Figure 4A Diagram of aex - 2 genomic structure . The boxes and lines represent exons and introns . Black and gray indicate coding sequence and UTRs . The white asterisk marks the location of the R232Q mutation site of sa3 allele . B - K Confocal images of AIY presynaptic site labeled with GFP : : RAB - 3 in wild type ( B ) , nlp - 40 ( tm4085 ) ( C ) , aex - 2 ( sa3 ) ( D ) , nlp - 40 ( tm4085 ) ; aex - 2 ( sa3 ) ( E ) over - expression NLP - 40 in aex - 2 ( sa3 ) mutants ( F ) and aex - 2 ( sa3 ) with a wild - type aex - 2 transgene driven by the endogenous promoter ( Paex - 2 ) ( G ) , neuron - specific promoter ( Prab - 3 ) ( H ) , AIY - specific promoter ( Pttx - 3 ) ( I ) intestinal - specific promoter ( Pges - 1 ) ( J ) or muscle - specific promoter ( Pmyo - 3 ) ( K ) . L - M Quantification of the fluorescence intensity of AIY GFP : : RAB - 3 for the indicated genotypes . Each dot represents one animal . Data were collected from at least three independent experiments . Transgenic data are averaged from at least two independent lines . tg : Paex - 2 : : aex - 2 , Pttx - 3 : : aex - 2 or Pges - 1 : : aex - 2 transgene , + : wild type ; - : mutant or without tg . The AIY synaptic region was quantified for the synaptic intensity analysis . N - Q ' Confocal images of Paex - 2 : : mNeonGreen of whole animal with bright field ( N ) , the head region labeled with Paex - 2 : : mNeonGreen and Pttx - 3 : : mCherry with green channel ( O ) , red channel ( P ) and the merged channels ( Q ) . ( O ' - Q ' ) are the cross sections corresponding to the dashed line sites .

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

Figure 4A Diagram of aex-2 genomic structure. The boxes and lines represent exons and introns. Black and gray indicate coding sequence and UTRs. The white asterisk marks the location of the R232Q mutation site of sa3 allele. B-K Confocal images of AIY presynaptic site labeled with GFP::RAB-3 in wild type(B), nlp-40(tm4085)(C), aex-2(sa3)(D), nlp-40(tm4085); aex-2(sa3) (E) over-expression NLP-40 in aex-2(sa3) mutants(F) and aex-2(sa3) with a wild-type aex-2 transgene driven by the endogenous promoter (Paex-2) (G), neuron-specific promoter (Prab-3) (H) , AIY-specific promoter (Pttx-3) (I) intestinal-specific promoter (Pges-1) (J) or muscle-specific promoter (Pmyo-3)(K). L-M Quantification of the fluorescence intensity of AIY GFP::RAB-3 for the indicated genotypes. Each dot represents one animal. Data were collected from at least three independent experiments. Transgenic data are averaged from at least two independent lines. tg: Paex-2::aex-2, Pttx-3::aex-2 or Pges-1::aex-2 transgene, +: wild type ; -: mutant or without tg. The AIY synaptic region was quantified for the synaptic intensity analysis.N-Q' Confocal images of Paex-2::mNeonGreen of whole animal with bright field (N), the head region labeled with Paex-2::mNeonGreen and Pttx-3::mCherry with green channel (O), red channel (P) and the merged channels (Q). (O'-Q') are the cross sections corresponding to the dashed line sites.


Figure 5A - A ' Representative confocal images showing inactive ( A ) and active ( A ' ) state of AIY : : GCaMP6s ( with mod - 1 promoter ) colabeled with mCherry ( with ttx - 3 promoter ) ( red ) . Dashed ovals mark the AIY zone 2 region where GCaMP was quantified . B Representative AIY calcium activity over 60 seconds for the indicated genotypes . C - D Quantification of the peak frequency and amplitude of AIY : : GCaMP6s in for the indicated genotypes .

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Figure 5A-A' Representative confocal images showing inactive (A) and active(A') state of AIY::GCaMP6s (with mod-1 promoter) colabeled with mCherry (with ttx-3 promoter) (red). Dashed ovals mark the AIY zone 2 region where GCaMP was quantified.B Representative AIY calcium activity over 60 seconds for the indicated genotypes. C-D Quantification of the peak frequency and amplitude of AIY::GCaMP6s in for the indicated genotypes.


Figure 6B - G Confocal images of Prab - 3 : : GFP : : RAB - 3 in the nerve region corresponding to the dashed box in ( A ) of wild type ( B ) , cwn - 2 ( ok895 ) ( C ) , cfz - 2 ( ok1201 ) ( D ) , pop - 1 ( hu9 ) ( E ) , nlp - 40 ( tm4085 ) ( F ) and aex - 2 ( sa3 ) ( G ) mutants . H - I Quantification of the GFP : : RAB - 3 intensity in the nerve ring synaptic region for the indicated genotypes . Each dot represents one animal . Data were collected from at least three independent experiments . Transgenic data are averaged from at least two independent lines .

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Figure 6B-G Confocal images of Prab-3::GFP::RAB-3 in the nerve region corresponding to the dashed box in (A) of wild type (B), cwn-2(ok895) (C), cfz-2(ok1201) (D), pop-1(hu9) (E), nlp-40(tm4085) (F) and aex-2(sa3) (G) mutants. H-I Quantification of the GFP::RAB-3 intensity in the nerve ring synaptic region for the indicated genotypes. Each dot represents one animal. Data were collected from at least three independent experiments. Transgenic data are averaged from at least two independent lines.


Figure 1B Endogenous USP45 was immunoprecipitated ( IP ) from wild ‐ type ( WT ) and USP45 knockout ( KO ) KBM7 cells lines . The immunoprecipitates were resolved on a polyacrylamide gel and stained with Instant Blue . The gel was divided into the indicated sections , and proteins identified by mass spectrometry are detailed in Supplementary Table S1 . The key proteins that we focus on in this study are indicated . C Endogenous USP45 was immunoprecipitated from the WT and KO KBM7 cell lines and subjected to immunoblotting with the indicated antibodies . D Endogenous USP45 was immunoprecipitated from WT and USP45 KO U2OS cells lines and analysed as in ( B ) . Mascot scores of the interactors are detailed in Supplementary Table S2 . E Endogenous USP45 was immunoprecipitated from the WT or KO U2OS cell lines and subjected to immunoblotting with the indicated antibodies . Similar results were obtained in at least two separate experiments .

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Figure 1B Endogenous USP45 was immunoprecipitated (IP) from wild‐type (WT) and USP45 knockout (KO) KBM7 cells lines. The immunoprecipitates were resolved on a polyacrylamide gel and stained with Instant Blue. The gel was divided into the indicated sections, and proteins identified by mass spectrometry are detailed in Supplementary Table S1. The key proteins that we focus on in this study are indicated.C Endogenous USP45 was immunoprecipitated from the WT and KO KBM7 cell lines and subjected to immunoblotting with the indicated antibodies.D Endogenous USP45 was immunoprecipitated from WT and USP45 KO U2OS cells lines and analysed as in (B). Mascot scores of the interactors are detailed in Supplementary Table S2.E Endogenous USP45 was immunoprecipitated from the WT or KO U2OS cell lines and subjected to immunoblotting with the indicated antibodies. Similar results were obtained in at least two separate experiments.


Figure 2A Extracts of HEK293 cells were analysed by gel filtration on a Superdex 200 10 / 300 GL preparative grade column ( GE Healthcare ) in buffer containing 0 . 15 M NaCl , and every second fraction denatured and analysed by immunoblotting with the indicated antibodies . The white dotted lines indicate that the samples were run on different polyacrylamide gels . B Endogenous USP45 or ERCC1 was immunoprecipitated from HEK293 cell lysates and subjected to immunoblotting with the indicated antibodies . C Yeast two ‐ hybrid ( Y2H ) assays were performed with a GAL4 DNA ‐ binding domain fusion and / or activation domain for each protein indicated in the table to detect interaction between these proteins . Cells grown on media lacking LEU , TRP and HIS ( to select for bait and prey plasmids ) or tested for lacZ reporter gene activity . D , E Endogenous SLX4 and ERCC1 were immunoprecipitated from extracts of WT or ERCC1 KO MEFs ( D ) , or WT or SLX4 KO MEFs ( E ) and subjected to immunoblotting with the indicated antibodies . F Extracts of WT or SLX4 KO MEFs were analysed by gel filtration as described in ( A ) . Similar results were obtained in at least two separate experiments .

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Figure 2A Extracts of HEK293 cells were analysed by gel filtration on a Superdex 200 10/300 GL preparative grade column (GE Healthcare) in buffer containing 0.15 M NaCl, and every second fraction denatured and analysed by immunoblotting with the indicated antibodies. The white dotted lines indicate that the samples were run on different polyacrylamide gels.B Endogenous USP45 or ERCC1 was immunoprecipitated from HEK293 cell lysates and subjected to immunoblotting with the indicated antibodies.C Yeast two‐hybrid (Y2H) assays were performed with a GAL4 DNA‐binding domain fusion and/or activation domain for each protein indicated in the table to detect interaction between these proteins. Cells grown on media lacking LEU, TRP and HIS (to select for bait and prey plasmids) or tested for lacZ reporter gene activity.D, E Endogenous SLX4 and ERCC1 were immunoprecipitated from extracts of WT or ERCC1 KO MEFs (D), or WT or SLX4 KO MEFs (E) and subjected to immunoblotting with the indicated antibodies.F Extracts of WT or SLX4 KO MEFs were analysed by gel filtration as described in (A). Similar results were obtained in at least two separate experiments.


Figure 3A HEK293 cells were transiently transfected with the indicated GFP fusion constructs encoding full ‐ length or indicated fragments of USP45 . Thirty ‐ six hours post ‐ transfection cells were lysed and GFP immunoprecipitated . The immunoprecipitates were resolved on a polyacrylamide gel and stained with Instant Blue or subjected to immunoblotting with the indicated antibodies . GFP empty vector was used as a negative control . C , D HEK293 cells were transiently transfected with constructs expressing the indicated wild ‐ type or mutant GFP - USP45 [ 1 - 62 ] fragment ( C ) or GFP full ‐ length USP45 ( D ) or empty GFP ( C and D ) as a control and processed as described in ( A ) . LICOR - Odyssey quantitation of results shown in ( C ) is shown in Supplementary Fig S4 . E As in ( C ) except HEK293 cells were transfected with the indicated N ‐ terminal Znf ‐ UBP domain ‐ containing DUBs . Similar results were obtained in at least two separate experiments .

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

Figure 3A HEK293 cells were transiently transfected with the indicated GFP fusion constructs encoding full‐length or indicated fragments of USP45. Thirty‐six hours post‐transfection cells were lysed and GFP immunoprecipitated. The immunoprecipitates were resolved on a polyacrylamide gel and stained with Instant Blue or subjected to immunoblotting with the indicated antibodies. GFP empty vector was used as a negative control.C, D HEK293 cells were transiently transfected with constructs expressing the indicated wild‐type or mutant GFP-USP45 [1-62] fragment (C) or GFP full‐length USP45 (D) or empty GFP (C and D) as a control and processed as described in (A). LICOR-Odyssey quantitation of results shown in (C) is shown in Supplementary Fig S4.E As in (C) except HEK293 cells were transfected with the indicated N‐terminal Znf‐UBP domain‐containing DUBs. Similar results were obtained in at least two separate experiments.


Figure 4A HEK293 cells were treated with the proteasome inhibitor lactacystin ( 5 μM ) or bortezomib ( 10 μM ) for the times indicated . Cells were lysed in the presence of 0 . 1 M N ‐ ethylmaleimide to inhibit deubiquitinylation and lysates were subjected to immunoblotting with indicated antibodies . B Wild ‐ type ( WT ) and USP45 knockout ( KO ) U2OS cells were lysed and subjected to immunoblotting as in ( A ) . C USP45 KO U2OS cells were transiently transfected with a construct encoding FLAG ‐ ERCC1 . Thirty ‐ six hours post ‐ transfection cells were treated with lactacystin ( 5 μM ) for 6 h and lysed with a buffer containing 0 . 1 M N ‐ ethylmaleimide . Flag ‐ ERCC1 was immunoprecipitated and treated in the absence or presence of 0 . 1 μg of recombinant USP45 wild ‐ type ( WT ) , non ‐ ERCC1 ‐ binding USP45 [ Asp25Ala , Glu26Ala ] ( AA ) or catalytically inactive USP45 [ Cys199Ala ] ( CA ) for 1 h . The reactions were terminated by addition of sample buffer and analysed by immunoblot and Coomassie staining . Similar results were obtained in at least two separate experiments .

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Figure 4A HEK293 cells were treated with the proteasome inhibitor lactacystin (5 μM) or bortezomib (10 μM) for the times indicated. Cells were lysed in the presence of 0.1 M N‐ethylmaleimide to inhibit deubiquitinylation and lysates were subjected to immunoblotting with indicated antibodies.B Wild‐type (WT) and USP45 knockout (KO) U2OS cells were lysed and subjected to immunoblotting as in (A).C USP45 KO U2OS cells were transiently transfected with a construct encoding FLAG‐ERCC1. Thirty‐six hours post‐transfection cells were treated with lactacystin (5 μM) for 6 h and lysed with a buffer containing 0.1 M N‐ethylmaleimide. Flag‐ERCC1 was immunoprecipitated and treated in the absence or presence of 0.1 μg of recombinant USP45 wild‐type (WT), non‐ERCC1‐binding USP45 [Asp25Ala, Glu26Ala] (AA) or catalytically inactive USP45 [Cys199Ala] (CA) for 1 h. The reactions were terminated by addition of sample buffer and analysed by immunoblot and Coomassie staining. Similar results were obtained in at least two separate experiments.


Figure 5A Clonogenic survival assays were carried out in wild ‐ type ( WT ) , USP45 knockout ( KO ) or USP45 KO cells re ‐ expressing wild ‐ type USP45 ( RESWT ) or catalytically inactive USP45 [ Cys199Ala ] ( RESCA ) . The cells were treated with the indicated doses of mitomycin C ( MMC ) 16 h . Medium was changed and number of colonies quantified after 10 days . Each data point is the average of 3 experiments undertaken with 6 replicates ± SD . B Control immunoblot analysis demonstrating that in the USP45 KO U2OS rescue cell lines , similar levels of wild ‐ type and catalytically inactive USP45 [ Cys199Ala ] were expressed . C , D As above , except rescue experiments are undertaken using the non ‐ ERCC1 ‐ binding USP45 [ Asp25Ala , Glu26Ala ] mutant . E , F As in ( A ) , except that DNA damage is induced using UV ‐ C irradiation ( E ) or 16 h hydroxyurea treatment ( F ) . Similar results were obtained in at two separate experiments .

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

Figure 5A Clonogenic survival assays were carried out in wild‐type (WT), USP45 knockout (KO) or USP45 KO cells re‐expressing wild‐type USP45 (RESWT) or catalytically inactive USP45 [Cys199Ala] (RESCA). The cells were treated with the indicated doses of mitomycin C (MMC) 16 h. Medium was changed and number of colonies quantified after 10 days. Each data point is the average of 3 experiments undertaken with 6 replicates ± SD.B Control immunoblot analysis demonstrating that in the USP45 KO U2OS rescue cell lines, similar levels of wild‐type and catalytically inactive USP45 [Cys199Ala] were expressed.C, D As above, except rescue experiments are undertaken using the non‐ERCC1‐binding USP45 [Asp25Ala, Glu26Ala] mutant.E, F As in (A), except that DNA damage is induced using UV‐C irradiation (E) or 16 h hydroxyurea treatment (F). Similar results were obtained in at two separate experiments.


Figure 6A Wild ‐ type ( WT ) , USP45 knockout ( KO ) or USP45 KO cells re ‐ expressing wild ‐ type USP45 ( RESWT ) or catalytically inactive USP45 [ Cys199Ala ] ( RESCA ) were used to analyse ERCC1 foci formation . Staining and analysis of endogenous ERCC1 foci formation was undertaken before ( left panel ) , or after DNA damage induction by mitomycin C ( 40 ng / ml , 16 h , middle panel ) or UV ( 20 J / m2 followed by 3 ‐ h recovery , right panel ) . Three independent experiments were performed in which 500 cells per experiment were analysed . Scale bar , 10 μm . B Proportion of cells displaying more than 10 endogenous ERCC1 foci were quantified . Three independent experiments were performed in which 500 cells per experiment were analysed . Results are the mean of 3 experiments ± SD . C The wild ‐ type and USP45 knockout U2OS cells were treated with mitomycin C ( 40 ng / ml , 16 h ) and total number of ERCC1 staining foci per cell was quantified in at least 50 independent cells at the indicated times . Similar results were obtained in two separate experiments . The data are presented as the average number of ERCC1 staining foci per cell . D As in ( C ) , except that γ ‐ H2AX staining foci were analysed . The data are presented as the average number of γ ‐ H2AX staining foci per cell .

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Figure 6A Wild‐type (WT), USP45 knockout (KO) or USP45 KO cells re‐expressing wild‐type USP45 (RESWT) or catalytically inactive USP45 [Cys199Ala] (RESCA) were used to analyse ERCC1 foci formation. Staining and analysis of endogenous ERCC1 foci formation was undertaken before (left panel), or after DNA damage induction by mitomycin C (40 ng/ml, 16 h, middle panel) or UV (20 J/m2 followed by 3‐h recovery, right panel). Three independent experiments were performed in which 500 cells per experiment were analysed. Scale bar, 10 μm.B Proportion of cells displaying more than 10 endogenous ERCC1 foci were quantified. Three independent experiments were performed in which 500 cells per experiment were analysed. Results are the mean of 3 experiments ± SD.C The wild‐type and USP45 knockout U2OS cells were treated with mitomycin C (40 ng/ml, 16 h) and total number of ERCC1 staining foci per cell was quantified in at least 50 independent cells at the indicated times. Similar results were obtained in two separate experiments. The data are presented as the average number of ERCC1 staining foci per cell.D As in (C), except that γ‐H2AX staining foci were analysed. The data are presented as the average number of γ‐H2AX staining foci per cell.


Figure 7A Representative immunofluorescence microscopy images of endogenous wild ‐ type ( WT ) and USP45 knockout ( KO ) U2OS cells treated with no drug , BrdU ( 10 μM , 24 h ) , psoralen or angelicin ( 25 μM , 3 h ) . Cells were fixed 10 min after laser micro ‐ irradiation and stained for localisation of endogenous USP45 , phosphorylated γ ‐ H2AX and DAPI . The right ‐ hand panel shows magnification of one micro ‐ irradiated cell . Similar results were obtained in at least three independent experiments . Scale bar , 10 μm .

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Figure 7A Representative immunofluorescence microscopy images of endogenous wild‐type (WT) and USP45 knockout (KO) U2OS cells treated with no drug, BrdU (10 μM, 24 h), psoralen or angelicin (25 μM, 3 h). Cells were fixed 10 min after laser micro‐irradiation and stained for localisation of endogenous USP45, phosphorylated γ‐H2AX and DAPI. The right‐hand panel shows magnification of one micro‐irradiated cell. Similar results were obtained in at least three independent experiments. Scale bar, 10 μm.


Figure 8A U2OS cells were transiently transfected with the GFP - USP45 or GFP alone . Thirty ‐ six hours post ‐ transfection cells were irradiated with UV ‐ C ( 150 J / m2 ) through a micropore filter ( lower panels ) or left untreated ( upper panels ) . Five minutes post ‐ irradiation , cells were fixed and co ‐ immunostained with antibodies recognising cyclobutane pyrimidine dimer ( CPD ) DNA lesions and GFP . The white arrows indicate co ‐ localisation between GFP - USP45 and CPD . Scale bar , 10 μm . B U2OS wild ‐ type ( WT ) and USP45 knockout ( KO ) cells were irradiated with UV ‐ C ( 20 J / m2 ) . At the indicated post ‐ irradiation times , genomic DNA was extracted and subjected to Southern dot blot analysis using antibodies recognising CPD and dsDNA total antibody . Data are shown from a triplicate experiment in which each blot is derived from independent cells . C Same as ( B ) but using a High Sensitivity CPD / Cyclobutane Pyrimidine Dimer Elisa kit ( NM ‐ MA ‐ K001 ) from Cosmo Bio . For the DNA damage detection , the manufacturer protocol was followed . Absorbance at 492 was measured that represent amount of CPDs in each sample at indicated times . Calf thymus DNA , UV ‐ Cirradiated ( 10 J / m2 ) and not irradiated was used as positive and negative samples , respectively .

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

Figure 8A U2OS cells were transiently transfected with the GFP-USP45 or GFP alone. Thirty‐six hours post‐transfection cells were irradiated with UV‐C (150 J/m2) through a micropore filter (lower panels) or left untreated (upper panels). Five minutes post‐irradiation, cells were fixed and co‐immunostained with antibodies recognising cyclobutane pyrimidine dimer (CPD) DNA lesions and GFP. The white arrows indicate co‐localisation between GFP-USP45 and CPD. Scale bar, 10 μm.B U2OS wild‐type (WT) and USP45 knockout (KO) cells were irradiated with UV‐C (20 J/m2). At the indicated post‐irradiation times, genomic DNA was extracted and subjected to Southern dot blot analysis using antibodies recognising CPD and dsDNA total antibody. Data are shown from a triplicate experiment in which each blot is derived from independent cells.C Same as (B) but using a High Sensitivity CPD/Cyclobutane Pyrimidine Dimer Elisa kit (NM‐MA‐K001) from Cosmo Bio. For the DNA damage detection, the manufacturer protocol was followed. Absorbance at 492 was measured that represent amount of CPDs in each sample at indicated times. Calf thymus DNA, UV‐Cirradiated (10 J/m2) and not irradiated was used as positive and negative samples, respectively.


Figure 9A Live ‐ cell fluorescence microscopy analysis of U2OS cells stably expressing either GFPNLS empty , wild ‐ type ( WT ) USP45 or catalytically inactive USP45 [ Cys199Ala ] . Cells were treated with BrdU ( 10 μM , 24 h ) , psoralen or angelicin ( 25 μM , 3 h ) and images captured at the indicated times after laser micro ‐ irradiation . The location of the micro ‐ irradiation stripe in each cell is indicated with a yellow line . Similar results were obtained in at least two independent experiments . Scale bar , 10 μm .

Figure 9A Live‐cell fluorescence microscopy analysis of U2OS cells stably expressing either GFPNLS empty, wild‐type (WT) USP45 or catalytically inactive USP45 [Cys199Ala]. Cells were treated with BrdU (10 μM, 24 h), psoralen or angelicin (25 μM, 3 h) and images captured at the indicated times after laser micro‐irradiation. The location of the micro‐irradiation stripe in each cell is indicated with a yellow line. Similar results were obtained in at least two independent experiments. Scale bar, 10 μm.


Figure 3 ( E ) In C . elegans , all conserved ciliary cluster genes with available mutants as well as genes of particular interest for which mutants were generated in the course of this study were screened using the dye - fill assay to test for cilium structural integrity . 12 amphid neurons in the head and 4 phasmid neurons in the tail , both featuring non - motile sensory cilia , stain with the lipophilic dye DiI . Failure to take up dye is an indicator of ciliary structural defects . IFT mutants such as osm - 5 ( p813 ) tend to display dye - fill null phenotypes , while other ciliary mutants present weaker phenotypes . Phenotypes were scored using a scale from 0 ( no dye - fill phenotype ) to 4 ( null ) . ( F ) Scatter plot displaying the phenotypes of mutants in IFT and transition zone components . Each point on scatter plots represents one gene , with values shifted slightly to avoid overlap . ( G ) Scatter plot displaying the phenotypes for all known ( grey ) and previously uncharacterised genes ( green ) within the ciliary cluster , plotted as in ( F ) .

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Figure 3(E) In C. elegans, all conserved ciliary cluster genes with available mutants as well as genes of particular interest for which mutants were generated in the course of this study were screened using the dye-fill assay to test for cilium structural integrity. 12 amphid neurons in the head and 4 phasmid neurons in the tail, both featuring non-motile sensory cilia, stain with the lipophilic dye DiI. Failure to take up dye is an indicator of ciliary structural defects. IFT mutants such as osm-5(p813) tend to display dye-fill null phenotypes, while other ciliary mutants present weaker phenotypes. Phenotypes were scored using a scale from 0 (no dye-fill phenotype) to 4 (null).(F) Scatter plot displaying the phenotypes of mutants in IFT and transition zone components. Each point on scatter plots represents one gene, with values shifted slightly to avoid overlap. (G) Scatter plot displaying the phenotypes for all known (grey) and previously uncharacterised genes (green) within the ciliary cluster, plotted as in (F).


Figure 4 ( B ) Schematic and confocal microscopy images of scolopidia in chordotonal organ of the fly . Each scolopidium contains two cilia with their ends embedded in a cap cell . Scolopidium outline , cilia and ciliary dilation can be visualized by DIC . Immunofluorescence micrographs with Sas - 4 and NompC antibodies used to mark basal bodies and ciliary tips , respectively . DIC images of flies depleted of candidate genes by RNAi reveal relatively few defects on par with IFT genes such as IFT140 , with the exception of misplaced dilations for Cep135 and CFAP97D1 ( arrows ) . Missing cilia ( asterisks ) as with SAS4 or IFT52 are not observed . NompC / Sas - 4 staining reveals more subtle phenotypes , including mislocalized NompC signal ( with CFAP97D1 , Cep135 , Elmod , Mapk15 and Tex9 , arrowheads ) and apparent ciliary structural defects ( Cep104 and Cables1 , arrows ) . At least 3 animals examined per condition . ( E ) Mature sperm can be dissected from the seminal vesicle and motility analyzed using high speed video capture in dark field microscopy . Sinusoidal motion can be seen in wild type . In RNAi depletions of certain known motility genes ( here IQUB and GAS8 ) , this motion is reduced or even absent . Arrowheads mark position of individual bends in sequential frames . At least 3 movies made from different animals per condition . ( F ) Spermatogenesis in wild - type and RNAi depletions of selected genes , highlighting the stage where morphogenesis first becomes noticeably abnormal . At least 6 testes from 3 different animals examined per condition . ( G ) Graph summarizing sperm defects observed for candidate genes chosen for further analysis , with selected known centriolar / ciliary genes shown for comparison . Yellow bar indicates stage where phenotypes first become apparent , blue bar indicates furthest progression observed . Any sperm accumulating in the seminal vesicle were scored for motility . The second graph plots the severity of sperm ciliary defects ( stage at which defects were first observed ) for all previously uncharacterized ciliary genes chosen for further analysis . Number of animals examined as in ( E , F ) .

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

Figure 4(B) Schematic and confocal microscopy images of scolopidia in chordotonal organ of the fly. Each scolopidium contains two cilia with their ends embedded in a cap cell. Scolopidium outline, cilia and ciliary dilation can be visualized by DIC. Immunofluorescence micrographs with Sas-4 and NompC antibodies used to mark basal bodies and ciliary tips, respectively. DIC images of flies depleted of candidate genes by RNAi reveal relatively few defects on par with IFT genes such as IFT140, with the exception of misplaced dilations for Cep135 and CFAP97D1 (arrows). Missing cilia (asterisks) as with SAS4 or IFT52 are not observed. NompC/Sas-4 staining reveals more subtle phenotypes, including mislocalized NompC signal (with CFAP97D1, Cep135, Elmod, Mapk15 and Tex9, arrowheads) and apparent ciliary structural defects (Cep104 and Cables1, arrows). At least 3 animals examined per condition.(E) Mature sperm can be dissected from the seminal vesicle and motility analyzed using high speed video capture in dark field microscopy. Sinusoidal motion can be seen in wild type. In RNAi depletions of certain known motility genes (here IQUB and GAS8), this motion is reduced or even absent. Arrowheads mark position of individual bends in sequential frames. At least 3 movies made from different animals per condition.(F) Spermatogenesis in wild-type and RNAi depletions of selected genes, highlighting the stage where morphogenesis first becomes noticeably abnormal. At least 6 testes from 3 different animals examined per condition.(G) Graph summarizing sperm defects observed for candidate genes chosen for further analysis, with selected known centriolar/ciliary genes shown for comparison. Yellow bar indicates stage where phenotypes first become apparent, blue bar indicates furthest progression observed. Any sperm accumulating in the seminal vesicle were scored for motility. The second graph plots the severity of sperm ciliary defects (stage at which defects were first observed) for all previously uncharacterized ciliary genes chosen for further analysis. Number of animals examined as in (E, F).


Figure 5 ( A , RNAi - mediated depletion / deletion of CABLES1 , CCDC6 , CEP104 in Drosophila chordotonal neurons ( A ) results in defects in ciliary ultrastructure similar to CEP135 . In neurons , there is a loss of 9 - fold symmetry towards the ciliary tips with displaced doublet microtubules ( arrowheads ) and axonemes frequently fail to reach the cap cell ( asterisks ) . At least 3 animals examined per condition and tissue . B ) RNAi - mediated depletion / deletion of CABLES1 , CCDC6 , CEP104 and TEX9 in Drosophila sperm ( B ) results in defects in ciliary ultrastructure similar to CEP135 . In sperm , there is a loss of the inner pair of microtubules ( asterisks ) , a phenotype hitherto only associated with CEP135 . At least 3 animals examined per condition and tissue . ( C ) GFP fusions to TEX9 , CCDC6 and CEP104 localize to the central lumen of sperm basal bodies marked by Ana1 , while CABLES1 only faintly localizes to basal bodies . ( D ) TEX9 , CCDC6 and CABLES1 are recruited to maturing basal bodies during Drosophila spermatogenesis . n > 10 basal body pairs for each developmental stage . ( E Localization interdependencies between CEP135 , CEP104 and the novel components TEX9 , CCDC6 and CABLES1 in spermatogenesis . CEP135 is required for the localization of TEX9 , CCDC6 and CEP104 to mature basal bodies ( E ) n > 6 basal body pairs per condition . Asterisks indicate statistically significant differences to the respective control ( t - test , * * P < 0 . 01 , * * * P < 0 . 001 ) . F ) Localization interdependencies between CEP135 , CEP104 and the novel components TEX9 , CCDC6 and CABLES1 in spermatogenesis . while CEP135 localization is independent of TEX9 , CCDC6 and CABLES1 at all stages of spermatogenesis ( F ) . Asterisks indicate statistically significant differences to the respective control ( t - test , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

Figure 5(A, RNAi-mediated depletion/deletion of CABLES1, CCDC6, CEP104 in Drosophila chordotonal neurons (A) results in defects in ciliary ultrastructure similar to CEP135. In neurons, there is a loss of 9-fold symmetry towards the ciliary tips with displaced doublet microtubules (arrowheads) and axonemes frequently fail to reach the cap cell (asterisks). At least 3 animals examined per condition and tissue.B) RNAi-mediated depletion/deletion of CABLES1, CCDC6, CEP104 and TEX9 in Drosophila sperm (B) results in defects in ciliary ultrastructure similar to CEP135. In sperm, there is a loss of the inner pair of microtubules (asterisks), a phenotype hitherto only associated with CEP135. At least 3 animals examined per condition and tissue.(C) GFP fusions to TEX9, CCDC6 and CEP104 localize to the central lumen of sperm basal bodies marked by Ana1, while CABLES1 only faintly localizes to basal bodies.(D) TEX9, CCDC6 and CABLES1 are recruited to maturing basal bodies during Drosophila spermatogenesis. n >10 basal body pairs for each developmental stage.(E Localization interdependencies between CEP135, CEP104 and the novel components TEX9, CCDC6 and CABLES1 in spermatogenesis. CEP135 is required for the localization of TEX9, CCDC6 and CEP104 to mature basal bodies (E) n>6 basal body pairs per condition. Asterisks indicate statistically significant differences to the respective control (t-test, **P < 0.01,***P<0.001).F) Localization interdependencies between CEP135, CEP104 and the novel components TEX9, CCDC6 and CABLES1 in spermatogenesis. while CEP135 localization is independent of TEX9, CCDC6 and CABLES1 at all stages of spermatogenesis (F). Asterisks indicate statistically significant differences to the respective control (t-test, **P < 0.01,***P<0.001).


Figure 6 ( B ) . RNAi - mediated depletion / deletion of Drosophila CFAP97D1 and CFAP299 results in defects in ciliary ultrastructure in chordotonal neurons , including broken axonemes ( asterisk ) and perturbed 9 - fold symmetry ( arrowheads ) . At least 3 animals examined per condition . ( C ) A GFP fusion to CFAP97D1 localizes to the ciliary dilation but not basal body in chordotonal neurons . Insets acquired in Airyscan mode . ( D ) RNAi - mediated depletion / deletion of CFAP299 results in failure of membrane deposition around the developing sperm axoneme ( arrowheads ) . At least 3 animals examined per condition .

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Figure 6(B). RNAi-mediated depletion/deletion of Drosophila CFAP97D1 and CFAP299 results in defects in ciliary ultrastructure in chordotonal neurons, including broken axonemes (asterisk) and perturbed 9-fold symmetry (arrowheads). At least 3 animals examined per condition.(C) A GFP fusion to CFAP97D1 localizes to the ciliary dilation but not basal body in chordotonal neurons. Insets acquired in Airyscan mode.(D) RNAi-mediated depletion/deletion of CFAP299 results in failure of membrane deposition around the developing sperm axoneme (arrowheads). At least 3 animals examined per condition.


Figure 7 ( A , B ) Full gene deletions of mapk - 15 and elmd - 1 in C . elegans result in both shortened cilia and shortened dendrites , combining the signature phenotypes of IFT and transition zone mutants ( here kap - 1 ; osm - 3 and ccep - 290 ; nphp - 4 double mutants , respectively ) . Representative images of phasmid ( tail ) neurons expressing OSM - 6 : GFP ( A ) and quantitation of cilium and dendrite lengths ( B ) in control and mutant animals as indicated . n > 20 animals for all conditions . Asterisks indicate statistically significant difference to wild - type ( cilium lengths , t - test , * * * P < 0 . 001 , dendrite lengths , Wilcoxon Mann - Whitney test , * * * P < 0 . 001 ) . mapk - 15 ; elmd - 1 double mutants do not display phenotypes stronger than either single mutant ( t - test and Wilcoxon Mann - Whitney test as above , not significant ) . mapk - 15 and elmd - 1 mutants display defects in ciliary ultrastructure . ( C ) Cross - sectional views through the amphid channel reveal defects in cilium extension in both mutants , with fewer cilia protruding into the channel ( asterisks ) . ( D ) Quantitation of cilium extension defects at the level of the proximal and distal amphid channel . mapk - 15 and elmd - 1 mutants display defects in ciliary ultrastructure . ( E ) Higher magnification views of individual cilia reveal defects ( indicated by arrowheads ) at the level of the proximal segment of the axoneme ( fewer than 9 doublet microtubules ) , transition zone ( disorganization of the central cylinder and overall architecture ) and ciliary base ( membrane blebs ) . Percentages reported in figure based on examination of 4 mapk - 15 and 7 elmd - 1 mutant animals . ( F ) Endogenous / Endogenous promoter GFP fusions to ELMD - 1 and MAPK - 15 localize to the ciliary base and periciliary membrane compartment in C . elegans phasmid neurons , the former marked by the IFT component CHE - 11 . Localization interdependencies between MAPK - 15 and ELMD - 1 . ( G ) Loss of ELMD - 1 results in a hyperaccumulation of MAPK - 15 at the C . elegans ciliary base , while loss of MAPK - 15 impairs localization of ELMD - 1 Percentages reported in figure based on examination of > 30 animals for each condition .

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Figure 7(A, B) Full gene deletions of mapk-15 and elmd-1 in C. elegans result in both shortened cilia and shortened dendrites, combining the signature phenotypes of IFT and transition zone mutants (here kap-1;osm-3 and ccep-290;nphp-4 double mutants, respectively). Representative images of phasmid (tail) neurons expressing OSM-6:GFP (A) and quantitation of cilium and dendrite lengths (B) in control and mutant animals as indicated. n >20 animals for all conditions. Asterisks indicate statistically significant difference to wild-type (cilium lengths, t-test, ***P < 0.001, dendrite lengths, Wilcoxon Mann-Whitney test, ***P < 0.001). mapk-15;elmd-1 double mutants do not display phenotypes stronger than either single mutant (t-test and Wilcoxon Mann-Whitney test as above, not significant).mapk-15 and elmd-1 mutants display defects in ciliary ultrastructure. (C) Cross-sectional views through the amphid channel reveal defects in cilium extension in both mutants, with fewer cilia protruding into the channel (asterisks). (D) Quantitation of cilium extension defects at the level of the proximal and distal amphid channel.mapk-15 and elmd-1 mutants display defects in ciliary ultrastructure. (E) Higher magnification views of individual cilia reveal defects (indicated by arrowheads) at the level of the proximal segment of the axoneme (fewer than 9 doublet microtubules), transition zone (disorganization of the central cylinder and overall architecture) and ciliary base (membrane blebs). Percentages reported in figure based on examination of 4 mapk-15 and 7 elmd-1 mutant animals.(F) Endogenous/Endogenous promoter GFP fusions to ELMD-1 and MAPK-15 localize to the ciliary base and periciliary membrane compartment in C. elegans phasmid neurons, the former marked by the IFT component CHE-11.Localization interdependencies between MAPK-15 and ELMD-1. (G) Loss of ELMD-1 results in a hyperaccumulation of MAPK-15 at the C. elegans ciliary base, while loss of MAPK-15 impairs localization of ELMD-1 Percentages reported in figure based on examination of >30 animals for each condition.


Figure 1B . Results of the sequential digestion workflow applied to a synthetic 7 - protein mix , compared to using trypsin alone in four replicates and parallel digestion with trypsin , AspN , chymotrypsin and GluC . A trypsin four replicate experiment shows a large overlap of the four datasets with little gain . Parallel digestions with trypsin , AspN , chymotrypsin and GluC demonstrates high complementarity but moderate gains over trypsin . Sequential digestion shows low overlap between the four datasets and the largest gain in unique residue pairs . C . Gains of repeated analysis ( trypsin only ) , parallel digestion and sequential digestion for the same data as shown in panel B . D . Crosslinked peptides obtained by sequential digestion of a synthetic 7 - protein mix are smaller than their corresponding tryptic peptides .

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Figure 1B. Results of the sequential digestion workflow applied to a synthetic 7-protein mix, compared to using trypsin alone in four replicates and parallel digestion with trypsin, AspN, chymotrypsin and GluC. A trypsin four replicate experiment shows a large overlap of the four datasets with little gain. Parallel digestions with trypsin, AspN, chymotrypsin and GluC demonstrates high complementarity but moderate gains over trypsin. Sequential digestion shows low overlap between the four datasets and the largest gain in unique residue pairs. C. Gains of repeated analysis (trypsin only), parallel digestion and sequential digestion for the same data as shown in panel B. D. Crosslinked peptides obtained by sequential digestion of a synthetic 7-protein mix are smaller than their corresponding tryptic peptides.


Figure 2B . The in vitro helicase loading assay demonstrates that an Mcm2 C - terminal deletion mutant supports complex assembly ( lanes 6 and 7 ) and blocks formation of the final helicase loading product ( lanes 8 and 9 ) . C . Overexpression analysis of Mcm2 - 7ΔC2 shows that this mutant causes dominant lethality , indicating that the C - terminus of Mcm2 is essential in cell survival .

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Figure 2B. The in vitro helicase loading assay demonstrates that an Mcm2 C-terminal deletion mutant supports complex assembly (lanes 6 and 7) and blocks formation of the final helicase loading product (lanes 8 and 9).C. Overexpression analysis of Mcm2-7ΔC2 shows that this mutant causes dominant lethality, indicating that the C-terminus of Mcm2 is essential in cell survival.


Figure 1A Immunofluorescence staining of KANK1 ( red ) and α - tubulin ( blue ) in Strep - GFP - CEP170B knock - in HeLa cells . Lower panels show enlargements of the peripheral ( 1 ) and central ( 2 ) cortical regions boxed in the upper panels . Data information : Scale bars , 5 μm . C Immunofluorescence staining of KANK1 in Strep - GFP - CEP170B knock - in HeLa cells treated with nocodazole . Lower panels show enlargements of boxed areas in the upper panels . D Immunofluorescence staining of CEP170B and α - tubulin in MCF7 cells . Lower panels show enlargements of boxed areas in the upper panels . Data information : Scale bars , 5 μm . E Images of fixed HeLa cells transiently expressing mCherry - CEP170B together with GFP - tagged KANK1 , KANK2 , liprin - α1 , liprin - β1 , ELKS , or LL5β . Insets show enlargements of the boxed areas . Data information : Scale bars , 5 μm . G Immunofluorescence staining of KANK1 , KANK2 , or liprin - α1 in control Strep - GFP - CEP170B knock - in HeLa cell line or indicated knockout ( KO ) cell lines that were generated based on the parental Strep - GFP - CEP170B knock - in HeLa cell line . Data information : Scale bars , 5

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Figure 1A Immunofluorescence staining of KANK1 (red) and α-tubulin (blue) in Strep-GFP-CEP170B knock-in HeLa cells. Lower panels show enlargements of the peripheral (1) and central (2) cortical regions boxed in the upper panels. Data information: Scale bars, 5 μm.C Immunofluorescence staining of KANK1 in Strep-GFP-CEP170B knock-in HeLa cells treated with nocodazole. Lower panels show enlargements of boxed areas in the upper panels. D Immunofluorescence staining of CEP170B and α-tubulin in MCF7 cells. Lower panels show enlargements of boxed areas in the upper panels. Data information: Scale bars, 5 μm.E Images of fixed HeLa cells transiently expressing mCherry-CEP170B together with GFP-tagged KANK1, KANK2, liprin-α1, liprin-β1, ELKS, or LL5β. Insets show enlargements of the boxed areas. Data information: Scale bars, 5 μm.G Immunofluorescence staining of KANK1, KANK2, or liprin-α1 in control Strep-GFP-CEP170B knock-in HeLa cell line or indicated knockout (KO) cell lines that were generated based on the parental Strep-GFP-CEP170B knock-in HeLa cell line. Data information: Scale bars, 5 μ


Figure 2B Streptavidin pull - down assay with extracts of HEK293T cells expressing Bio - GFP - tagged liprin - α1 full length ( FL ) or its indicated fragments , analyzed by Western blotting with indicated antibodies . The Bio - GFP - tagged bait proteins were detected by IRDye ® 680RD Streptavidin . D Streptavidin pull down assay with extracts of HEK293T cells expressing Bio - GFP - tagged wild - type ( WT ) liprin - α1 fragment N1 or its NR / AA mutant , analyzed by Western blotting with indicated antibodies . The Bio - GFP - tagged bait proteins were detected by IRDye ® 680RD Streptavidin . E Streptavidin pull down assay with extracts of HEK293T cells expressing Bio - GFP - tagged WT liprin - α1 N1 fragment or its NR / AA mutant ( bait ) together with GFP - CEP170B ( prey ) , analyzed by Western blotting with GFP antibody . F Immunofluorescence staining of liprin - β1 in liprin - α1 KO HeLa cells transiently transfected with mCherry - tagged liprin - α1 full length or its NR / AA mutant . G , H Averaged intensity distributions for WT or NR / AA versions of mCherry - liprin - α1 ( G ) and liprin - β1 ( H ) for the experiments shown in ( F ) . Distance from the cell edge is shown on the horizontal axis . n = 25 - 29 cells from three technical replicates . Data represent mean ± SEM . Data information : Scale bars , 5 μm . J , K Immunofluorescence staining of α - tubulin ( blue ) in liprin - α1 KO / Strep - GFP - CEP170B knock - in double - engineered HeLa cell line transiently transfected with mCherry - tagged liprin - α1 FL ( J ) , fragment N1 ( K ) , or their NR / AA mutant counterparts ( J , K ) . Data information : Scale bars , 5 μm .

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Figure 2B Streptavidin pull-down assay with extracts of HEK293T cells expressing Bio-GFP-tagged liprin-α1 full length (FL) or its indicated fragments, analyzed by Western blotting with indicated antibodies. The Bio-GFP-tagged bait proteins were detected by IRDye® 680RD Streptavidin.D Streptavidin pull down assay with extracts of HEK293T cells expressing Bio-GFP-tagged wild-type (WT) liprin-α1 fragment N1 or its NR/AA mutant, analyzed by Western blotting with indicated antibodies. The Bio-GFP-tagged bait proteins were detected by IRDye® 680RD Streptavidin. E Streptavidin pull down assay with extracts of HEK293T cells expressing Bio-GFP-tagged WT liprin-α1 N1 fragment or its NR/AA mutant (bait) together with GFP-CEP170B (prey), analyzed by Western blotting with GFP antibody.F Immunofluorescence staining of liprin-β1 in liprin-α1 KO HeLa cells transiently transfected with mCherry-tagged liprin-α1 full length or its NR/AA mutant. G, H Averaged intensity distributions for WT or NR/AA versions of mCherry-liprin-α1 (G) and liprin-β1 (H) for the experiments shown in (F). Distance from the cell edge is shown on the horizontal axis. n = 25-29 cells from three technical replicates. Data represent mean ± SEM. Data information: Scale bars, 5 μm.J, K Immunofluorescence staining of α-tubulin (blue) in liprin-α1 KO/Strep-GFP-CEP170B knock-in double-engineered HeLa cell line transiently transfected with mCherry-tagged liprin-α1 FL (J), fragment N1 (K), or their NR/AA mutant counterparts (J, K). Data information: Scale bars, 5 μm.


Figure 3A TIRF microscopy time - lapse images on the left show the accumulation of CEP170B along the distal segments of MT lattices in the cortical region of Strep - GFP - CEP170B knock - in HeLa cell line transiently transfected with EB3 - TagRFP . Representative kymographs on the right show an MT plus end undergoing very short alternating growth and shrinking episodes ( corresponding to the one marked as " 1 " in the images ; upper right panel ) and an MT plus end undergoing longer depolymerization followed by rescues ( corresponding to the one marked as " 2 " in the images ; bottom right panel ) . Scale bars : horizontal , 2 μm ; vertical , 30 s . Data information : Scale bars , 5 μm . D Immunofluorescence staining of CAMSAP2 ( red ) and α - tubulin ( blue ) in HeLa cells transiently transfected with GFP control or GFP - CEP170B . Lower panels show enlargements of boxed areas . E Quantification of the length of CAMSAP2 stretches for the experiments shown in ( D ) . n = 12 cells from three technical replicates . Data information : Unless otherwise stated , Data represent mean ± SD . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; two - tailed t test . Scale bars , 5 μm . I TIRF images showing distribution of TagRFP - Rab6 vesicles in indicated HeLa cell lines . Seven consecutive frames ( 0 . 3s / frame ) were merged into a single image . J Representative images of TagRFP - Rab6 vesicle tracks in indicated HeLa cell lines . Data information : Scale bars , 5 μm . K - M Cartoon representation and quantification of the radiality of TagRFP - Rab6 vesicle tracks in indicated HeLa cell lines ( K , L ) . Center : Golgi center ; Start : starting position of the vesicle ; End : ending position of the vesicle . A 0 ° or 180 ° angle means that the vesicle moves radially toward the cell periphery or center of the Golgi , respectively , with the Golgi center , starting position and ending position of the vesicle in a straight line . Percentage of tracks with the angle ranging from 0 ° to 45 ° is shown in ( M ) . n = 10 , 000 vesicle tracks in 8 - 9 cells from two technical replicates in ( L ) ; n = 8 - 9 cells from two technical replicates in ( M ) . Data information : Unless otherwise stated , Data represent mean ± SD . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; two - tailed t test .

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

Figure 3A TIRF microscopy time-lapse images on the left show the accumulation of CEP170B along the distal segments of MT lattices in the cortical region of Strep-GFP-CEP170B knock-in HeLa cell line transiently transfected with EB3-TagRFP. Representative kymographs on the right show an MT plus end undergoing very short alternating growth and shrinking episodes (corresponding to the one marked as "1" in the images; upper right panel) and an MT plus end undergoing longer depolymerization followed by rescues (corresponding to the one marked as "2" in the images; bottom right panel ). Scale bars: horizontal, 2 μm; vertical, 30 s. Data information: Scale bars, 5 μm.D Immunofluorescence staining of CAMSAP2 (red) and α-tubulin (blue) in HeLa cells transiently transfected with GFP control or GFP-CEP170B. Lower panels show enlargements of boxed areas. E Quantification of the length of CAMSAP2 stretches for the experiments shown in (D). n = 12 cells from three technical replicates. Data information: Unless otherwise stated, Data represent mean ± SD. *, P<0.05; **, P < 0.01; ***, P < 0.001; two-tailed t test. Scale bars, 5 μm.I TIRF images showing distribution of TagRFP-Rab6 vesicles in indicated HeLa cell lines. Seven consecutive frames (0.3s / frame) were merged into a single image. J Representative images of TagRFP-Rab6 vesicle tracks in indicated HeLa cell lines. Data information: Scale bars, 5 μm.K-M Cartoon representation and quantification of the radiality of TagRFP-Rab6 vesicle tracks in indicated HeLa cell lines (K, L). Center: Golgi center; Start: starting position of the vesicle; End: ending position of the vesicle. A 0° or 180° angle means that the vesicle moves radially toward the cell periphery or center of the Golgi, respectively, with the Golgi center, starting position and ending position of the vesicle in a straight line. Percentage of tracks with the angle ranging from 0° to 45° is shown in (M). n = 10,000 vesicle tracks in 8-9 cells from two technical replicates in (L); n = 8-9 cells from two technical replicates in (M). Data information: Unless otherwise stated, Data represent mean ± SD. *, P<0.05; **, P < 0.01; ***, P < 0.001; two-tailed t test.


Figure 4C Immunofluorescence staining of CAMSAP2 ( green ) and α - tubulin ( red ) in control or CEP170B KO MCF7 monolayer . Left panels : basal plane ; middle panels : apical plane ; right panels : side view . D Immunofluorescence staining of α - tubulin ( red ) in control CAMSAP3 - GFP knock - in Caco - 2 monolayer or CEP170B KO / CAMSAP3 - GFP knock - in double engineered Caco - 2 monolayer . Left panels : basal plane ; middle panels : apical plane ; right panels : side view . Data information : Scale bars : 5 μmG Caco - 2 cells were cultured in Matrigel for 5 days , and subsequently treated with Cholera toxin for 12 hours . Phase - contrast images of thus formed control or CEP170B KO Caco - 2 cysts were shown . H , I Representative images and quantification of four categories of lumen formation for experiments shown in ( G ) . n = 100 cysts from three technical replicates . J Quantification of the diameter of control and CEP170B KO Caco - 2 cysts . n = 30 cysts from three technical replicates . Data information : Data represents the mean ± SD . * * * , P < 0 . 001 ; two - tailed t test . Scale bars : 25

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

Figure 4C Immunofluorescence staining of CAMSAP2 (green) and α-tubulin (red) in control or CEP170B KO MCF7 monolayer. Left panels: basal plane; middle panels: apical plane; right panels: side view. D Immunofluorescence staining of α-tubulin (red) in control CAMSAP3-GFP knock-in Caco-2 monolayer or CEP170B KO/CAMSAP3-GFP knock-in double engineered Caco-2 monolayer. Left panels: basal plane; middle panels: apical plane; right panels: side view. Data information: Scale bars: 5 μmG Caco-2 cells were cultured in Matrigel for 5 days, and subsequently treated with Cholera toxin for 12 hours. Phase-contrast images of thus formed control or CEP170B KO Caco-2 cysts were shown. H, I Representative images and quantification of four categories of lumen formation for experiments shown in (G). n = 100 cysts from three technical replicates. J Quantification of the diameter of control and CEP170B KO Caco-2 cysts. n = 30 cysts from three technical replicates. Data information: Data represents the mean ± SD. ***, P < 0.001; two-tailed t test. Scale bars: 25 μm


Figure 5A , TIRF images of live KIF2A - GFP or KIF2C - GFP knock - in HeLa cells transiently transfected with mCherry - KANK1 ( A ) Insets show enlargements of boxed areas . Data information : Scale bars : 5 μm . F Immunofluorescence staining of CAMSAP2 ( green ) and α - tubulin ( red ) in control or indicated KO HeLa cell lines . Insets show enlargements of boxed areas . G Averaged intensity distributions for CAMSAP2 in the indicated cell lines for the experiments shown in ( F ) . Distance from the cell edge is shown on the horizontal axis . n = 38 - 40 cells from three technical replicates . H Quantification of the length of CAMSAP2 stretches for the experiments shown in ( F ) . n = 11 - 14 cells from three technical replicates . Data information : Data represent the mean ± SD . * , P < 0 . 05 ; * * * , P < 0 . 001 ; two - tailed t test . Scale bars : 5 μm . K , L Averaged intensity distributions for KIF2A ( K ) or CEP170B ( L ) in the indicated cell lines for the experiments shown in ( I , J ) . Distance from the cell edge is shown on the horizontal axis . n = 20 - 38 cells from three technical replicates . Data information : Data represent the mean ± SD . * , P < 0 . 05 ; * * * , P < 0 . 001 ; two - tailed t test . M , N Quantification of intensities of KIF2A at the cell edge ( M ) or CEP170B along cortical MT ( N ) in the indicated cell lines for the experiments shown in ( I , J ) . n = 22 - 26 cells from three technical replicates in ( M ) ; n = 16 cells from three technical replicates in ( N ) . Data information : Data represent the mean ± SD . * , P < 0 . 05 ; * * * , P < 0 . 001 ; two - tailed t test .

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

Figure 5A, TIRF images of live KIF2A-GFP or KIF2C-GFP knock-in HeLa cells transiently transfected with mCherry-KANK1 (A) Insets show enlargements of boxed areas. Data information: Scale bars: 5 μm.F Immunofluorescence staining of CAMSAP2 (green) and α-tubulin (red) in control or indicated KO HeLa cell lines. Insets show enlargements of boxed areas. G Averaged intensity distributions for CAMSAP2 in the indicated cell lines for the experiments shown in (F). Distance from the cell edge is shown on the horizontal axis. n = 38-40 cells from three technical replicates. H Quantification of the length of CAMSAP2 stretches for the experiments shown in (F). n = 11-14 cells from three technical replicates. Data information: Data represent the mean ± SD. *, P<0.05; ***, P < 0.001; two-tailed t test. Scale bars: 5 μm.K, L Averaged intensity distributions for KIF2A (K) or CEP170B (L) in the indicated cell lines for the experiments shown in (I, J). Distance from the cell edge is shown on the horizontal axis. n = 20-38 cells from three technical replicates. Data information: Data represent the mean ± SD. *, P<0.05; ***, P < 0.001; two-tailed t test.M, N Quantification of intensities of KIF2A at the cell edge (M) or CEP170B along cortical MT(N) in the indicated cell lines for the experiments shown in (I, J). n = 22-26 cells from three technical replicates in (M); n = 16 cells from three technical replicates in (N). Data information: Data represent the mean ± SD. *, P<0.05; ***, P < 0.001; two-tailed t test.


Figure 6A Images and corresponding kymographs of GFP - tagged full length CEP170B tracking or blocking dynamic MT minus ends at different concentrations . B , C Quantification of MT minus - end growth rate ( B ) or plus - end depolymerization rate ( C ) in the presence of GFP - CEP170B at indicated concentrations . n = 20 - 30 MTs from 3 independent experiments . N . D . , not detected . Data information : Data represent mean ± SD . Scale bars : horizontal , 2 μm ; vertical , 1 min . J Quantification of MT minus - end growth rate in the presence of indicated CEP170B fragments at indicated concentrations . n = 20 - 43 MTs from 3 independent experiments . N . D . , not detected . Data information : Data represent mean ± SD . L Images and corresponding kymographs showing the behavior of indicated GFP - tagged CEP170B fragments on dynamic MTs at 10 nM . M Quantification of intensity of indicated CEP170B fragments along MT lattice at 10 nM . n = 16 MTs from 2 independent experiments . N . D . , not detected . N , O Quantification of MT plus - end depolymerization rate ( N ) or minus - end growth rate ( O ) in the presence of indicated CEP170B fragments at 10 nM . n = 20 - 30 MTs from 2 independent experiments . Data information : Data represent mean ± SD . Scale bars : horizontal , 2 μm ; vertical , 1 min .

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

Figure 6A Images and corresponding kymographs of GFP-tagged full length CEP170B tracking or blocking dynamic MT minus ends at different concentrations. B, C Quantification of MT minus-end growth rate (B) or plus-end depolymerization rate (C) in the presence of GFP-CEP170B at indicated concentrations. n = 20-30 MTs from 3 independent experiments. N.D., not detected. Data information: Data represent mean ± SD. Scale bars: horizontal, 2 μm; vertical, 1 min.J Quantification of MT minus-end growth rate in the presence of indicated CEP170B fragments at indicated concentrations. n = 20-43 MTs from 3 independent experiments. N.D., not detected. Data information: Data represent mean ± SD.L Images and corresponding kymographs showing the behavior of indicated GFP-tagged CEP170B fragments on dynamic MTs at 10 nM. M Quantification of intensity of indicated CEP170B fragments along MT lattice at 10 nM. n = 16 MTs from 2 independent experiments. N.D., not detected. N, O Quantification of MT plus-end depolymerization rate (N) or minus-end growth rate (O) in the presence of indicated CEP170B fragments at 10 nM. n = 20-30 MTs from 2 independent experiments. Data information: Data represent mean ± SD. Scale bars: horizontal, 2 μm; vertical, 1 min.


Figure 7A Images and corresponding kymographs showing that KIF2A - SNAP labelled with Alexa Fluor 647 ( shown here as green ) tracks growing MT minus ends at 0 . 5 - 2 . 5 nM but induces depolymerization from minus - end at 5 nM . Quantification of MT minus - end ( B ) or plus - end ( C ) catastrophe frequency in the presence of KIF2A - SNAP at indicated concentrations . N . A . , not available . n = 20 - 39 MTs from 2 - 3 independent experiments . Data information : Data represent mean ± SD . Scale bars : horizontal , 2 μm ; vertical , 1 min . J - L Images and corresponding kymographs showing the behaviors of 30 nM TagBFP - CAMSAP2 alone , 3 nM GFP - CEP170B alone , 5 nM KIF2A - SNAP alone , 30 nM TagBFP - CAMSAP3 alone ( J ) , 30 nM TagBFP - CAMSAP2 in combination with 3 nM GFP - CEP170B ( K ) , or 30 nM TagBFP - CAMSAP2 in combination with 5 nM KIF2A - SNAP ( L ) on dynamic MTs . Data information : Data represent mean ± SD . M Quantification of MT minus - end depolymerization rate in the presence of GFP - CEP170B and KIF2A - SNAP at indicated concentrations . n = 22 - 35 MTs from 2 - 3 independent experiments . Data information : Data represent mean ± SD . P , Q Kymographs showing that 5 nM KIF2A - SNAP in complex with 3 nM but not 1 nM GFP - CEP170B could depolymerize preassembled MT stretches decorated by 30 nM TagBFP - CAMSAP2 ( P ) or TagBFP - CAMSAP3 ( Q ) in the flow - in experiments . Red arrows indicate time points of flow - in . Data information : Scale bars : horizontal , 2 μm ; vertical , 1 min .

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

Figure 7A Images and corresponding kymographs showing that KIF2A-SNAP labelled with Alexa Fluor 647 (shown here as green) tracks growing MT minus ends at 0.5-2.5 nM but induces depolymerization from minus-end at 5 nM. Quantification of MT minus-end (B) or plus-end (C) catastrophe frequency in the presence of KIF2A-SNAP at indicated concentrations. N.A., not available. n = 20-39 MTs from 2-3 independent experiments. Data information: Data represent mean ± SD. Scale bars: horizontal, 2 μm; vertical, 1 min.J-L Images and corresponding kymographs showing the behaviors of 30 nM TagBFP-CAMSAP2 alone, 3 nM GFP-CEP170B alone, 5 nM KIF2A-SNAP alone, 30 nM TagBFP-CAMSAP3 alone (J), 30 nM TagBFP-CAMSAP2 in combination with 3 nM GFP-CEP170B (K), or 30 nM TagBFP-CAMSAP2 in combination with 5 nM KIF2A-SNAP (L) on dynamic MTs. Data information: Data represent mean ± SD.M Quantification of MT minus-end depolymerization rate in the presence of GFP- CEP170B and KIF2A-SNAP at indicated concentrations. n = 22-35 MTs from 2-3 independent experiments. Data information: Data represent mean ± SD.P, Q Kymographs showing that 5 nM KIF2A-SNAP in complex with 3 nM but not 1 nM GFP-CEP170B could depolymerize preassembled MT stretches decorated by 30 nM TagBFP-CAMSAP2 (P) or TagBFP-CAMSAP3 (Q) in the flow-in experiments. Red arrows indicate time points of flow-in. Data information: Scale bars: horizontal, 2 μm; vertical, 1 min.


Figure 1Representative images of euploid control or ArCK cells interacting with NK cells . The NK cell - mediated killing was measured at a 2 . 5 : 1 ( NK cells : target cells ) ratio and was recorded by live cell imaging for 36 hours at a 30 - min interval . TO - PRO - 3 ( 1 μM ) was added to the medium at the same time of NK cell addition to measure cell membrane integrity . Phase contrast ( top ) and TO - PRO - 3 signal ( bottom ) from the same field were presented . Arrowheads indicate ArCK cell death . All images were acquired at the same exposure time and light intensity . Scale bar 20 μM . NK cell - mediated cytotoxicity across various NK cell to target cell ratios . Either euploid control ( Ctrl ) or ArCK cells were co - cultured with NK cells at the indicated NK cell : target cell ratios . The cumulative killing of target cells was measured . n = 3 biological replicates ; mean ± SEM . p < 0 . 0001 for all four NK cell : target cell ratios , KS test .

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

Figure 1Representative images of euploid control or ArCK cells interacting with NK cells. The NK cell-mediated killing was measured at a 2.5:1 (NK cells: target cells) ratio and was recorded by live cell imaging for 36 hours at a 30-min interval. TO-PRO-3 (1 μM) was added to the medium at the same time of NK cell addition to measure cell membrane integrity. Phase contrast (top) and TO-PRO-3 signal (bottom) from the same field were presented. Arrowheads indicate ArCK cell death. All images were acquired at the same exposure time and light intensity. Scale bar 20 μM.NK cell-mediated cytotoxicity across various NK cell to target cell ratios. Either euploid control (Ctrl) or ArCK cells were co-cultured with NK cells at the indicated NK cell: target cell ratios. The cumulative killing of target cells was measured. n=3 biological replicates; mean± SEM. p< 0.0001 for all four NK cell: target cell ratios, KS test.


Figure 2NK cell - mediated killing for doxorubicin ( Doxo ) , palbociclib ( Palbo ) , and nutlin3 ( Nutlin ) treated samples ( NK cell : target cell = 2 . 5 : 1 ) . n = 3 biological replicates ; mean ± SEM . Ctrl vs . Doxo , p < 0 . 0001 ; Ctrl vs . Palbo , p < 0 . 0001 ; Ctrl vs . Nutlin , p < 0 . 0001 ; KS test . NK cell - mediated cytotoxicity towards torin1 - treated cells . Torin1 treated ( Torin ) cells were generated as described in ( F ) and the NK cell killing assay was performed as described in Figure 1 . n = 3 biological replicates ; mean ± SEM . Ctrl vs . Torin , p = 0 . 79 , not significant ( n . s . ) ; KS test .

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

Figure 2NK cell-mediated killing for doxorubicin (Doxo), palbociclib (Palbo), and nutlin3 (Nutlin) treated samples (NK cell: target cell =2.5:1). n=3 biological replicates; mean± SEM. Ctrl vs. Doxo, p< 0.0001; Ctrl vs. Palbo, p< 0.0001; Ctrl vs. Nutlin, p< 0.0001; KS test.NK cell-mediated cytotoxicity towards torin1-treated cells. Torin1 treated (Torin) cells were generated as described in (F) and the NK cell killing assay was performed as described in Figure 1. n=3 biological replicates; mean± SEM. Ctrl vs. Torin, p= 0.79, not significant (n.s.); KS test.


Figure 3 γ - H2AX foci were analyzed in ArCK and G1 arrested cells ( generated as described in Figure 2 ) . At least 50 cells were analyzed per condition per replicate . n = 2 biological replicates ; individual values and mean are shown . The distribution of the foci number in each treated condition was compared to that of control using Kolmogorov - Smirnov test . Ctrl vs . Doxo , p < 0 . 0001 ; Ctrl vs . Palbo , p = 0 . 07 , n . s . ; Ctrl vs . Nutlin , p = 0 . 11 , n . s . ; Ctrl vs . ArCK , p = 0 . 0007 . Secreted cytokine and interferon levels were determined in cell supernatants . Media were collected after 36 hours of incubation with cells grown as described in Figure 1 and 2 . Cytokine and interferon levels were shown as fold change normalized to euploid control cells . Individual values and mean are shown ; n = 2 biological replicates . NK cell medium was incubated with either euploid control or ArCK cells for 12 hours . At the time of NK cell addition , media were switched between ArCK and euploid control cells ( Ctrl ) . NK cell killing was measured as described in Figure 1C . For reference , NK cell killing of ArCK and euploid control cells ( Ctrl ) without medium switch were performed side by side and plotted on the graph . Black , euploid control cells without medium switch ; red , ArCK cells without medium switch ; blue , euploid control cells in ArCK cell condition medium ; green , ArCK cells in euploid control cell condition medium . n = 3 biological replicates ; mean ± SEM . Ctrl vs . ArCK , p < 0 . 0001 ; Ctrl vs . Ctrl in ArCK med , p < 0 . 0001 ; Ctrl vs . ArCK in Ctrl med , p < 0 . 0001 ; ArCK vs . ArCK in Ctrl med , p = 0 . 0002 ; KS test .

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Figure 3   γ-H2AX foci were analyzed in ArCK and G1 arrested cells (generated as described in Figure 2). At least 50 cells were analyzed per condition per replicate. n=2 biological replicates; individual values and mean are shown. The distribution of the foci number in each treated condition was compared to that of control using Kolmogorov-Smirnov test. Ctrl vs. Doxo, p< 0.0001; Ctrl vs. Palbo, p= 0.07, n.s.; Ctrl vs. Nutlin, p= 0.11, n.s.; Ctrl vs. ArCK, p= 0.0007.   Secreted cytokine and interferon levels were determined in cell supernatants. Media were collected after 36 hours of incubation with cells grown as described in Figure 1 and 2. Cytokine and interferon levels were shown as fold change normalized to euploid control cells. Individual values and mean are shown; n=2 biological replicates.NK cell medium was incubated with either euploid control or ArCK cells for 12 hours. At the time of NK cell addition, media were switched between ArCK and euploid control cells (Ctrl). NK cell killing was measured as described in Figure 1C. For reference, NK cell killing of ArCK and euploid control cells (Ctrl) without medium switch were performed side by side and plotted on the graph. Black, euploid control cells without medium switch; red, ArCK cells without medium switch; blue, euploid control cells in ArCK cell condition medium; green, ArCK cells in euploid control cell condition medium. n=3 biological replicates; mean± SEM. Ctrl vs. ArCK, p< 0.0001; Ctrl vs. Ctrl in ArCK med, p< 0.0001; Ctrl vs. ArCK in Ctrl med, p< 0.0001; ArCK vs. ArCK in Ctrl med, p= 0.0002; KS test.


Figure 4RT - qPCR quantifying NF - κB downstream target gene expression in ArCK and euploid control ( Ctrl ) cells . n = 6 biological replicates ; mean ± SEM . IL - 1b , * p = 0 . 029 ; IL - 6 , * p = 0 . 032 ; IL - 8 , * * p = 0 . 008 ; unpaired t - test .

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Figure 4RT-qPCR quantifying NF-κB downstream target gene expression in ArCK and euploid control (Ctrl) cells. n=6 biological replicates; mean± SEM. IL-1b, *p= 0.029; IL-6, *p= 0.032; IL-8, **p= 0.008; unpaired t-test.


Figure 5ArCK or euploid proliferating control cells were treated with either DMSO or the NF - κB inhibitor BMS - 345541 ( 5 μM ) for 48 hours before assessing NK cell - mediated cytotoxicity . The drug was washed out during the NK cell co - culture assay . n = 3 biological replicates ; mean ± SEM . ArCK vs ArCK NF - κB inhibitor , p < 0 . 0001 ; KS test . ( H , I ) The effect of inactivating both RELA and RELB on NK cell - mediated cytotoxicity in 7 - day doxorubicin ( H ) and nutlin3 - treated ( I ) cells . n2 biological replicates ; Doxo - c1 vs . Doxo RELA RELB - c1 , p = 0 . 02 . Nutlin - c1 vs . Nutlin RELA RELB - c1 , p = 0 . 44 , n . s . ; KS test .

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Figure 5ArCK or euploid proliferating control cells were treated with either DMSO or the NF-κB inhibitor BMS-345541 (5 μM) for 48 hours before assessing NK cell-mediated cytotoxicity. The drug was washed out during the NK cell co-culture assay. n=3 biological replicates; mean± SEM. ArCK vs ArCK NF-κB inhibitor, p< 0.0001; KS test. (H, I) The effect of inactivating both RELA and RELB on NK cell-mediated cytotoxicity in 7-day doxorubicin (H) and nutlin3-treated (I) cells. n2 biological replicates; Doxo-c1 vs. Doxo RELA RELB-c1, p = 0.02. Nutlin-c1 vs. Nutlin RELA RELB-c1, p = 0.44, n.s.; KS test.


Figure 6HCT116 ( left ) or DLD1 cells ( right ) were treated with either DMSO or the Mps1 inhibitor reversine ( 500 nM ) for 48 hours before assessing NK cell - mediated cytotoxicity . The drug was washed out during the NK cell co - culture assay . n = 3 biological replicates ; mean ± SEM . HCT116 vs . HCT116 Mps1 inhibitor , p = 0 . 44 , n . s . ; DLD1 vs . DLD1 Mps1 inhibitor , p = 0 . 44 , n . s . ; KS test .

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Figure 6HCT116 (left) or DLD1 cells (right) were treated with either DMSO or the Mps1 inhibitor reversine (500 nM) for 48 hours before assessing NK cell-mediated cytotoxicity. The drug was washed out during the NK cell co-culture assay. n=3 biological replicates; mean±SEM. HCT116 vs. HCT116 Mps1 inhibitor, p= 0.44, n.s.; DLD1 vs. DLD1 Mps1 inhibitor, p= 0.44, n.s.; KS test.


Figure 4 ( A ) Binding assay for Hsc70 binding in the presence of WT DNAJB1 : control of DNAJB1 / Hsc70 , αSyn fibres + DNAJB1 / Hsc70 , αSyn fibres + DNAJB1 / Hsc70 / Apg2 , control of DNAJB1 / Hsc70 / Apg2 . The ' * ' highlights the condition where an increase of Hsc70 in the bound fraction is observed . P , pellet ; S , supernatant . The amount of Hsc70 recruited to the fibres is shown in the pellet fractions . ( B ) Binding assay for Hsc70 binding in the presence of the truncated ΔJ - DNAJB1 , lacking the J domain . The gel layout is similar to the previous one with ΔJ - DNAJB1 replacing WT DNAJB1 . Both Apg2 and J domain are required for enhancement of Hsc70 binding . ( C ) Histogram of Hsc70 bound fraction in each sample ( N = 3 independent experiments , WT : WT DNAJB1 , ΔJ : ΔJ - DNAJB1 mutant , - Apg2 : condition with fibres + DNAJB1 / Hsc70 , + Apg2 : condition with fibres + DNAJB1 / Hsc70 / Apg2 ) . A 2 - way ANOVA was performed with Tukey ' s multiple comparisons test ( * * : P = 0 . 003 ; * * * * : P < 0 . 0001 ) . Mean Hsc70 binding values with SD are shown . ( D ) TIRF microscopy images of the labelled αSyn fibres and Hsc70 in the absence or in the presence of Apg2 . Scale bar , 1 µm . ( E , F ) Quantitation of fluorescence intensity of Hsc70 ( E ) and αSyn ( F ) in the two conditions ( + / - Apg2 , N = 3 independent experiments , n = 65 fibres ) . Mean Hsc70 and αSyn fluorescence intensities with SD are shown . ( G ) Quantitation of differences in Hsc70 binding between the two ends of the fibres , normalised to the local αSyn intensity , in the absence and presence of Apg2 ( N = 3 independent experiments , n = 54 fibres ) . Mean Hsc70 fluorescence intensities with SD are shown . Data information : ( E - G ) For each plot , data were tested for normality by performing a Shapiro - Wilk test . As the data did not display a normal distribution , they were analysed using a two - tailed Mann - Whitney test ( * * * * : P < 0 . 0001 , ns : not significant ) .

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Figure 4(A) Binding assay for Hsc70 binding in the presence of WT DNAJB1: control of DNAJB1/Hsc70, αSyn fibres + DNAJB1/Hsc70, αSyn fibres + DNAJB1/Hsc70/Apg2, control of DNAJB1/Hsc70/Apg2. The '*' highlights the condition where an increase of Hsc70 in the bound fraction is observed. P, pellet; S, supernatant. The amount of Hsc70 recruited to the fibres is shown in the pellet fractions. (B) Binding assay for Hsc70 binding in the presence of the truncated ΔJ-DNAJB1, lacking the J domain. The gel layout is similar to the previous one with ΔJ-DNAJB1 replacing WT DNAJB1. Both Apg2 and J domain are required for enhancement of Hsc70 binding. (C) Histogram of Hsc70 bound fraction in each sample (N = 3 independent experiments, WT: WT DNAJB1, ΔJ: ΔJ-DNAJB1 mutant, -Apg2: condition with fibres + DNAJB1/Hsc70, +Apg2: condition with fibres + DNAJB1/Hsc70/Apg2). A 2-way ANOVA was performed with Tukey's multiple comparisons test (**: P=0.003; ****: P<0.0001). Mean Hsc70 binding values with SD are shown.(D) TIRF microscopy images of the labelled αSyn fibres and Hsc70 in the absence or in the presence of Apg2. Scale bar, 1 µm. (E, F) Quantitation of fluorescence intensity of Hsc70 (E) and αSyn (F) in the two conditions (+/- Apg2, N = 3 independent experiments, n = 65 fibres). Mean Hsc70 and αSyn fluorescence intensities with SD are shown. (G) Quantitation of differences in Hsc70 binding between the two ends of the fibres, normalised to the local αSyn intensity, in the absence and presence of Apg2 (N = 3 independent experiments, n = 54 fibres). Mean Hsc70 fluorescence intensities with SD are shown. Data information: (E-G) For each plot, data were tested for normality by performing a Shapiro-Wilk test. As the data did not display a normal distribution, they were analysed using a two-tailed Mann-Whitney test (****: P<0.0001, ns: not significant).


Figure 5Binding assay with different dilutions of Apg2 incubated with DNAJB1 / Hsc70 and ( A ) with αSyn fibres or ( B ) without αSyn fibres ( control ) . 5 mM ATP was present in the buffer throughout .

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Figure 5Binding assay with different dilutions of Apg2 incubated with DNAJB1/Hsc70 and (A) with αSyn fibres or (B) without αSyn fibres (control). 5 mM ATP was present in the buffer throughout.


figf1 ( a ) Expression of ATG16L1 in a set of different tissues as detected by RT - PCR ( IEC , intestinal epithelial cells ) . The corresponding β - actin control ( with a 518 - bp amplicon size ) is given below . b ) Amounts of ATG16L1 in colonic mucosal biopsy specimens . Proteins ( 15 μg ) from rectal mucosal biopsies of Crohn disease patients and normal controls ( N ) were separated by denaturing SDS - PAGE , transferred onto polyvinylidine fluoride ( PVDF ) membranes and probed for the presence of ATG16L1 using a specific primary antibody and horseradish peroxidase - coupled secondary antibody . The abundant 68 . 2 - kDa band corresponds to the predicted molecular weight of the isoform encoded by GenBank AY398617 ( UniProt accession number Q676U5 ) . This major protein form was used for three - dimensional modeling of the ATG16L1 protein ( Fig . 2 ) . The 48 . 5 - kDa band corresponds to GenBank EF079890 , which contains exon 9 ( harboring rs2241880 ) in the same reading frame as the major form . ( c - i ) Representative immunohistochemistry results ( out of six normal controls and six individuals with Crohn disease ) from colonic mucosal tissue samples from normal controls for genotype AA ( c ) and GG ( f ) and individuals with Crohn disease for genotype AA ( d ) and GG ( g ) are shown to demonstrate the cellular localization of the ATG16L1 protein . Cellular expression patterns in mononuclear cells ( h ) and intestinal epithelial cells ( i ) of a Crohn disease sample ( rs2241880AA ) are shown at higher magnification . e shows a Crohn disease sample without the primary antibody , used as a control . Bar represents 10 μm in c - g .

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

figf1(a) Expression of ATG16L1 in a set of different tissues as detected by RT-PCR (IEC, intestinal epithelial cells). The corresponding β-actin control (with a 518-bp amplicon size) is given below.b) Amounts of ATG16L1 in colonic mucosal biopsy specimens. Proteins (15 μg) from rectal mucosal biopsies of Crohn disease patients and normal controls (N) were separated by denaturing SDS-PAGE, transferred onto polyvinylidine fluoride (PVDF) membranes and probed for the presence of ATG16L1 using a specific primary antibody and horseradish peroxidase-coupled secondary antibody. The abundant 68.2-kDa band corresponds to the predicted molecular weight of the isoform encoded by GenBank AY398617 (UniProt accession number Q676U5). This major protein form was used for three-dimensional modeling of the ATG16L1 protein (Fig. 2). The 48.5-kDa band corresponds to GenBank EF079890, which contains exon 9 (harboring rs2241880) in the same reading frame as the major form.(c-i) Representative immunohistochemistry results (out of six normal controls and six individuals with Crohn disease) from colonic mucosal tissue samples from normal controls for genotype AA (c) and GG (f) and individuals with Crohn disease for genotype AA (d) and GG (g) are shown to demonstrate the cellular localization of the ATG16L1 protein. Cellular expression patterns in mononuclear cells (h) and intestinal epithelial cells (i) of a Crohn disease sample (rs2241880AA) are shown at higher magnification. e shows a Crohn disease sample without the primary antibody, used as a control. Bar represents 10 μm in c-g.


figf2The 32 β - strands forming an eight - bladed β - propeller are numbered as in Supplementary Note . The location of the variant amino acid T300A in strand β3 , corresponding to rs2241880 , is marked in yellow .

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figf2The 32 β-strands forming an eight-bladed β-propeller are numbered as in Supplementary Note. The location of the variant amino acid T300A in strand β3, corresponding to rs2241880, is marked in yellow.