( H ) Relative in HT29 subject to nutrient starvation for 24 h . Atg7 knockdown increased survival . increase in survival following Atg7 knockdown was significantly reversed by simultaneously depleting β ‐ catenin ( double knockdown of Atg7 β ‐ catenin ) . Data are mean ± s . e . m . of three independent experiments performed in triplicate , * * * P0 . 001 ; * * P0 . 01 . Source data for this figure is available on 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 . Purified were resolved on a 4 - 12 % gel , revealed by silver , 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.


containing Rfa1 - TAP were synchronized at G1 , S , G2 / M phase used for TAP purification . Rfa1 - TAP associated complexes were analyzed by Western blot using CBP , Rfa2 , Rtt105 antibodies ( left panel ) . 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 visualized by Coomassie Brilliant Blue ( CBB ) . 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 ( FL ) Rtt105 deletion mutants ( Schematic of Rtt105 truncations is shown in upper panel ) were purified used to pull down Rfa1 . GST were used as a negative control . Isolated complexes were resolved on 15 % SDS - PAGE gels visualized by CBB .

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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 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 rtt105 ∆ released into HU - medium . average Rfa1 ChIP - Seq read density from released into HU - medium around ACS sites . ACS : ARS consensus .

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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 released at 16 ° C for 72 min . average Rfa2 ChIP - Seq read density surrounding ACS sites from 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 of progression through S phase . G1 - arrested were released into fresh YPD medium to allow progression through S phase at standard growth ( 25 ° C ) . Equal amounts of were collected at each time point . Rfa1 ChIP were performed using IgG beads for TAP - tag . ARS305 ARS607 , early fired replication origins ; ARS607 + 14kb , a corresponding 14kb away region of ARS607 . mean 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.


localization of Rfa1 is altered in rtt105 ∆ mutant . Rfa1 tagged with GFP at its C terminus was used to analyze localization of Rfa1 - GFP fusion ( Green ) envelop is visualized with Nup49 - mCherry fusion ( Red ) using 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 localization of Rfa1 in rtt105 ∆ mutant . wild type RFA1 gene was fused with an SV40 large T Antigen localization ( NLS ) at its 5 ' - end a GFP gene at its 3 ' end to obtain a construct , driven by RFA1 promoter to express NLS - RFA1 - GFP fusion . engineered construct was then transformed into WT or rtt105 ∆ mutant yeast to replace its endogenous RFA1 expression . resulting yeast were then visualized under microscope , GFP signals enriched in nuclei were scored . RFA1 - GFP lacking NLS was transformed into rtt105 ∆ rfa1 ∆ mutant as a control . DAPI indicates .

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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 localization of Rfa1 could not rescue RPA binding defects at replication regions in rtt105 ∆ . of Rfa2 ChIP - over total input was calculated . mean standard error ( SE ) of three biological replicates were shown . Statistical significance was evaluated on Student ' s t - ( * : 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 Rtt105 - RPA were resolved on 15 % SDS - PAGE gels analyzed by silver .

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


EMSA was performed to analyze ssDNA binding ability of RPA or Rtt105 - RPA . Cy3 - labeled 30nt ssDNA was used as substrate , reaction mixtures were resolved on native PAGE gels . of 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 was mixed with a 30nt - ssDNA in a 2 : 1 , reaction product were subjected to gel filtration chromatography . Equal volume of fraction 5 - 10 were resolved on SDS - PAGE for detecting by Ruby ( 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 , bound were resolved on SDS - PAGE visualized by silver .

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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 . of two patterns in each reaction were plotted in ( C ) . Please note a fraction of ssDNA shows both patterns . Rtt105 increases rate of ssDNA stretching upon RPA binding . Stretching rate was defined as change of ssDNA upon binding to RPA - mCherry in unit time as shown in ( B , arrow ) . Rtt105 increases extent of ssDNA stretching upon RPA binding . increment = at 8 minutes / 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 released into YPD medium containing HU for 45 min . average BrdU IP - Seq read density around fired ACS from released into YPD medium containing HU for 45 min sites . Snapshots of BrdU IP - Seq peaks at ARS305 from released into YPD medium at 16 ° C for 72 min . average BrdU IP - Seq read density around ACS sites from 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 rtt105 ∆ , of with Rad52 - foci is reported . Error bars indicate one standard deviation from three independent experiments with at least 150 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 rtt105 ∆ by immunoblotting of extracts with anti - Rad53 antibodies . Ponceau S ( Pon S ) 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 ) rtt105 ∆ mutant strains integrated with yWSS439 - 5ori ∆ yeast artificial chromosomes ( YACs ) . GCRs rates in 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 ∆ in two different backgrounds were assayed on normal growth media ( YPD ) on media containing indicated damaging agents , methyl - methane sulfonate ( MMS ) , camptothecin ( CPT ) , bleomycin ( Bleo ) , 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 , SHARPIN co - localize with Htt - Q97 . SH - SY5Y co - expressing Htt - Q25 - GFP or Htt - Q97 - GFP ( green ) 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 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 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 in cultured SH - SY5Y ( A ) Autofluorescent lipofuscin appears in 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 in cultured R6 / 2 mouse cortex 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 in cultured human HD frontal cortex ( C ) . Autofluorescent lipofuscin appears in 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 , M1 ubiquitin antibody was preincubated with recombinant tetra - ubiquitin linked via M1 ( 4 x M1 ubiquitin ) or K63 ( 4 x K63 ubiquitin ) .

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

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(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 analyzed by immunocytochemistry using an antibody against HA . DAPI ( blue ) . Scale bar , 10 µm .

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

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(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 . WT HAP1 ( control ) or HOIP KO HAP1 reconstituted with either WT HOIP or HOIP ∆ PUB were analyzed by immunocytochemistry . Data are displayed as mean ± SD were analyzed by one - way ANOVA followed by Bonferroni Multiple Comparison , n = 5 . Data information : * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

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

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(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 phospho - c - Jun in neurons . 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 were analyzed by 2 independent Mann - Whitney U - , n = 3 . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

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(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 silenced for HOIP . SH - SY5Y transiently transfected with Htt - Q25 or Htt - Q97 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 . of Htt - expressing with phospho - c - Jun is on Student ' s t from 3 independent experiments . Data are displayed as mean ± SD unless otherwise stated . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

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

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

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

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

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

(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 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 ) plasmids indicated . were lysed under denaturing conditions ( 1 % SDS ) , followed by immunoprecipitation of Htt via HA tag ( 1 % Triton X - 100 , 0 . 1 % SDS ) . Immunopurified were detected by Western blotting using M1 ubiquitin - specific antibody 1E3 anti - HA .

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 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 B-MEASURED_VAR 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 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

(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 was performed as described in ( F ) with HEK293T transfected with Htt - Q97 - HA control siRNA or OTULIN siRNA .

O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR 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 expressing Htt - Q97 - HA or Htt - Q97 - GFP were lysed under denaturing conditions in 1 . 5 % SDS . After centrifugation , pellets containing SDS - insoluble ( SDS - insoluble fraction ) were dissolved in formic acid . Formic acid - dissolved were analyzed by immunoblotting using M1 ubiquitin - specific 1F11 / 3F5 / Y102L antibody .

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

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

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

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

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

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

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

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

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

(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 - SHARPIN - specific mRNA is decreased in R6 / 2 striatum . of mRNA from striatum of 9 - 12 old R6 / 2 mice non - transgenic . Data are displayed as mean ± SD with n = 6 - 9 biological replicates from unpaired two - Student ' s t - . * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

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

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 B-MEASURED_VAR O O O O B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O 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 formed by Ataxin - 3 - Q84 , SOD1 G85R , TDP - 43 Q331K or Optineurin R96L . SH - SY5Y co - expressing disease - associated ( green ) HOIP ( red ) were analyzed by immunocytochemistry ; DAPI ( blue ) . Scale bar , 20

O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O 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 B-MEASURED_VAR O O O O O O O O O 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 under endogenous LUBAC expression . M1 - linked ubiquitin ( red ) disease - associated ( green ) were analyzed by immunocytochemistry in SH - SY5Y using 1E3 antibody ( Millipore ) ( B ) DAPI ( blue ) . Scale bar , 20 µm .

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

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

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

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

O O O O O O O O O O O O O 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 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 O O O 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 fraction of with Htt - Q97 . SH - SY5Y were transfected with Htt - Q97 either control siRNA , HOIP siRNA or OTULIN siRNA . Data are displayed as mean ± SD were analyzed by one - way ANOVA followed by Tukey ' s Multiple Comparison , n = 9 . Expression of HOIP OTULIN were analyzed by Western blotting . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 .

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

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

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

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

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

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

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

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

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

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

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) of Ca2 + ions after ligand ( C10ORF99 ) stimulation of GPR15 in human , mouse , Japanese quail , painted turtle . YM . 254890 inhibits Ca2 + ion in transfected with GPR15 receptors of vertebrates . fold - change in calcium was calculated on ( excitation emission wavelengths : 490 520 nm , respectively ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) of Ca2 + ions after ligand ( C10ORF99 ) stimulation of GPR15 in coelacanth , Asian bony tongue , their mutants . ( k , l ) of Ca2 + ions after ligand ( C10ORF99 ) stimulating GPR15 in Japanese quail its mutants . fold - change in calcium was calculated on ( excitation emission wavelengths : 490 520 nm , respectively ) .

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 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

(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 ) migration of HEK293 expressing mGPR15 , qGPR15 , tGPR15 , cGPR15 , aGPR15 , their mutants induced by ligand C10ORF99 . Data information : 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 - ) .

O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 O O O O O O O O O O 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 ) migration of CD4 + from GPR15 - knockout mice , engineered to express mGPR15 , qGPR15 , tGPR15 , cGPR15 , aGPR15 , their mutants induced by ligand C10ORF99 . Data information : 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 - ) . Ad - NC represents adeno - - gene - delivery vector as negative control .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 small intestine ( SI ) colon of Gallus gallus Lonchura striata . ( b ) CCR9 expression in small intestine ( SI ) colon of Chrysemys picta bellii Pelodiscus sinensis . ( c ) CCR9 expression in small intestine ( SI ) colon of Rana catesbiana Shaw Rana nigromaculata . ( d ) GPR15 expression in small intestine ( SI ) colon of Gallus gallus Lonchura striata . ( e ) GPR15 expression in small intestine ( SI ) colon of Chrysemys picta bellii Pelodiscus sinensis . ( f ) C10ORF99 expression in small intestine ( SI ) colon of Gallus gallus Lonchura striata . ( g ) C10ORF99 expression in small intestine ( SI ) colon of Chrysemys picta bellii Pelodiscus sinensis . ( h ) GPR15 C10ORF99 expression in small intestine ( SI ) colon of Rana catesbeiana Shaw Rana nigromaculata . Data information : 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 - ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O 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 from chicken small intestine ( SI ) colon . Data information : 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 - ) .

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

(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 ) migration of CD4 + from chicken small intestine ( SI ) colon induced by ligand C10ORF99 . Data information : 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 - ) .

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

(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).


expression of GPR15 , C10ORF99 , FOXP3 in mice fed with corn , chicken , mixture of corn plus chicken , commercial food chow 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 from colon tissues from three mice . Data information : 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 O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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).


expression of IL - 10 in mice fed with corn , chicken , mixture of corn plus chicken , commercial food chow 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 from colon tissues from three mice . Data information : 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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).


( f , g ) SCFAs acetic acid content in dietary experiments . Faecal samples from mice on indicated diets were analysed for SCFAs by chromatography . Data information : 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f, g) SCFAs and acetic acid content in dietary experiments. Faecal samples from mice on the indicated diets were analysed for SCFAs by gas chromatography. 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).


( h ) TNF - α IL - 6 in faecal samples from mice on indicated diet demonstrated no colitis in mouse groups . IL - 6 TNF - α production , as determined by performing enzyme - linked immunosorbent assays . Data information : 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 O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h) TNF-α and IL-6 levels in faecal samples from mice on the indicated diet demonstrated no colitis in all mouse groups. IL-6 and TNF-α protein production levels, as determined by performing enzyme-linked immunosorbent assays. 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).


( i ) Luciferase activities of constructs with mouse C10ORF99 promoter in HEK293 . Data information : 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 O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(i) Luciferase activities of constructs with the mouse C10ORF99 promoter in HEK293 cells. 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).


Luciferase activities of constructs with Japanese quail C10ORF99 promoter in HEK293 . Data information : 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 O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Luciferase activities of constructs with the Japanese quail C10ORF99 promoter in HEK293 cells. 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).


( A ) Single confocal sections of HeLa infected with GFP - expressing S . Typhimurium strains ( blue ) for 10 h , fixed immunolabelled for LAMP1 ( green ) ubiquitin ( Ub , red ) ( false coloured , scale bars , 5 µm ) . far right panels show merged of LAMP1 , ubiquitin Salmonella . Arrows indicate SCV - associated ubiquitinated .

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

(A) Single confocal sections of HeLa cells infected with GFP-expressing S. Typhimurium strains (blue) for 10 h, fixed and immunolabelled for LAMP1 (green) and ubiquitin (Ub, red) (false coloured, scale bars, 5 µm). The far right panels show merged images of LAMP1, ubiquitin and Salmonella. Arrows indicate SCV-associated ubiquitinated aggregates.


B ) of of infected containing SCV - associated ubiquitinated . were processed as in ( A ) analysed by microscopy .

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 percentage of infected cells containing SCV-associated ubiquitinated aggregates. Cells were processed as in (A) and analysed by fluorescence microscopy.


( C ) Kinetics of formation of SCV - associated ubiquitinated . were infected with GFP - expressing S . Typhimurium strains for indicated amount of time , processed as in ( A ) .

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

(C) Kinetics of the formation of SCV-associated ubiquitinated aggregates. Cells were infected with GFP-expressing S. Typhimurium strains for the indicated amount of time, and processed as in (A).


( D ) of of infected with SCV - associated ubiquitinated in HeLa infected with S . Typhimurium strains for 14 h . were processed as in ( A ) analysed by microscopy . For quantifications , a minimum of 50 infected were counted per experiment values are mean ± from at least 3 independent experiments . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

(D) Quantification of the percentage of infected cells with SCV-associated ubiquitinated aggregates in HeLa cells infected with S. Typhimurium strains for 14 h. Cells were processed as in (A) and analysed by fluorescence microscopy. For all quantifications, a minimum of 50 infected cells were counted per experiment and values are the mean ± SEM from at least 3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001.


A ) Immunoelectron microscopy of HeLa infected with S . Typhimurium strains for 12 h ( ubiquitin - 10 nm gold particles ; scale bars , 0 . 5 µm ) . Arrows indicate ubiquitin accumulations near within vacuoles .

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

A) Immunoelectron microscopy of HeLa cells infected with S. Typhimurium strains for 12 h (ubiquitin - 10 nm gold particles; scale bars, 0.5 µm). Arrows indicate ubiquitin accumulations near bacteria within vacuoles.


( B ) Single confocal sections of HeLa infected with GFP - expressing ΔsseL mutant ( blue ) for 10 h immunolabelled for ubiquitin ( chain types - Ub , red ) lysine - 48 - linked ( K48 - Ub , green ) or lysine - 63 - linked ( K63 - Ub , green ) ubiquitin chains ( false coloured , scale bars , 5 µm ) . Arrows indicate SCV - associated ubiquitin K63 - Ub labelling .

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

(B) Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue) for 10 h and immunolabelled for ubiquitin (all chain types - Ub, red) and lysine-48-linked (K48-Ub, green) or lysine-63-linked (K63-Ub, green) ubiquitin chains (false coloured, scale bars, 5 µm). Arrows indicate SCV-associated ubiquitin and K63-Ub labelling.


SseL DUB activity reduces recruitment of ubiquitin autophagic markers to Salmonella microcolonies . Single confocal sections of HeLa infected with GFP - expressing ΔsseL mutant ( blue ) for 12 h immunolabelled for ubiquitin ( Ub , red ) ( A ) p62 ( green ) or ( B ) LC3 ( green ) ( false coloured , scale bars , 5 µm ) . far right panels show merged of p62 or LC3 , ubiquitin Salmonella . Arrows indicate SCV - associated ubiquitin labelling with p62 or LC3 .

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SseL DUB activity reduces the recruitment of ubiquitin and autophagic markers to Salmonella microcolonies.Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue) for 12 h and immunolabelled for ubiquitin (Ub, red) and (A) p62 (green) or (B) LC3 (green) (false coloured, scale bars, 5 µm). The far right panels show merged images of p62 or LC3, ubiquitin and Salmonella. Arrows indicate SCV-associated ubiquitin labelling with p62 or LC3.


( C - E ) . of relative of ( C ) ubiquitin , ( D ) p62 ( E ) LC3 per microcolony ( see Materials Methods ) . were processed as in ( A ) a minimum of 30 microcolonies analysed for each infection per experiment . Results from representative experiments showing relative mean ± are shown . Similar results were obtained in at least 3 independent experiments . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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(C-E). Quantification of the relative fluorescence intensity of (C) ubiquitin, (D) p62 and (E) LC3 per microcolony (see Materials and Methods). Cells were processed as in (A) and a minimum of 30 microcolonies analysed for each bacterial infection per experiment. Results from representative experiments showing the relative mean fluorescence intensity ± SEM are shown. Similar results were obtained in at least 3 independent experiments. * p<0.05; ** p<0.01; *** p<0.001.


( A ) of of infected with SCV - associated ubiquitinated . HeLa were infected with ΔsseL mutant for 12 h . At 8 h post - infection , were subjected to indicated treatments for 4 h before fixation immunolabelling . A minimum of 50 were counted per condition in each experiment values are mean ± of at least 3 independent experiments . p values are relative to mean value of mock - treated . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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(A) Quantification of the percentage of infected cells with SCV-associated ubiquitinated aggregates. HeLa cells were infected with ΔsseL mutant bacteria for 12 h. At 8 h post-infection, cells were subjected to the indicated treatments for 4 h before fixation and immunolabelling. A minimum of 50 cells were counted per condition in each experiment and values are the mean ± SEM of at least 3 independent experiments. p values are relative to the mean value of mock-treated cells. * p<0.05; ** p<0.01; *** p<0.001.


( B ) Single confocal sections of HeLa infected with GFP - expressing ΔsseL mutant ( blue ) , treated processed as in ( A ) analysed by microscopy for ubiquitin ( Ub , red ) LC3 ( green ) ( scale bars , 5 µm ) . far right panels show merged of LC3 , ubiquitin Salmonella .

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(B) Single confocal sections of HeLa cells infected with GFP-expressing ΔsseL mutant bacteria (blue), treated and processed as in (A) and analysed by fluorescence microscopy for ubiquitin (Ub, red) and LC3 (green) (scale bars, 5 µm). The far right panels show merged images of LC3, ubiquitin and Salmonella.


( A ) Single confocal sections of RAW264 . 7 macrophages infected with GFP - expressing strains of S . Typhimurium ( green ) for 10 h immunolabelled for ubiquitin ( Ub , red ) ( scale bars , 5 µm ) .

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(A) Single confocal sections of RAW264.7 macrophages infected with GFP-expressing strains of S. Typhimurium (green) for 10 h and immunolabelled for ubiquitin (Ub, red) (scale bars, 5 µm).


( B ) of infected macrophages containing ubiquitinated 10 h after uptake . were processed as in ( A ) analysed by microscopy . A minimum of 50 were counted for each infection per experiment values are mean ± of at least 3 independent experiments . * p < 0 . 05 ; * * * p < 0 . 001 . Uninfected ( uninf ) were from same wells as infected therefore were exposed to extracellular . ( C ) Immunoelectron microscopy of RAW264 . 7macrophages infected with ΔsseL mutant for 12 h . Arrows arrowheads indicate ubiquitin ( 15 nm gold particles ) p62 ( 10 nm gold particles ) , respectively ( scale bar , 0 . 5 µm ) .

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(B) Quantification of infected macrophages containing ubiquitinated aggregates 10 h after bacterial uptake. Cells were processed as in (A) and analysed by fluorescence microscopy. A minimum of 50 cells were counted for each bacterial infection per experiment and values are the mean ± SEM of at least 3 independent experiments. * p<0.05; *** p<0.001. Uninfected cells (uninf) were from the same wells as infected and therefore were exposed to extracellular bacteria. (C) Immunoelectron microscopy of RAW264.7macrophages infected with the ΔsseL mutant bacteria for 12 h. Arrows and arrowheads indicate ubiquitin (15 nm gold particles) and p62 (10 nm gold particles), respectively (scale bar, 0.5 µm).


( D E ) Single confocal sections of RAW264 . 7 macrophages infected with GFP - expressing strains of S . Typhimurium ( blue ) immunolabelled for ubiquitin ( Ub , red ) ( D ) p62 ( green ) or ( E ) LC3 ( green ) . far right panels show merged of p62 or LC3 , ubiquitin Salmonella . Arrows indicate Arrows indicate SCV - associated ubiquitin labelling with p62 or LC3 ( scale bars , 5 µm ) . outlines nuclei are delineated by lines .

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(D and E) Single confocal sections of RAW264.7 macrophages infected with GFP-expressing strains of S. Typhimurium (blue) and immunolabelled for ubiquitin (Ub, red) and (D) p62 (green) or (E) LC3 (green). The far right panels show merged images of p62 or LC3, ubiquitin and Salmonella. Arrows indicate Arrows indicate SCV-associated ubiquitin labelling with p62 or LC3 (scale bars, 5 µm). Cell outlines and nuclei are delineated by white lines.


( A ) of large ( > 2 µm ) , ubiquitinated aggresome - like induced structures ( ALIS ) in macrophages . bone marrow - derived macrophages ( BMM ) were infected with indicated GFP - expressing S . Typhimurium strains for 10 h , immunolabelled for ubiquitin analysed by confocal microscopy . A minimum of 50 were counted for each infection per experiment values are mean ± of 3 independent experiments . Uninfected ( uninf ) were from same wells as infected therefore were exposed to extracellular . * p < 0 . 05 ; * * p < 0 . 01 .

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(A) Quantification of large (>2 µm), ubiquitinated aggresome-like induced structures (ALIS) in macrophages. Primary bone marrow-derived macrophages (BMM) were infected with the indicated GFP-expressing S. Typhimurium strains for 10 h, immunolabelled for ubiquitin and analysed by confocal microscopy. A minimum of 50 cells were counted for each bacterial infection per experiment and values are the mean ± SEM of 3 independent experiments. Uninfected cells (uninf) were from the same wells as infected and therefore were exposed to extracellular bacteria. * p<0.05; ** p<0.01.


( B ) of number of puromycin - induced ubiquitin inclusions in . HeLa were transfected with a vector expressing myc - SseL or myc - SseLC / A for 16 h followed by treatment with puromycin ( 5 µg / ml ) for 4 h immunolabelled with anti - myc , anti - p62 anti - ubiquitin . 50 were counted per condition in each experiment . Values are means ± of at least 3 independent experiments . * * p < 0 . 01 .

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(B) Quantification of the number of puromycin-induced ubiquitin inclusions in cells. HeLa cells were transfected with a vector expressing myc-SseL or myc-SseLC/A for 16 h followed by treatment with puromycin (5 µg/ml) for 4 h and immunolabelled with anti-myc, anti-p62 and anti-ubiquitin. 50 individual cells were counted per condition in each experiment. Values are the means ± SEM of at least 3 independent experiments. ** p<0.01.


( C ) Single confocal sections of HeLa transfected with a vector expressing myc - SseL for 16 h followed by treatment with puromycin ( 5 µg / ml ) for 4 h immunolabelled with anti - myc ( blue ) , anti - p62 ( green ) anti - ubiquitin ( Ub , red ) ( scale bars , 5 µm ) . lower panels show a merged image of p62 , ubiquitin myc - SseL . Arrows indicate ubiquitin p62 present in untransfected arrowheads indicate expressing myc -

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(C) Single confocal sections of HeLa cells transfected with a vector expressing myc-SseL for 16 h followed by treatment with puromycin (5 µg/ml) for 4 h and immunolabelled with anti-myc (blue), anti-p62 (green) and anti-ubiquitin (Ub, red) (scale bars, 5 µm). The lower panels show a merged image of p62, ubiquitin and myc-SseL. Arrows indicate ubiquitin and p62 aggregates present in untransfected cells and arrowheads indicate cells expressing myc-SseL


SseL interacts with p62 autophagic substrates . ( A B ) Co - immunoprecipitation from RAW264 . 7 macrophages infected with ΔsseL mutant expressing SseL - HA or SseLC / A - HA for 10 h . ( A ) Infectedcell lysates immunoprecipated fractions were probed with anti - HA , anti - ubiquitin anti - p62 antibodies . ( B ) At 7 h post - infection , infected were subjected to mock , 3 - MA or starvation ( Stv ) treatments for 3 h before harvesting processing as described in ( A ) . lysates immunoprecipiations were probed with anti - HA anti - ubiquitin antibodies .

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SseL interacts with p62 and autophagic substrates.(A and B) Co-immunoprecipitation from RAW264.7 macrophages infected with ΔsseL mutant bacteria expressing SseL-HA or SseLC/A-HA for 10 h. (A) Infectedcell lysates and immunoprecipated fractions were probed with anti-HA, anti-ubiquitin and anti-p62 antibodies. (B) At 7 h post-infection, infected cells were subjected to mock, 3-MA or starvation (Stv) treatments for 3 h before harvesting and processing as described in (A). Cell lysates and immunoprecipiations were probed with anti-HA and anti-ubiquitin antibodies.


( A ) Lipidated LC3B ( LC3 - II ) in RAW264 . 7 macrophages infected with indicated strains of S . Typhimurium for 12 h . were left untreated ( − ) or subjected to 10 mM NH4Cl treatment ( + ) for 2 h before harvesting .

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(A) Lipidated LC3B (LC3-II) levels in RAW264.7 macrophages infected with the indicated strains of S. Typhimurium for 12 h. Cells were left untreated (−) or subjected to 10 mM NH4Cl treatment (+) for 2 h before harvesting.


( B ) of relative LC3 II in infected . Denistometry of bands was analysed using Image J software . of LC3 - II / tubulin signal was normalized to 1 for wild - type untreated infections . graph shows mean relative LC3 II / tubulin ratios ± for 3 independent experiments .

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(B) Quantification of relative LC3 II levels in infected cells. Denistometry of bands was analysed using Image J software. The ratio of LC3-II/tubulin signal intensity was normalized to 1 for wild-type untreated infections. The graph shows the mean relative LC3 II/tubulin ratios ± SEM for 3 independent experiments.


C ) RAW264 . 7 or ( D ) murine bone marrow - derived macrophages were infected with indicated strains of S . Typhimurium for ( C ) 10 h or ( D ) 16 h replication was monitored using flow cytometry . Results are represented as fold replication normalized to wt ; values are means ± of at least 3 independent experiments . * p < 0 . 05 ; * * p < 0 . 01 .

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C) RAW264.7 or (D) murine bone marrow-derived primary macrophages were infected with the indicated strains of S. Typhimurium for (C) 10 h or (D) 16 h and bacterial replication was monitored using flow cytometry. Results are represented as fold replication normalized to wt; values are the means ± SEM of at least 3 independent experiments. * p<0.05; ** p<0.01.


( B , C ) of WT L - HRD KO mice at of 12 . ( n = 5 for each group ) Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(B, C) Body weight of the WT and L-HRD KO mice at the age of 12 weeks. (n=5 for each group) Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( D ) heights tibia lengths of WT L - HRD KO mice . ( n = 5 for each group ) Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(D) Body heights and tibia lengths of the WT and L-HRD KO mice. (n=5 for each group) Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( E ) Phosphorylation of STAT5 in WT HRD1 LKO liver .

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(E) Phosphorylation of STAT5 in WT and HRD1 LKO liver.


( E ) Hepatic growth hormone signaling target genes mRNA in WT L - HRD1 KO mice . ( n = 5 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) .

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(E) Hepatic growth hormone signaling target genes mRNA levels in the WT and L-HRD1 KO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.).


Hepatic growth hormone signaling target genes mRNA in WT L - HRD1 KO mice . ( n = 5 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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Hepatic growth hormone signaling target genes mRNA levels in the WT and L-HRD1 KO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( A ) Volcano plot of differential genes between WT HRD1 LKO livers .

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(A) Volcano plot of differential genes between WT and HRD1 LKO livers.


( B ) Hepatic Fgf21 mRNA in WT L - HRD1 KO mice . ( n = 5 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(B) Hepatic Fgf21 mRNA in the WT and L-HRD1 KO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( C ) Serum FGF21 in WT L - HRD1 KO mice . ( n = 5 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(C) Serum FGF21 protein levels in the WT and L-HRD1 KO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( D ) Correlation of gene expression ( Red : Upregulated genes Blue : Downrelated genes ) between HRD1 LKO mice FGF21 transgenic mice .

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(D) Correlation analysis of the gene expression (Red: Upregulated genes and Blue: Downrelated genes) between HRD1 LKO mice and FGF21 transgenic mice.


( E ) of mated with proven stud at onset of puberty in WT L - HRD KO mice . ( n = 10 for each group ) Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(E) Percentage of mated with proven stud males and age at onset of puberty in the female WT and L-HRD KO mice. (n=10 for each group) Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( F ) Examples of ovarian histology from WT HRD1 LKO mice .

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(F) Examples of ovarian histology from WT and HRD1 LKO mice.


( G ) Representative examples of estrus cycles in WT L - HRD1 KO mice as determined by vaginal cytology .

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(G) Representative examples of estrus cycles in WT and L-HRD1 KO mice as determined by vaginal cytology.


( H ) of feeding , water intake activity in time . ( n = 8 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(H) Percentage of feeding, water intake and activity in the light time. (n=8 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( I ) H & E stain of adipose tissue WT HRD1 LKO mice 14 after HFD feeding .

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(I) H&E stain of white adipose tissue WT and HRD1 LKO mice 14 weeks after HFD feeding.


( J ) Ucp1 Dio2 mRNA of adipose tissue from ( I ) ( n = 5 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(J) Ucp1 and Dio2 mRNA levels of adipose tissue from (I) (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( B ) Volcano plot of differential between WT HRD1 LKO livers .

(B) Volcano plot of differential proteins between WT and HRD1 LKO livers.


( F ) of IRE1α , NRF1 , RXRβ , NOTCH1 , SIRT3 GAPDH in liver of WT HRD1 LKO mice under refed condition . ( n = 6 for each group ) .

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(F) Protein levels of IRE1α, NRF1, RXRβ, NOTCH1, SIRT3 and GAPDH in the liver of WT and HRD1 LKO mice under the refed condition. (n=6 for each group).


Hepatic CREBH in WT L - HRD1 KO mice . ( n = 6 for each group ) .

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Hepatic CREBH protein levels in the WT and L-HRD1 KO mice. (n=6 for each group).


Hepatic CREBH mRNA ( B ) in WT L - HRD1 KO mice . ( n = 6 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) .

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Hepatic CREBH mRNA (B) levels in the WT and L-HRD1 KO mice. (n=6 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.).


( C ) Western Blot CREBH stability after HRD1 over - expression . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(C) Western Blot analysis CREBH protein stability after HRD1 over-expression. Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( D ) Western Blot of hepatic CREBH HRD1 in WT HRD1 LKO mice . mice were fasted 16 hours refed for another 4 hours .

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(D) Western Blot analysis of hepatic CREBH and HRD1 protein levels in WT and HRD1 LKO mice. The mice were fasted 16 hours and refed for another 4 hours.


( E ) Western Blot interaction of CREBH HRD1 after immunoprecipitates of Flag - agarose in transfected HEK293T .

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(E) Western Blot analysis interaction of CREBH and HRD1 after immunoprecipitates of Flag-agarose in transfected HEK293T.


( F ) Endogenous interaction between CREBH HRD1 in liver .

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(F) Endogenous interaction between CREBH and HRD1 in liver.


( G ) Western Blot ubiquitination of CREBH after immunoprecipitates of Flag - agarose in transfected HEK293T .

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(G) Western Blot analysis ubiquitination of CREBH after immunoprecipitates of Flag-agarose in transfected HEK293T.


( H ) Hepatic CREBH ubiquitination level in WT L - HRD1 KO

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(H) Hepatic CREBH ubiquitination level in the WT and L-HRD1 KO mice


( I ) Relative of CREBH , HRD1 ubiquitination of CREBH . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(I) Relative protein levels of CREBH, HRD1 and ubiquitination of CREBH. Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( J ) hepatocytes were isolated from WT HRD1 LKO mice Western Blot CREBH stability .

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(J) Primary hepatocytes were isolated from WT and HRD1 LKO mice and Western Blot analysis CREBH protein stability.


( L ) Interactions of full - C - terminal of HRD1 with CREBH were measured by Co - IP . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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

(L) Interactions of full-length and C-terminal of HRD1 with CREBH were measured by Co-IP. Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( B ) Interactions of full - activated form of CREBH with HRD1 were measured by Co - IP .

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) Interactions of full-length and activated form of CREBH with HRD1 were measured by Co-IP.


( C , D ) Ubiquitination of CREBH CREBH K294R were measured after co - transfected C - terminal of HRD1 .

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

(C, D) Ubiquitination of CREBH and CREBH K294R were measured after co-transfected C-terminal of HRD1.