sp1 is decreased by linear ubiquitination. HEK293T cells were co-transfected with Htt-Q97-HA and the siRNAs indicated. Htt-Q97-GFP was transfected as a control for specific immunoprecipitation of HA-Htt-Q97. Nuclear (left panel) and cytosolic fractions (middle panel) were subjected to an anti-HA immunoprecipitation followed by Western blotting using an anti-Sp1 antibody. Right panel: Input controls (whole cell lysates).


( B ) Quantification of the interaction between Htt - Q97 and Sp1 in nuclear fractions . Shown is the relative amount of Sp1 co - purifying with Htt - Q97 . Signal intensities were normalized to nuclear Sp1 . Data are displayed as mean ± SD and were analyzed by 2 independent Mann - Whitney U - tests , n = 4 . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR B-GENEPROD O O O O O O O O O O O O O O O O O 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) Quantification of the interaction between Htt-Q97 and Sp1 in nuclear fractions. Shown is the relative amount of Sp1 co-purifying with Htt-Q97. Signal intensities were normalized to nuclear Sp1. Data are displayed as mean ± SD and were analyzed by 2 independent Mann-Whitney U-tests, n = 4. *p ≤ 0.05, ***p ≤ 0.001.


( C , D ) Impairment of Sp1 - mediated transcription by Htt - Q97 is modified by HOIP . ( C ) Shown is the relative luciferase activity in HEK293T cells expressing Htt - Q97 - HA , a Sp1 luciferase reporter construct and HOIP cDNA . Data are displayed as mean ± SD and were analyzed by Kruskal - Wallis test followed by Dunn ' s Multiple Comparison Test , n = 9 - 11 . ( D ) Shown is the relative luciferase activity in HEK293T cells expressing Htt - Q97 - HA , a Sp1 luciferase reporter construct and HOIP siRNA . Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Tukey ' s Multiple Comparison Test , n = 9 . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) Impairment of Sp1-mediated transcription by Htt-Q97 is modified by HOIP. (C) Shown is the relative luciferase activity in HEK293T cells expressing Htt-Q97-HA, a Sp1 luciferase reporter construct and HOIP cDNA. Data are displayed as mean ± SD and were analyzed by Kruskal-Wallis test followed by Dunn's Multiple Comparison Test, n = 9-11. (D) Shown is the relative luciferase activity in HEK293T cells expressing Htt-Q97-HA, a Sp1 luciferase reporter construct and HOIP siRNA. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9. *p ≤ 0.05, ***p ≤ 0.001.


( E ) Sp1 silencing decreases HOIP and SHARPIN protein expression . SH - SY5Y cells were transfected with control or Sp1 - specific siRNAs and analyzed for protein expression 2 days after transfection by immunoblotting ( left panel ) . Quantification of normalized Sp1 , HOIP and SHARPIN protein expression levels ( right panel ) . Data are displayed as mean ± SEM with n = 5 from unpaired two - tailed Student ' s t - tests . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O 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) Sp1 silencing decreases HOIP and SHARPIN protein expression. SH-SY5Y cells were transfected with control or Sp1-specific siRNAs and analyzed for protein expression 2 days after transfection by immunoblotting (left panel). Quantification of normalized Sp1, HOIP and SHARPIN protein expression levels (right panel). Data are displayed as mean ± SEM with n=5 from unpaired two-tailed Student's t-tests. *p ≤ 0.05, ***p ≤ 0.001.


( F ) HOIP - , HOIL - 1L - and SHARPIN - specific mRNA is decreased in R6 / 2 striatum . Quantification of mRNA from the striatum of 9 - 12 weeks old R6 / 2 mice and non - transgenic littermates . Data are displayed as mean ± SD with n = 6 - 9 biological replicates from unpaired two - tailed Student ' s t - tests . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O 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) HOIP-, HOIL-1L- and SHARPIN-specific mRNA is decreased in R6/2 striatum. Quantification of mRNA from the striatum of 9-12 weeks old R6/2 mice and non-transgenic littermates. Data are displayed as mean ± SD with n = 6-9 biological replicates from unpaired two-tailed Student's t-tests. *p ≤ 0.05, ***p ≤ 0.001.


( G ) HOIP protein levels are decreased in human HD brain . Total protein lysates from human brain ( anterior cingulate cortex ) analyzed by immunoblotting using antibodies against HOIP and βIII - tubulin . HOIP - positive signals were normalized to βIII - tubulin . To verify the specificity of the HOIP antibody , WT and HOIP KO HAP1 cells were analyzed in parallel on a different gel . Data are displayed as mean ± SEM with n = 5 for control and n = 7 for HD patients from unpaired two - tailed Student ' s t - tests . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM B-DISEASE B-TISSUE O O O O O B-ORGANISM B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-CELL_LINE O O O O 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-DISEASE B-ORGANISM O O O O 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) HOIP protein levels are decreased in human HD brain. Total protein lysates from human brain (anterior cingulate cortex) analyzed by immunoblotting using antibodies against HOIP and βIII-tubulin. HOIP-positive signals were normalized to βIII-tubulin. To verify the specificity of the HOIP antibody, WT and HOIP KO HAP1 cells were analyzed in parallel on a different gel. Data are displayed as mean ± SEM with n=5 for control and n=7 for HD patients from unpaired two-tailed Student's t-tests. *p ≤ 0.05, ***p ≤ 0.001.


( A ) HOIP is recruited to disease - associated protein aggregates formed by Ataxin - 3 - Q84 , SOD1 G85R , TDP - 43 Q331K or Optineurin R96L . SH - SY5Y cells co - expressing disease - associated proteins ( green ) and HOIP ( red ) were analyzed by immunocytochemistry ; DAPI ( blue ) . Scale bar , 20

O O O B-GENEPROD O O O B-DISEASE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-DISEASE O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O

(A) HOIP is recruited to disease-associated protein aggregates formed by Ataxin-3-Q84, SOD1 G85R, TDP-43 Q331K or Optineurin R96L. SH-SY5Y cells co-expressing disease-associated proteins (green) and HOIP (red) were analyzed by immunocytochemistry; DAPI (blue). Scale bar, 20 µ


Linear ubiquitin accumulates at disease - associated protein aggregates under endogenous LUBAC expression . M1 - linked ubiquitin ( red ) and disease - associated proteins ( green ) were analyzed by immunocytochemistry in SH - SY5Y cells using the 1E3 antibody ( Millipore ) ( B ) DAPI ( blue ) . Scale bar , 20 µm .

B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-DISEASE O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-DISEASE O O O O O O O O O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

Linear ubiquitin accumulates at disease-associated protein aggregates under endogenous LUBAC expression. M1-linked ubiquitin (red) and disease-associated proteins (green) were analyzed by immunocytochemistry in SH-SY5Y cells using the 1E3 antibody (Millipore) (B) DAPI (blue). Scale bar, 20 µm.


Linear ubiquitin accumulates at disease - associated protein aggregates under endogenous LUBAC expression . M1 - linked ubiquitin ( red ) and disease - associated proteins ( green ) were analyzed by immunocytochemistry in primary striatal neurons using the 1F11 / 3F5 / Y102L antibody ( Genentech ) ( C ) . DAPI ( blue ) . Scale bar , 20 µm .

B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-DISEASE O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-DISEASE O O O O O O O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

Linear ubiquitin accumulates at disease-associated protein aggregates under endogenous LUBAC expression. M1-linked ubiquitin (red) and disease-associated proteins (green) were analyzed by immunocytochemistry in primary striatal neurons using the 1F11/3F5/Y102L antibody (Genentech) (C). DAPI (blue). Scale bar, 20 µm.


( D ) SDS - soluble SOD G85R and TDP - 43 Q331K species are modified by linear ubiquitin chains . HEK293T cells were co - transfected with disease - associated proteins and either the plasmids indicated or with control siRNA or OTULIN siRNA . 48 h after transfection cells were lysed under denaturing conditions ( 1 % SDS ) , followed by immunoprecipitation of SOD G85R and TDP - 43 Q331K via the HA tag ( 1 % Triton X - 100 , 0 . 1 % SDS ) . Immunopurified proteins were detected by Western blotting using the M1 ubiquitin - specific antibody 1E3 .

O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_LINE O O O O O O B-DISEASE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O

(D) SDS-soluble SOD G85R and TDP-43 Q331K species are modified by linear ubiquitin chains. HEK293T cells were co-transfected with disease-associated proteins and either the plasmids indicated or with control siRNA or OTULIN siRNA. 48 h after transfection cells were lysed under denaturing conditions (1% SDS), followed by immunoprecipitation of SOD G85R and TDP-43 Q331K via the HA tag (1% Triton X-100, 0.1% SDS). Immunopurified proteins were detected by Western blotting using the M1 ubiquitin-specific antibody 1E3.


( A ) Linear ubiquitination reduces the fraction of cells with Htt - Q97 aggregates . SH - SY5Y cells were transfected with Htt - Q97 and either control siRNA , HOIP siRNA or OTULIN siRNA . Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Tukey ' s Multiple Comparison Test , n = 9 . Expression levels of HOIP and OTULIN were analyzed by Western blotting . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O 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) Linear ubiquitination reduces the fraction of cells with Htt-Q97 aggregates. SH-SY5Y cells were transfected with Htt-Q97 and either control siRNA, HOIP siRNA or OTULIN siRNA. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 9. Expression levels of HOIP and OTULIN were analyzed by Western blotting. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.


( B ) Linear ubiquitination reduces the fraction of cells with Htt - Q97 aggregates . SH - SY5Y cells were transfected with Htt - Q97 and either WT HOIP or the catalytically inactive HOIP mutant C885A . Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Tukey s Multiple Comparison Test , n = 5 . Expression levels of WT and mutant HOIP and OTULIN were analyzed by Western blotting . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O 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) Linear ubiquitination reduces the fraction of cells with Htt-Q97 aggregates. SH-SY5Y cells were transfected with Htt-Q97 and either WT HOIP or the catalytically inactive HOIP mutant C885A. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey s Multiple Comparison Test, n = 5. Expression levels of WT and mutant HOIP and OTULIN were analyzed by Western blotting. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.


( C ) The effect of HOIP on Htt - Q97 aggregation is independent of NF - κB signaling . SH - SY5Y cells transiently transfected with Htt - Q97 and either control vector ( CO ) or WT HOIP plus the NF - κB super - repressor IκB - 2S / A were analyzed by immunocytochemistry . Data are mean ± SD with n = 5 from an unpaired two - tailed Student ' s t - test . Expression levels of WT HOIP and IκB - 2S / A were analyzed by Western blotting . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) The effect of HOIP on Htt-Q97 aggregation is independent of NF-κB signaling. SH-SY5Y cells transiently transfected with Htt-Q97 and either control vector (CO) or WT HOIP plus the NF-κB super-repressor IκB-2S/A were analyzed by immunocytochemistry. Data are mean ± SD with n=5 from an unpaired two-tailed Student's t-test. Expression levels of WT HOIP and IκB-2S/A were analyzed by Western blotting. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.


( D ) Catalytically active HOIP decreases the number of cells with Htt - Q97 aggregates independently of autophagy . WT and ATG5 KO MEFs were transiently transfected with Htt - Q97 - GFP and either control vector ( CO ) , HOIP or HOIP C885A . Cells were treated 24 h after transfection with the proteasomal inhibitor MG132 ( 1 µM , 16 h ) . Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Tukey ' s Multiple Comparison Test , n = 7 . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

O O O O O B-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Catalytically active HOIP decreases the number of cells with Htt-Q97 aggregates independently of autophagy. WT and ATG5 KO MEFs were transiently transfected with Htt-Q97-GFP and either control vector (CO), HOIP or HOIP C885A. Cells were treated 24 h after transfection with the proteasomal inhibitor MG132 (1 µM, 16 h). Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Tukey's Multiple Comparison Test, n = 7. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.


( E ) Linear ubiquitination reduces the fraction of cells with TDP - 43 Q331K aggregates . SH - SY5Y cells were transfected with TDP - 43 Q331K and either control siRNA , HOIP siRNA or OTULIN siRNA . Data are displayed as mean ± SD and were analyzed by Kruskal - Wallis test , followed by Dunn ' s Multiple Comparison Test , n = 7 . Expression levels of HOIP and OTULIN were analyzed by Western blotting . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O 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) Linear ubiquitination reduces the fraction of cells with TDP-43 Q331K aggregates. SH-SY5Y cells were transfected with TDP-43 Q331K and either control siRNA, HOIP siRNA or OTULIN siRNA. Data are displayed as mean ± SD and were analyzed by Kruskal-Wallis test, followed by Dunn's Multiple Comparison Test, n = 7. Expression levels of HOIP and OTULIN were analyzed by Western blotting. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.


( F ) Catalytically active HOIP decreases the number of cells with TDP - 43 aggregates dependent on proteasomal degradation . SH - SY5Y cells were transfected with TDP - 43 Q331K and either control vector ( CO ) , HOIP or HOIP C885A . Cells were treated 48 h after transfection with the proteasomal inhibitor MG132 ( 1 µM ) or the p97 / VCP inhibitor NSM - 873 ( 1 µM ) for 3 h . Data are displayed as mean ± SD and were analyzed by one - way ANOVA followed by Dunnett ' s Multiple Comparison Test , n = 5 . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

O O O O O B-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Catalytically active HOIP decreases the number of cells with TDP-43 aggregates dependent on proteasomal degradation. SH-SY5Y cells were transfected with TDP-43 Q331K and either control vector (CO), HOIP or HOIP C885A. Cells were treated 48 h after transfection with the proteasomal inhibitor MG132 (1 µM) or the p97/VCP inhibitor NSM-873 (1 µM) for 3 h. Data are displayed as mean ± SD and were analyzed by one-way ANOVA followed by Dunnett's Multiple Comparison Test, n = 5. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.


( G ) Inhibition of the proetasome and p97 / VCP do not have additive effects on the degradation of misfolded TDP - 43 species . SH - SY5Y cells expressing TDP - 43 Q331K were treated 48 h after transfection with MG132 ( 1 µM ) and / or NSM - 873 ( 1 µM ) for 3 h . All data are mean ± SD , data were analyzed by one - way ANOVA followed by Bonferroni ' s Multiple Comparison Test , n = 5 . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

O O O O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Inhibition of the proetasome and p97/VCP do not have additive effects on the degradation of misfolded TDP-43 species. SH-SY5Y cells expressing TDP-43 Q331K were treated 48 h after transfection with MG132 (1 µM) and/or NSM-873 (1 µM) for 3 h. All data are mean ± SD, data were analyzed by one-way ANOVA followed by Bonferroni's Multiple Comparison Test, n = 5. Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.


( h ) The cellular levels of Ca2 + ions after ligand ( C10ORF99 ) stimulation of GPR15 in human , mouse , Japanese quail , painted turtle . YM . 254890 inhibits Ca2 + ion levels in cells transfected with GPR15 receptors of vertebrates . The fold - change in calcium levels was calculated based on the fluorescence intensity ( excitation and emission wavelengths : 490 and 520 nm , respectively ) .

O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O B-GENEPROD O B-ORGANISM O B-ORGANISM O B-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

(h) The cellular levels of Ca2+ ions after ligand (C10ORF99) stimulation of GPR15 in human, mouse, Japanese quail, painted turtle. YM.254890 inhibits Ca2+ ion levels in cells transfected with GPR15 receptors of vertebrates. The fold-change in calcium levels was calculated based on the fluorescence intensity (excitation and emission wavelengths: 490 and 520 nm, respectively).


( i , j ) The cellular levels of Ca2 + ions after ligand ( C10ORF99 ) stimulation of GPR15 in coelacanth , Asian bony tongue , and their mutants . ( k , l ) The cellular levels of Ca2 + ions after ligand ( C10ORF99 ) stimulating GPR15 in Japanese quail and its mutants . The fold - change in calcium levels was calculated based on the fluorescence intensity ( excitation and emission wavelengths : 490 and 520 nm , respectively ) .

O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O B-GENEPROD O B-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O B-GENEPROD O B-ORGANISM I-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

(i, j) The cellular levels of Ca2+ ions after ligand (C10ORF99) stimulation of GPR15 in coelacanth, Asian bony tongue, and their mutants. (k, l) The cellular levels of Ca2+ ions after ligand (C10ORF99) stimulating GPR15 in Japanese quail and its mutants. The fold-change in calcium levels was calculated based on the fluorescence intensity (excitation and emission wavelengths: 490 and 520 nm, respectively).


( a - h ) The migration of HEK293 cells expressing mGPR15 , qGPR15 , tGPR15 , cGPR15 , aGPR15 , and their mutants induced by ligand C10ORF99 . Data information : All data shown are generated from at least three technical replicates . Error bars indicate SD ( n = 3 ) . * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ( t - test ) .

O O O O O O B-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-h) The migration of HEK293 cells expressing mGPR15, qGPR15, tGPR15, cGPR15, aGPR15, and their mutants induced by ligand C10ORF99. Data information: All data shown are generated from at least three technical replicates. Error bars indicate SD (n=3). ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05 (t-test).


( i - p ) The migration of CD4 + cells from GPR15 - knockout mice , engineered to express mGPR15 , qGPR15 , tGPR15 , cGPR15 , aGPR15 , and their mutants induced by ligand C10ORF99 . Data information : All data shown are generated from at least three technical replicates . Error bars indicate SD ( n = 3 ) . * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ( t - test ) . Ad - NC represents adeno - virus - based gene - delivery vector as negative control .

O O O O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O B-ORGANISM O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O

(i-p) The migration of CD4+ cells from GPR15-knockout mice, engineered to express mGPR15, qGPR15, tGPR15, cGPR15, aGPR15, and their mutants induced by ligand C10ORF99. Data information: All data shown are generated from at least three technical replicates. Error bars indicate SD (n=3). ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05 (t-test). Ad-NC represents adeno-virus-based gene-delivery vector as negative control.


( a ) CCR9 expression in the small intestine ( SI ) and colon of Gallus gallus and Lonchura striata . ( b ) CCR9 expression in the small intestine ( SI ) and colon of Chrysemys picta bellii and Pelodiscus sinensis . ( c ) CCR9 expression in the small intestine ( SI ) and colon of Rana catesbiana Shaw and Rana nigromaculata . ( d ) GPR15 expression in the small intestine ( SI ) and colon of Gallus gallus and Lonchura striata . ( e ) GPR15 expression in the small intestine ( SI ) and colon of Chrysemys picta bellii and Pelodiscus sinensis . ( f ) C10ORF99 expression in the small intestine ( SI ) and colon of Gallus gallus and Lonchura striata . ( g ) C10ORF99 expression in the small intestine ( SI ) and colon of Chrysemys picta bellii and Pelodiscus sinensis . ( h ) GPR15 and C10ORF99 expression in the small intestine ( SI ) and colon of Rana catesbeiana Shaw and Rana nigromaculata . Data information : All data shown are representative of at least three independent technical replicates . Error bars indicate SD ( n = 3 ) . UD , undetermined , * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; NS : no significance ( t - test ) .

O O O B-GENEPROD B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE O B-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM O O O O B-GENEPROD B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM O O O O B-GENEPROD B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM O O O O B-GENEPROD B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE O B-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM O O O O B-GENEPROD B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM O O O O B-GENEPROD B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE O B-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM O O O O B-GENEPROD B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CCR9 expression in the small intestine (SI) and colon of Gallus gallus and Lonchura striata. (b) CCR9 expression in the small intestine (SI) and colon of Chrysemys picta bellii and Pelodiscus sinensis. (c) CCR9 expression in the small intestine (SI) and colon of Rana catesbiana Shaw and Rana nigromaculata. (d) GPR15 expression in the small intestine (SI) and colon of Gallus gallus and Lonchura striata. (e) GPR15 expression in the small intestine (SI) and colon of Chrysemys picta bellii and Pelodiscus sinensis. (f) C10ORF99 expression in the small intestine (SI) and colon of Gallus gallus and Lonchura striata. (g) C10ORF99 expression in the small intestine (SI) and colon of Chrysemys picta bellii and Pelodiscus sinensis. (h) GPR15 and C10ORF99 expression in the small intestine (SI) and colon of Rana catesbeiana Shaw and Rana nigromaculata. Data information: All data shown are representative of at least three independent technical replicates. Error bars indicate SD (n=3). UD, undetermined, ***P-value < 0.001; **P-value < 0.01; NS: no significance (t-test).


( i ) GPR15 expression in CD4 + T cell from chicken small intestine ( SI ) and colon . Data information : All data shown are representative of at least three independent technical replicates . Error bars indicate SD ( n = 3 ) . UD , undetermined , * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; NS : no significance ( t - test ) .

O O O B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-ORGANISM B-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(i) GPR15 expression in CD4+ T cell from chicken small intestine (SI) and colon. Data information: All data shown are representative of at least three independent technical replicates. Error bars indicate SD (n=3). UD, undetermined, ***P-value < 0.001; **P-value < 0.01; NS: no significance (t-test).


( j ) The migration of CD4 + cells from chicken small intestine ( SI ) and colon induced by ligand C10ORF99 . Data information : All data shown are representative of at least three independent technical replicates . Error bars indicate SD ( n = 3 ) . UD , undetermined , * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; NS : no significance ( t - test ) .

O O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-ORGANISM B-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The migration of CD4+ cells from chicken small intestine (SI) and colon induced by ligand C10ORF99. Data information: All data shown are representative of at least three independent technical replicates. Error bars indicate SD (n=3). UD, undetermined, ***P-value < 0.001; **P-value < 0.01; NS: no significance (t-test).


The expression levels of GPR15 , C10ORF99 , FOXP3 in mice fed with corn , chicken , and mixture of corn plus chicken , commercial food chow and a positive control ( propionate added to commercial food chow ) at different times ( weanling mice were defined as w - 1 ) in colonic tissue were determined by q - RT - PCR . Each symbol or bar represents pooled RNA from colon tissues from three mice . Data information : All data shown are representative of at least three independent biological replicates . Error bars indicate SD ( n = 3 ) . * * * * P - value < 0 . 0001 * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ; NS : no significance ( One - way ANOVA ) .

O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM O O B-ORGANISM O B-ORGANISM O O O O B-ORGANISM O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM O O O O O O O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-TISSUE O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 expression levels of GPR15, C10ORF99, FOXP3 in mice fed with corn, chicken, and mixture of corn plus chicken, commercial food chow and a positive control (propionate added to commercial food chow) at different times (weanling mice were defined as w-1) in colonic tissue were determined by q-RT-PCR. Each symbol or bar represents pooled RNA from colon tissues from three mice. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).


The expression levels of IL - 10 in mice fed with corn , chicken , and mixture of corn plus chicken , commercial food chow and a positive control ( propionate added to commercial food chow ) at different times ( weanling mice were defined as w - 1 ) in colonic tissue were determined by q - RT - PCR . Each symbol or bar represents pooled RNA from colon tissues from three mice . Data information : All data shown are representative of at least three independent biological replicates . Error bars indicate SD ( n = 3 ) . * * * * P - value < 0 . 0001 * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ; NS : no significance ( One - way ANOVA ) .

O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O B-ORGANISM O B-ORGANISM O O O O B-ORGANISM O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM O O O O O O O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-TISSUE O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 expression levels of IL-10 in mice fed with corn, chicken, and mixture of corn plus chicken, commercial food chow and a positive control (propionate added to commercial food chow) at different times (weanling mice were defined as w-1) in colonic tissue were determined by q-RT-PCR. Each symbol or bar represents pooled RNA from colon tissues from three mice. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).