( B ) Top , experimental design . Left , densitometric analysis of IKBα protein levels in synchronized A549 cells detected over time after release . Bars represent ZM values normalized with respect to DMSO . n = 3 independent experiments , data represent mean values ± SEM . Right , time course immunoblot analysis of synchronized A549 cells , released in DMSO or ZM ( 2μM ) . Data information : The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 1 ( * ) , p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) .

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

(B) Top, experimental design. Left, densitometric analysis of IKBα protein levels in synchronized A549 cells detected over time after release. Bars represent ZM values normalized with respect to DMSO. n=3 independent experiments, data represent mean values ± SEM. Right, time course immunoblot analysis of synchronized A549 cells, released in DMSO or ZM (2μM). Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.001 (***), p ≤ 0.0001 (****).


( D , E ) ( D ) Western blot analysis confirming lack of PIDD1 expression in A549 cells and ( E ) MDM2 processing after induction of cytokinesis failure using ZM ( 2µM ) for 48h . Data information : The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 1 ( * ) , p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E) (D) Western blot analysis confirming lack of PIDD1 expression in A549 cells and (E) MDM2 processing after induction of cytokinesis failure using ZM (2µM) for 48h. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.001 (***), p ≤ 0.0001 (****).


( F ) Live - cell imaging analysis of NF - κB EGFP - reporter activity in CRISPR - edited polyclonal A549κB cells . NF - κB activity of ZM - treated cells is represented as fold - change with respect their untreated counterparts ( DMSO - treated cells , grey lines ) . n = 5 independent experiments . Error bars represent ± SEM . Data information : The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 1 ( * ) , p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Live-cell imaging analysis of NF-κB EGFP-reporter activity in CRISPR-edited polyclonal A549κB cells. NF-κB activity of ZM-treated cells is represented as fold-change with respect their untreated counterparts (DMSO-treated cells, grey lines). n=5 independent experiments. Error bars represent ± SEM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.001 (***), p ≤ 0.0001 (****).


( H ) Live - cell imaging analysis of NF - κB EGFP - reporter activity in CRISPR - edited polyclonal RPE1kB cells analyzed as in as in F . Data information : The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 1 ( * ) , p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Live-cell imaging analysis of NF-κB EGFP-reporter activity in CRISPR-edited polyclonal RPE1kB cells analyzed as in as in F. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.001 (***), p ≤ 0.0001 (****).


( A ) Representative immunofluorescence images of γ - tubulin staining to mark and enumerate centrosomes in A549κB reporter cells . Cells were pre - treated for 24 h with centrinone ( 125nM ) or solvent control ( DMSO ) and cultured further in medium either containing DMSO or ZM to promote cytokinesis failure . Size bar : 10µM . Data information : The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) .

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

(A) Representative immunofluorescence images of γ-tubulin staining to mark and enumerate centrosomes in A549κB reporter cells. Cells were pre-treated for 24 h with centrinone (125nM) or solvent control (DMSO) and cultured further in medium either containing DMSO or ZM to promote cytokinesis failure. Size bar: 10µM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0.001 (***), p ≤ 0.0001 (****).


( B ) Cells treated as indicated in A were analyzed for NF - κB - driven EGFP - reporter activation by IncuCyte live - cell imaging . Left , representative pictures of live - cell imaging NF - κB activation after treatment with DMSO , ZM ( 2μM , 48 h ) or TNF ( 20 ng / mL , 24h ) . Size bar : 20µm . Right , EGFP MFI quantification of ZM - treated cells was plotted as fold - change expression relative to DMSO - treated controls . Data points represent means of n = 4 biological replicates ± SEM . Data information : The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) .

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

(B) Cells treated as indicated in A were analyzed for NF-κB-driven EGFP-reporter activation by IncuCyte live-cell imaging. Left, representative pictures of live-cell imaging NF-κB activation after treatment with DMSO, ZM (2μM, 48 h) or TNF (20 ng/mL, 24h). Size bar: 20µm. Right, EGFP MFI quantification of ZM-treated cells was plotted as fold-change expression relative to DMSO-treated controls. Data points represent means of n=4 biological replicates ± SEM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0.001 (***), p ≤ 0.0001 (****).


( C ) Left , representative immunofluorescence images of supernumerary centrosomes originated from Dox - inducible PLK4 overexpression in CRISPR - edited polyclonal U2OSPLK4 cells . Size scale bar : 10µm . Right , representative cell cycle profiles of U2OSPLK4 control and PIDD1 - deficient cells . Data information : The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left, representative immunofluorescence images of supernumerary centrosomes originated from Dox-inducible PLK4 overexpression in CRISPR-edited polyclonal U2OSPLK4 cells. Size scale bar: 10µm. Right, representative cell cycle profiles of U2OSPLK4 control and PIDD1-deficient cells. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0.001 (***), p ≤ 0.0001 (****).


( E ) Live - cell imaging analysis of NF - κB EGFP - reporter activity in CRISPR - edited polyclonal PIDD1 - competent or - deficient U2OSκB - PLK4 and A549κB - PLK4 cells . NF - κB activity of ZM , Dox or TNF - treated cells is represented as fold - change with respect to their untreated counterparts . Bars represent means of n = 5 independent experiments ± SEM . Data information : The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) .

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

(E) Live-cell imaging analysis of NF-κB EGFP-reporter activity in CRISPR-edited polyclonal PIDD1-competent or -deficient U2OSκB-PLK4 and A549κB-PLK4 cells. NF-κB activity of ZM, Dox or TNF-treated cells is represented as fold-change with respect to their untreated counterparts. Bars represent means of n=5 independent experiments ± SEM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0.001 (***), p ≤ 0.0001 (****).


( F ) Representative immunofluorescence images of A549 cells co - stained with the indicated antibodies and documenting PIDD1 localization at centrosomes . Scale bars : 10 μm . ( G ) Luciferase reporter analysis of NF - κB activity in CRISPR - edited SCLT1 - competent or - deficient A549 cells . Luciferase activity was normalized respect renilla control . n = 3 independent experiments ; represented mean values ± SEM . Data information : The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) .

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

(F) Representative immunofluorescence images of A549 cells co-stained with the indicated antibodies and documenting PIDD1 localization at centrosomes. Scale bars: 10 μm. (G) Luciferase reporter analysis of NF-κB activity in CRISPR-edited SCLT1- competent or -deficient A549 cells. Luciferase activity was normalized respect renilla control. n=3 independent experiments; represented mean values ± SEM. Data information: The statistical significance was determined using two-way ANOVA; p ≤ 0.001 (***), p ≤ 0.0001 (****).


( B ) Left , immunoblot of CRISPR - edited polyclonal RIPK1 - or NEMO - deficient A549κB derivatives . Right , luciferase assay of A549 cells indicated genotypes , treated for 48h with either ZM ( 2 μM ) or DHCB ( 4 μM ) . n = 4 independent experiments . Data are represented as the mean values ± SEM . The statistical significance was determined using two - way ANOVA ; p < 0 . 01 ( * * ) ; p < 0 . 001 ( * * * ) ; p < 0 . 0001 ( * * * * ) .

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

(B) Left, immunoblot of CRISPR-edited polyclonal RIPK1- or NEMO-deficient A549κB derivatives. Right, luciferase assay of A549 cells indicated genotypes, treated for 48h with either ZM (2 μM) or DHCB (4 μM). n=4 independent experiments. Data are represented as the mean values ± SEM. The statistical significance was determined using two-way ANOVA; p< 0.01 (**);p< 0.001 (***); p< 0.0001 (****).


( C ) Representative confocal fluorescence images documenting centrosome accumulation , as identified by CEP63 staining in 293T - Trex - Flip - in cells . Time course analysis was performed in cells synchronized in G1 / S after a 24h single thymidine block and release in either DMSO or ZM ( 2 μM ) . Cells were fixed at the indicated times for IF analyses . Scale bars : 10 μm .

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

(C) Representative confocal fluorescence images documenting centrosome accumulation, as identified by CEP63 staining in 293T-Trex-Flip-in cells. Time course analysis was performed in cells synchronized in G1/S after a 24h single thymidine block and release in either DMSO or ZM (2 μM). Cells were fixed at the indicated times for IF analyses. Scale bars: 10 μm.


( D ) Different time point immunoprecipitations ( IP ) after release in ZM ( 2 μM ) of endogenous RAIDD , RIPK1 and NEMO proteins in PIDD1 - deficient 293T - Trex - Flip - in cells re - expressing a FLAG - tagged PIDD1 - FL protein . One representative experiment out of three is shown .

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

(D) Different time point immunoprecipitations (IP) after release in ZM (2 μM) of endogenous RAIDD, RIPK1 and NEMO proteins in PIDD1-deficient 293T-Trex-Flip-in cells re-expressing a FLAG-tagged PIDD1-FL protein. One representative experiment out of three is shown.


( E ) Analysis of NEMO - PIDDosome formation in PIDD1 - deficient 293Trex - Flip - In cells re - expressing different PIDD1 mutants depicted in G . Cells were treated as in C and processed for FLAG - IP 11h after release into DMSO or ZM ( 2 μM ) . One out of two independent experiments is shown .

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

(E) Analysis of NEMO-PIDDosome formation in PIDD1-deficient 293Trex-Flip-In cells re-expressing different PIDD1 mutants depicted in G. Cells were treated as in C and processed for FLAG-IP 11h after release into DMSO or ZM (2 μM). One out of two independent experiments is shown.


( F ) 293T cells were transiently transfected with constructs allowing expression of GFP , a full length ( FL ) single ( PIDD1 - FL _ FLAG ) or double - tagged ( HA _ PIDD1 - FL _ FLAG ) version of PIDD1 , or an HA - tagged N - terminal fragment of PIDD1 ( HA _ PIDD1 - N ) . Co - immunoprecipitation was performed to probe for interaction with endogenous RIPK1 after either anti - FLAG or anti - HA pull - down . One out of two independent experiments is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were transiently transfected with constructs allowing expression of GFP, a full length (FL) single (PIDD1-FL_FLAG) or double-tagged (HA_PIDD1-FL_FLAG) version of PIDD1, or an HA-tagged N-terminal fragment of PIDD1 (HA_PIDD1-N). Co-immunoprecipitation was performed to probe for interaction with endogenous RIPK1 after either anti-FLAG or anti-HA pull-down. One out of two independent experiments is shown.


Primary MEF from Pidd1 - / - mice were treated for 72h with ZM or DMSO and subjected to mRNA - sequencing analysis . ( B ) qRT - PCR analysis confirm expression of different pro - inflammatory genes in Pidd1 + / + but not Pidd1 - / - MEF . Hprt1 , β - actin and Gapdh were used as housekeeping gene expression controls . n = 3 independent samples ; data represent mean values ± SEM . The statistical significance was determined using two - way ANOVA test ; p ≤ 0 . 01 ( * * ) , p ≤ 0 . 001 ( * * * ) .

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

Primary MEF from Pidd1-/- mice were treated for 72h with ZM or DMSO and subjected to mRNA-sequencing analysis. (B) qRT-PCR analysis confirm expression of different pro-inflammatory genes in Pidd1+/+ but not Pidd1-/- MEF. Hprt1, β-actin and Gapdh were used as housekeeping gene expression controls. n=3 independent samples; data represent mean values ± SEM. The statistical significance was determined using two-way ANOVA test; p ≤ 0.01 (**), p ≤ 0.001 (***).


Primary MEF from Pidd1 - / - mice were treated for 72h with ZM or DMSO and subjected to mRNA - sequencing analysis . ( D ) qRT - qPCR analysis of macrophage activation markers indicative of a pro - inflammatory state . Relative expression values are plotted as the fold - change expression relative to BMDM stimulated with conditioned medium from untreated MEFPLK4 . β - actin was used as housekeeping gene for normalization . Bars represent mean ± SEM ( n = 9 ) biological replicates . The statistical significance was determined using two - way ANOVA ; p ≤ 0 . 1 ( * ) , p ≤ 0 . 01 ( * * ) , p ≤ 0 . 001 ( * * * ) , p ≤ 0 . 0001 ( * * * * ) .

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

Primary MEF from Pidd1-/- mice were treated for 72h with ZM or DMSO and subjected to mRNA-sequencing analysis. (D) qRT-qPCR analysis of macrophage activation markers indicative of a pro-inflammatory state. Relative expression values are plotted as the fold-change expression relative to BMDM stimulated with conditioned medium from untreated MEFPLK4. β-actin was used as housekeeping gene for normalization. Bars represent mean ± SEM (n=9) biological replicates. The statistical significance was determined using two-way ANOVA; p ≤ 0. 1 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****).


A549EGFP cells were treated with DMSO or ZM ( 2μM ) for 72h , followed by co - culture with NK - 92 cells and live - cell imaging analysis . ( A ) Representative images of target A549EGFP cells pre - treated for 72 h with ZM ( 2µM ) and co - cultured subsequently with NK - 92 cells in a 1 : 10 target to effector ratio . Interactions were recorded by live - cell imaging for 18 h at a 1 min interval . White arrow indicate polyploid cell undergoing cell death . EGFP release coincides with loss of cell membrane integrity . All images were acquired at the same exposure time and light intensity . Scale bar : 10 µm . Data information : The statistical significance was determined using two - tailed t - test ; P < 0 . 001 ( * * ) ; P < 0 . 001 ( * * * ) .

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

A549EGFP cells were treated with DMSO or ZM (2μM) for 72h, followed by co-culture with NK-92 cells and live-cell imaging analysis. (A) Representative images of target A549EGFP cells pre-treated for 72 h with ZM (2µM) and co-cultured subsequently with NK-92 cells in a 1:10 target to effector ratio. Interactions were recorded by live-cell imaging for 18 h at a 1 min interval. White arrow indicate polyploid cell undergoing cell death. EGFP release coincides with loss of cell membrane integrity. All images were acquired at the same exposure time and light intensity. Scale bar: 10 µm. Data information: The statistical significance was determined using two-tailed t-test; P < 0.001 (**); P < 0.001 (***).


A549EGFP cells were treated with DMSO or ZM ( 2μM ) for 72h , followed by co - culture with NK - 92 cells and live - cell imaging analysis . ( D ) Confluence of ZM pre - treated A549EGFP cells expressing or lacking PIDD1 was measured over time in presence of NK cells and normalized with respect to their DMSO treated counterparts . Middle , NK cell - mediated killing interpolated from cell confluence graphs at 50 h . n ≥ 4 independent experiments performed in duplicates , mean ± SEM . ( E ) Confluence measurement over time of A549EGFP derivatives impaired in either NEMO - PIDDosome ( RIPK1 KO ) or Caspase - 2 - PIDDosome formation ( RAIDD KO ) . Middle , NK cell - mediated killing interpolated from cell confluence graphs at 50 h . n ≥ 3 independent experiments ; mean ± SEM ; unpaired t - test . ( F ) Confluence analysis of Dox pre - treated A549EGFP cells overexpressing PLK4 was performed over time in the presence of NK cells and normalized with respect to their DMSO treated controls . n = 5 independent experiments ; mean ± SEM . ( G ) Confluence analysis of centriole - depleted and ZM pre - treated A549EGFP cells cultured in presence of NK cells . Values are normalized with respect to their DMSO ± Centrinone ( 125nM ) treated controls . n = 4 independent experiments ; mean ± SEM . Data information : The statistical significance was determined using two - tailed t - test ; P < 0 . 001 ( * * ) ; P < 0 . 001 ( * * * ) .

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

A549EGFP cells were treated with DMSO or ZM (2μM) for 72h, followed by co-culture with NK-92 cells and live-cell imaging analysis. (D) Confluence of ZM pre-treated A549EGFP cells expressing or lacking PIDD1 was measured over time in presence of NK cells and normalized with respect to their DMSO treated counterparts. Middle, NK cell-mediated killing interpolated from cell confluence graphs at 50 h. n≥4 independent experiments performed in duplicates, mean ± SEM. (E) Confluence measurement over time of A549EGFP derivatives impaired in either NEMO-PIDDosome (RIPK1 KO) or Caspase-2-PIDDosome formation (RAIDD KO). Middle, NK cell-mediated killing interpolated from cell confluence graphs at 50 h. n≥3 independent experiments; mean ± SEM; unpaired t-test. (F) Confluence analysis of Dox pre-treated A549EGFP cells overexpressing PLK4 was performed over time in the presence of NK cells and normalized with respect to their DMSO treated controls. n=5 independent experiments; mean ± SEM. (G) Confluence analysis of centriole-depleted and ZM pre-treated A549EGFP cells cultured in presence of NK cells. Values are normalized with respect to their DMSO ± Centrinone (125nM) treated controls. n=4 independent experiments; mean ± SEM. Data information: The statistical significance was determined using two-tailed t-test; P < 0.001 (**); P < 0.001 (***).


Phenotypes of wild type and nine microtubule - associated gene mutants . Comparison of the filament and stigma phenotypes of wild type and nine microtubule - associated gene mutants .

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

Phenotypes of wild type and nine microtubule-associated gene mutants. Comparison of the filament and stigma phenotypes of wild type and nine microtubule-associated gene mutants.


Microtubule organization in wild type stamen filament cells . ( D - F ) Microtubule organization in the stamen filament cells of lue1 and ktn80 . 1234 . Arrows indicate microtubule crossovers . Dotted lines indicate abnormal microtubules .

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

Microtubule organization in wild type stamen filament cells. (D-F) Microtubule organization in the stamen filament cells of lue1 and ktn80.1234. Arrows indicate microtubule crossovers. Dotted lines indicate abnormal microtubules.


Time - lapse images showing that KTN1 is associated with microtubule severing at crossover sites in wild type filament cells . GFP - labeled KTN1 fluorescent particles are pseudo - colored in red , and microtubules ( labeled with mCherry - TUB6 ) are shown in green . Yellow arrows indicate KTN1 at microtubule crossover sites . Asterisks indicate the location at which a microtubule has been severed at a crossover site . Time - lapse images showing that KTN1 cannot be recruited to crossover sites and no severing was observed in ktn80 . 1234 filament cells . Yellow open arrows indicate microtubule crossovers , which remain stable for a long time .

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

Time-lapse images showing that KTN1 is associated with microtubule severing at crossover sites in wild type filament cells. GFP-labeled KTN1 fluorescent particles are pseudo-colored in red, and microtubules (labeled with mCherry-TUB6) are shown in green. Yellow arrows indicate KTN1 at microtubule crossover sites. Asterisks indicate the location at which a microtubule has been severed at a crossover site. Time-lapse images showing that KTN1 cannot be recruited to crossover sites and no severing was observed in ktn80.1234 filament cells. Yellow open arrows indicate microtubule crossovers, which remain stable for a long time.


Comparison of the morphologies of the stamen filament cells in wild type and qui - 2 .

Comparison of the morphologies of the stamen filament cells in wild type and qui-2.


EMSA showing that BZR1 binds to the E - box of the KTN80 . 4 promoter regions in vitro . Nonlabelled ( free ) native probe in 10 - to 100 - fold molar excess relative to the biotin - labeled native probe was used as a cold competitor . The mutant probe was used as a negative control .

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

EMSA showing that BZR1 binds to the E-box of the KTN80.4 promoter regions in vitro. Nonlabelled (free) native probe in 10- to 100-fold molar excess relative to the biotin-labeled native probe was used as a cold competitor. The mutant probe was used as a negative control.


Time - lapse images showing that KTN1 is associated with microtubule severing at crossover sites in ( A ) wild type ( B qui - 2 filament cells . GFP - labeled KTN1 fluorescent particles are pseudo - colored in red , and microtubules ( labeled with mCherry - TUB6 ) are shown in green . Yellow arrows indicate KTN1 at the microtubule crossover sites . Asterisks indicate the location at which a microtubule has been severed at a crossover site in A and

Time-lapse images showing that KTN1 is associated with microtubule severing at crossover sites in (A) wild type (B qui-2 filament cells. GFP-labeled KTN1 fluorescent particles are pseudo-colored in red, and microtubules (labeled with mCherry-TUB6) are shown in green. Yellow arrows indicate KTN1 at the microtubule crossover sites. Asterisks indicate the location at which a microtubule has been severed at a crossover site in A and B


Time - lapse images showing that KTN1 is associated with microtubule severing at crossover sites C ) qui - 2 filament cells . GFP - labeled KTN1 fluorescent particles are pseudo - colored in red , and microtubules ( labeled with mCherry - TUB6 ) are shown in green . Yellow arrows indicate KTN1 at the microtubule crossover sites . or the location at which a microtubule has not been severed at a crossover site in C .

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Time-lapse images showing that KTN1 is associated with microtubule severing at crossover sites C) qui-2 filament cells. GFP-labeled KTN1 fluorescent particles are pseudo-colored in red, and microtubules (labeled with mCherry-TUB6) are shown in green. Yellow arrows indicate KTN1 at the microtubule crossover sites. or the location at which a microtubule has not been severed at a crossover site in C.


A ) Assessment of the detection limit of synthetic SARS - CoV - 2 by the CRISPR - based assay , inferred from fluorescence values after 30 min . Background subtracted fluorescence represents sample minus control fluorescence without target .

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A) Assessment of the detection limit of synthetic SARS-CoV-2 by the CRISPR-based assay, inferred from fluorescence values after 30 min. Background subtracted fluorescence represents sample minus control fluorescence without target.


Detection of synthetic SARS - CoV - 2 RNA using commercially available paper strips ( lateral - flow ) in buffer ( B )

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Detection of synthetic SARS-CoV-2 RNA using commercially available paper strips (lateral-flow) in buffer (B)


Detection of synthetic SARS - CoV - 2 RNA using commercially available paper strips ( lateral - flow ) in saliva ( C ) .

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Detection of synthetic SARS-CoV-2 RNA using commercially available paper strips (lateral-flow) in saliva (C).


( A - D ) Serum NfL concentrations of preataxic ( green ) and ataxic ( red ) SCA3 subjects and controls ( blue ) were measured in two independent cohorts , each with a different Simoa approach : cohort # 1 , recruited by the ESMI consortium ( A , C ) , and cohort # 2 , recruited by the EuroSCA / RiSCA consortium ( B , D ) . Boxes show the ranges between lower and upper quartiles , the central bands show the medians , and the whiskers show data within 1 . 5 ∙ IQR of the median , with dots representing outliers . Groups were compared with Mann Whitney U tests ( * * * p < . 001 , * * p < . 01 , ns p ≥ . 05 , two - tailed , Bonferroni - corrected ; In the scatter plots , the individual NfL values were plotted as a function of subjects ' age . The dashed grey lines visualise the optimal cut - offs for differentiating ataxic SCA3 subjects from controls in each cohort ( cohort # 1 : 20 . 0 pg / ml , 98 . 7 % sensitivity , 92 . 2 % specificity ; cohort # 2 : 50 . 9 pg / ml , 92 . 6 % sensitivity , 100 % specificity ; cut - offs were derived by maximising Youden ' s index irrespective of age ) . Note the logarithmic scale of the y - axes .

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(A-D) Serum NfL concentrations of preataxic (green) and ataxic (red) SCA3 subjects and controls (blue) were measured in two independent cohorts, each with a different Simoa approach: cohort #1, recruited by the ESMI consortium (A, C), and cohort #2, recruited by the EuroSCA/RiSCA consortium (B, D). Boxes show the ranges between lower and upper quartiles, the central bands show the medians, and the whiskers show data within 1.5∙IQR of the median, with dots representing outliers. Groups were compared with Mann Whitney U tests (*** p<.001, ** p<.01, ns p≥.05, two-tailed, Bonferroni-corrected; In the scatter plots, the individual NfL values were plotted as a function of subjects' age. The dashed grey lines visualise the optimal cut-offs for differentiating ataxic SCA3 subjects from controls in each cohort (cohort #1: 20.0 pg/ml, 98.7% sensitivity, 92.2% specificity; cohort #2: 50.9 pg/ml, 92.6% sensitivity, 100% specificity; cut-offs were derived by maximising Youden's index irrespective of age). Note the logarithmic scale of the y-axes.


( E , F ) Serum pNfH levels of preataxic and ataxic SCA3 subjects and controls were also measured in both cohorts , each with a different Simoa approach ( two - tailed Mann Whitney U tests , Bonferroni - corrected ;

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(E, F) Serum pNfH levels of preataxic and ataxic SCA3 subjects and controls were also measured in both cohorts, each with a different Simoa approach (two-tailed Mann Whitney U tests, Bonferroni-corrected;


( A ) Serum NfL levels of ataxic SCA3 subjects significantly correlated with disease severity , as quantified by the Scale for the Rating and Assessment of Ataxia ( SARA ) score ( r = 0 . 43 , p < . 001 , Pearson ' s correlation , two - tailed test ) .

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(A) Serum NfL levels of ataxic SCA3 subjects significantly correlated with disease severity, as quantified by the Scale for the Rating and Assessment of Ataxia (SARA) score (r=0.43, p<.001, Pearson's correlation, two-tailed test).


( B ) The cross - sectional NfL levels also reflected longitudinal disease progression , as quantified by the annual SARA score change ( available for 35 subjects ) . Boxes show the ranges between lower and upper quartiles , the central bands show the medians , and the whiskers show data within 1 . 5 ∙ IQR of the median , with dots representing outliers . Subjects with high disease progression ( annual SARA score increase ≥ 0 . 71 points / year , n = 18 , median split ) had significantly higher serum levels of NfL than subjects with low disease progression

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(B) The cross-sectional NfL levels also reflected longitudinal disease progression, as quantified by the annual SARA score change (available for 35 subjects). Boxes show the ranges between lower and upper quartiles, the central bands show the medians, and the whiskers show data within 1.5∙IQR of the median, with dots representing outliers. Subjects with high disease progression (annual SARA score increase ≥0.71 points/year, n=18, median split) had significantly higher serum levels of NfL than subjects with low disease progression


( B ) The cross - sectional NfL levels also reflected longitudinal disease progression , as quantified by the annual SARA score change ( available for 35 subjects ) . Boxes show the ranges between lower and upper quartiles , the central bands show the medians , and the whiskers show data within 1 . 5 ∙ IQR of the median , with dots representing outliers . Subjects with high disease progression ( annual SARA score increase ≥ 0 . 71 points / year , n = 18 , median split ) had significantly higher serum levels of NfL than subjects with low disease

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( C ) We modelled serum NfL levels ( log - transformed ) in SCA3 carriers ( n = 123 ) with the predictors age and ATXN3 CAG repeat length , their squares and all possible interactions ( for details , see Results and Methods ) . The highly significant predictors ( age , its square , and repeat length , and the intercept , all p < . 001 , explained variance : R2 = 0 . 37 ) were used to generate the diagram . For a given age , each increase in CAG repeat count was associated with higher NfL concentrations . The steepness of the slopes declined with increasing age . In controls ( black , n = 125 ) , the relation between NfL level ( log - transformed ) and age was linear .

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(C) We modelled serum NfL levels (log-transformed) in SCA3 carriers (n=123) with the predictors age and ATXN3 CAG repeat length, their squares and all possible interactions (for details, see Results and Methods). The highly significant predictors (age, its square, and repeat length, and the intercept, all p<.001, explained variance: R2=0.37) were used to generate the diagram. For a given age, each increase in CAG repeat count was associated with higher NfL concentrations. The steepness of the slopes declined with increasing age. In controls (black, n=125), the relation between NfL level (log-transformed) and age was linear.


( A ) Serum NfL levels in preataxic SCA3 subjects ( cross - sectional data ) were plotted over the time from the individually estimated ataxia onset . NfL levels increased significantly with proximity to the estimated onset ( F ( 1 , 21 ) = 39 . 68 , p < . 001 , R2 = 0 . 64 ) . For benchmarking the levels of preataxic subjects , the blue dashed line visualises the optimal cut - off for differentiating ataxic SCA3 subjects from controls in the pooled cohort ( 20 . 3 pg / ml , 99 . 0 % sensitivity , 95 . 2 % specificity ) .

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(A) Serum NfL levels in preataxic SCA3 subjects (cross-sectional data) were plotted over the time from the individually estimated ataxia onset. NfL levels increased significantly with proximity to the estimated onset (F(1,21)=39.68, p<.001, R2=0.64). For benchmarking the levels of preataxic subjects, the blue dashed line visualises the optimal cut-off for differentiating ataxic SCA3 subjects from controls in the pooled cohort (20.3 pg/ml, 99.0% sensitivity, 95.2% specificity).


( B ) To determine the time at which NfL levels become significantly increased in the preataxic stage of SCA3 , NfL levels of preataxic carriers need to be related to NfL levels of controls at the same age , as NfL levels physiologically increase with age . For each preataxic subject , the difference between the measured NfL value ( green dot ) and the NfL value predicted for control subjects of the same age ( solid blue line ) was visualised by the length of the vertical green line . Standardisation of this difference relative to the NfL distribution in controls

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(B) To determine the time at which NfL levels become significantly increased in the preataxic stage of SCA3, NfL levels of preataxic carriers need to be related to NfL levels of controls at the same age, as NfL levels physiologically increase with age. For each preataxic subject, the difference between the measured NfL value (green dot) and the NfL value predicted for control subjects of the same age (solid blue line) was visualised by the length of the vertical green line. Standardisation of this difference relative to the NfL distribution in controls


( B ) To determine the time at which NfL levels become significantly increased in the preataxic stage of SCA3 , NfL levels of preataxic carriers need to be related to NfL levels of controls at the same age , as NfL levels physiologically increase with age . For each preataxic subject , the difference between the measured NfL value ( green dot ) and the NfL value predicted for control subjects of the same age ( solid blue line ) was visualised by the length of the vertical green line . Standardisation of this difference relative to the NfL distribution in

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( C ) In preataxic SCA3 subjects , NfL z - scores increased significantly ( F ( 1 , 21 ) = 30 . 78 , p < . 001 , R2 = 0 . 58 ) with subjects approaching the expected age of onset . NfL levels of preataxic subjects were significantly increased compared to controls ( i . e . z - score > 1 . 96 ) 7 . 5 years before the expected onset , indicated by the non - overlapping 95 % confidence intervals of SCA3 subjects ( black solid line ) and controls ( blue dashed line ) .

(C) In preataxic SCA3 subjects, NfL z-scores increased significantly (F(1,21)=30.78, p<.001, R2=0.58) with subjects approaching the expected age of onset. NfL levels of preataxic subjects were significantly increased compared to controls (i.e. z-score >1.96) 7.5 years before the expected onset, indicated by the non-overlapping 95% confidence intervals of SCA3 subjects (black solid line) and controls (blue dashed line).


( A , B ) Longitudinal intraindividual stability of NfL ( A ) and pNfH ( B ) serum levels over 6 weeks , assessed in both ataxic ( red ) and preataxic ( green ) SCA3 and control ( blue ) subjects ( n = 21 ) . Data of the same individuals are linked by lines . ICC , intraclass correlation coefficient ( estimate and 95 % CI ) .

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(A, B) Longitudinal intraindividual stability of NfL (A) and pNfH (B) serum levels over 6 weeks, assessed in both ataxic (red) and preataxic (green) SCA3 and control (blue) subjects (n=21). Data of the same individuals are linked by lines. ICC, intraclass correlation coefficient (estimate and 95% CI).


( C ) Sample size estimations were performed for future intervention trials using the reduction of Nf levels towards control levels as outcome measure . The estimated number of subjects per study arm is plotted over the assumed therapeutic effect for lowering the Nf level in SCA3 subjects to levels observed in healthy controls ( NfL : dashed line , pNfH : dotted line ) .

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(C) Sample size estimations were performed for future intervention trials using the reduction of Nf levels towards control levels as outcome measure. The estimated number of subjects per study arm is plotted over the assumed therapeutic effect for lowering the Nf level in SCA3 subjects to levels observed in healthy controls (NfL: dashed line, pNfH: dotted line).


( A - B ) Plasma concentrations of NfL ( A ) and pNfH ( B ) were measured in the 304Q knock - in SCA3 mouse model . Heterozygous animals become symptomatic at ≈ 8 months of age .

(A-B) Plasma concentrations of NfL (A) and pNfH (B) were measured in the 304Q knock-in SCA3 mouse model. Heterozygous animals become symptomatic at ≈8 months of age.


( C - F ) Tissue levels of soluble and aggregated mutant ataxin - 3 were measured in cerebellum ( C , E ) and frontal lobe ( D , F ) .

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(C-F) Tissue levels of soluble and aggregated mutant ataxin-3 were measured in cerebellum (C, E) and frontal lobe (D, F).


The figure shows the degree to which each variable differs between heterozygous SCA3 mice and wildtype controls for a given age . For each variable , we quantified the deviation of hetero zygous mice from controls of the same age by calculating the effect size r of the group comparison ( independent t - tests ) . An effect size of 0 indicates that the value in heterozygous mice is not different from controls , while an effect size of 1 would indicate strong abnormality . Note that the drop in the effect size of Nf levels from 12 months to 18 months thus does not imply a putative reduction of Nf levels , but rather the loss of the effect in mutants relative to controls , as controls show strong age - related Nf increases at 18 months of age . Negative effect size values were set to 0 .

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The figure shows the degree to which each variable differs between heterozygous SCA3 mice and wildtype controls for a given age. For each variable, we quantified the deviation of heterozygous mice from controls of the same age by calculating the effect size r of the group comparison (independent t-tests). An effect size of 0 indicates that the value in heterozygous mice is not different from controls, while an effect size of 1 would indicate strong abnormality. Note that the drop in the effect size of Nf levels from 12 months to 18 months thus does not imply a putative reduction of Nf levels, but rather the loss of the effect in mutants relative to controls, as controls show strong age-related Nf increases at 18 months of age. Negative effect size values were set to 0.


( A ) We assessed samples of cerebellar cortex , deep cerebellar nuclei , frontal cortex , pons and pyramidal tract by immunohistochemistry with ataxin - 3 staining , comparing heterozygous 304Q SCA3 mice at 2 , 6 , 12 and 18 months of age with wildtype animals at 18 months . ML : molecular layer , PC : Purkinje cells , GL : granular layer , scale bars : 20 µm .

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(A) We assessed samples of cerebellar cortex, deep cerebellar nuclei, frontal cortex, pons and pyramidal tract by immunohistochemistry with ataxin-3 staining, comparing heterozygous 304Q SCA3 mice at 2, 6, 12 and 18 months of age with wildtype animals at 18 months. ML: molecular layer, PC: Purkinje cells, GL: granular layer, scale bars: 20 µm.


( B ) We histologically assessed the number and integrity of Purkinje cells in the cerebellar cortex by NfL , pNfH , Nissl and calbindin staining , comparing heterozygous 304Q SCA3 mice at 2 , 6 , 12 and 18 months of age with wildtype animals . ML : molecular layer , PC : Purkinje cells , GL : granular layer , scale bars : 20 µm .

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(B) We histologically assessed the number and integrity of Purkinje cells in the cerebellar cortex by NfL, pNfH, Nissl and calbindin staining, comparing heterozygous 304Q SCA3 mice at 2, 6, 12 and 18 months of age with wildtype animals. ML: molecular layer, PC: Purkinje cells, GL: granular layer, scale bars: 20 µm.


( B ) We histologically assessed the number and integrity of Purkinje cells in the cerebellar cortex by NfL , pNfH , Nissl and calbindin staining , comparing heterozygous 304Q SCA3 mice at 2 , 6 , 12 and 18 months of age with wildtype animals .

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(B) We histologically assessed the number and integrity of Purkinje cells in the cerebellar cortex by NfL, pNfH, Nissl and calbindin staining, comparing heterozygous 304Q SCA3 mice at 2, 6, 12 and 18 months of age with wildtype animals.


Figure 4 - MAP2K1 is a predictive marker for inhibitors targeting EGFR Effect - size heat maps of six drugs ( see titles of panels ) targeting EGFR . It is evident that the different drugs showed different profiles but also that high MAP2K1 expression ( blue / red gradient across cell lines ) was consistently associated with drug resistance ( dark blue / yellow gradient across cell lines ; AUC : area under the curve ; see main text and Appendix for details ) . See also Figure EV5 .

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Figure 4 - MAP2K1 is a predictive marker for inhibitors targeting EGFR Effect-size heat maps of six drugs (see titles of panels) targeting EGFR. It is evident that the different drugs showed different profiles but also that high MAP2K1 expression (blue/red gradient across cell lines) was consistently associated with drug resistance (dark blue/yellow gradient across cell lines; AUC: area under the curve; see main text and Appendix for details). See also Figure EV5.


( A ) Effect - size heat maps of two drugs ( one from two different drug sensitivity screens ) targeting MEK1 / 2 show consistent association of high MERTK expression with drug resistance . The colour scheme is the same as in Figure 4 .

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(A) Effect-size heat maps of two drugs (one from two different drug sensitivity screens) targeting MEK1/2 show consistent association of high MERTK expression with drug resistance. The colour scheme is the same as in Figure 4.


( C ) Dose - response curves of two drugs for which high MERTK ( left panels ) or ACVR2A ( right panels ) expression was predicted to confer drug resistance . For each drug , three cell lines predicted to be sensitive ( dark blue ) and three cell lines predicted to be resistant ( yellow ) were assayed for viability . The experimental data validated that cell lines predicted to be more sensitive to a drug indeed showed this phenotype ( data represents the average of three technical replicates ; see Appendix for details ) .

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(C) Dose-response curves of two drugs for which high MERTK (left panels) or ACVR2A (right panels) expression was predicted to confer drug resistance. For each drug, three cell lines predicted to be sensitive (dark blue) and three cell lines predicted to be resistant (yellow) were assayed for viability. The experimental data validated that cell lines predicted to be more sensitive to a drug indeed showed this phenotype (data represents the average of three technical replicates; see Appendix for details).


( D ) Boxplots summarizing the data shown in panel ( C ) using the area under the curve ( AUC ) as a measure for drug sensitivity . High AUC values indicate drug resistance . Again , it is evident that cell lines predicted to be more resistant to a certain drug were in fact significantly ( p ≤ 0 . 05 , one - sided Mann - Whitney test ) more resistant than cell lines predicted to be more sensitive .

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(D) Boxplots summarizing the data shown in panel (C) using the area under the curve (AUC) as a measure for drug sensitivity. High AUC values indicate drug resistance. Again, it is evident that cell lines predicted to be more resistant to a certain drug were in fact significantly (p≤0.05, one-sided Mann-Whitney test) more resistant than cell lines predicted to be more sensitive.


( E ) Western blot of and cells , visualising successful knockout by CRISPR / Cas9 .

(E) Western blot of and cells, visualising successful knockout by CRISPR/Cas9.


( F ) Dose - response curve of RDEA119 ( MEK1 / 2 inhibitor ) in and cells . The knockout is more sensitive to MEK1 / 2 inhibition than the wild type . See also Figure EV6 .

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(F) Dose-response curve of RDEA119 (MEK1/2 inhibitor) in and cells. The knockout is more sensitive to MEK1/2 inhibition than the wild type. See also Figure EV6.


( A ) Western Blot visualising MERTK expression in two negative ( RKO , CC07 ) and two positive control cell lines ( CaCo - 2 ; HT55 ) .

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(A) Western Blot visualising MERTK expression in two negative (RKO, CC07) and two positive control cell lines (CaCo-2; HT55).


( B ) IHC staining of MERTK expression in RKO ( negative control ) and CaCo - 2 ( positive control ) cells .

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(B) IHC staining of MERTK expression in RKO (negative control) and CaCo-2 (positive control) cells.


( C ) IHC staining of three representative TMAs from patients enrolled in the QUASAR 2 clinical trial and the corresponding quantification of the signal by pathologists .

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(C) IHC staining of three representative TMAs from patients enrolled in the QUASAR 2 clinical trial and the corresponding quantification of the signal by pathologists.


( D ) Kaplan - Meier plots showing worse overall , disease - free and recurrence - free survival of patients with high cytoplasmic / membranous expression of MERTK .

(D) Kaplan-Meier plots showing worse overall, disease-free and recurrence-free survival of patients with high cytoplasmic/membranous expression of MERTK.


A - H Effect of CCCP on the localisation of Noc and NocNΔ10 . Cellular localisation of Noc ‐ mYFP ( DWA206 ) and NocNΔ10 ‐ mYFP ( DWA382 ) either with no additions ( NA ) or after CCCP treatment ( 5 min ; 100 μM ) , as indicated . Scale bar , 2 . 5 μm .

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A-H Effect of CCCP on the localisation of Noc and NocNΔ10. Cellular localisation of Noc‐mYFP (DWA206) and NocNΔ10‐mYFP (DWA382) either with no additions (NA) or after CCCP treatment (5 min; 100 μM), as indicated. Scale bar, 2.5 μm.


I - L Effect of Noc overproduction on cell division . Exponentially growing cultures of DWA119 ( Δnoc , Pspac ( hy ) ‐ noc ) and DWA282 ( Δnoc , Pspac ( hy ) ‐ nocNΔ10 ) were examined after growth for 1 h with 1 mM IPTG . Cell membranes and DNA were stained with FM5 ‐ 95 and DAPI , respectively . Insets show corresponding phase contrast light microscopy images . Scale bar , 5 μm .

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I-L Effect of Noc overproduction on cell division. Exponentially growing cultures of DWA119 (Δnoc, Pspac(hy)‐noc) and DWA282 (Δnoc, Pspac(hy)‐nocNΔ10) were examined after growth for 1 h with 1 mM IPTG. Cell membranes and DNA were stained with FM5‐95 and DAPI, respectively. Insets show corresponding phase contrast light microscopy images. Scale bar, 5 μm.


M Complementation of noc in a Δnoc ΔminCD background . Strains DWA564 ( Δnoc , ΔminCD , Pxyl ‐ noc ‐ myfp ) and 566 ( Δnoc , ΔminCD , Pxyl ‐ nocNΔ10 ‐ myfp ) were streaked on nutrient agar ( NA ) plates in the presence of 0 . 5 % w / v xylose and incubated at 30 and 39 ° C , as indicated .

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M Complementation of noc in a Δnoc ΔminCD background. Strains DWA564 (Δnoc, ΔminCD, Pxyl‐noc‐myfp) and 566 (Δnoc, ΔminCD, Pxyl‐nocNΔ10‐myfp) were streaked on nutrient agar (NA) plates in the presence of 0.5% w/v xylose and incubated at 30 and 39°C, as indicated.


N Effect of Noc and NocNΔ10 overproduction on sporulation . Strains DWA119 ( Δnoc , Pspac ( hy ) ‐ noc ) and DWA282 ( Δnoc , Pspac ( hy ) ‐ nocNΔ10 ) were streaked on NA plates in the absence and presence of 1 mM IPTG , as indicated , and photographed after 48 h at 37 ° C .

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N Effect of Noc and NocNΔ10 overproduction on sporulation. Strains DWA119 (Δnoc, Pspac(hy)‐noc) and DWA282 (Δnoc, Pspac(hy)‐nocNΔ10) were streaked on NA plates in the absence and presence of 1 mM IPTG, as indicated, and photographed after 48 h at 37°C.


O Western blot analysis of cellular fractions ( T , total ; C , cytosolic ; M , membrane ) of strains DWA119 and 282 expressing Noc or NocNΔ10 , respectively . Proteins were detected using polyclonal antibodies against Noc , DnaA and PBP2B . Antibodies were used at a dilution of 1 : 10 , 000 .

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

O Western blot analysis of cellular fractions (T, total; C, cytosolic; M, membrane) of strains DWA119 and 282 expressing Noc or NocNΔ10, respectively. Proteins were detected using polyclonal antibodies against Noc, DnaA and PBP2B. Antibodies were used at a dilution of 1:10,000.


C - L Effects of N ‐ terminal substitutions on Noc localisation , in strains : DWA211 ( F5E ) , 318 ( F9E ) , 316 ( K2E ) , 212 ( R7E ) , 322 ( F5A ) , 323 ( F8A ) , 325 ( F9A ) , 206 ( WT ) , 328 ( S4A ) and 329 ( S4L ) , as indicated . Insets show the corresponding phase contrast light microscopy images . Scale bar , 5 μm .

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C-L Effects of N‐terminal substitutions on Noc localisation, in strains: DWA211 (F5E), 318 (F9E), 316 (K2E), 212 (R7E), 322 (F5A), 323 (F8A), 325 (F9A), 206 (WT), 328 (S4A) and 329 (S4L), as indicated. Insets show the corresponding phase contrast light microscopy images. Scale bar, 5 μm.


B Growth of strains DWA350 ( Δnoc ΔminCD ) and DWA307 ( Δnoc ΔminCD , Pxyl ‐ HCVAH ‐ NocNΔ10 ) on nutrient agar plates at 30 and 48 ° C in the absence and presence of 0 . 5 % w / v xylose , as indicated .

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B Growth of strains DWA350 (Δnoc ΔminCD) and DWA307 (Δnoc ΔminCD, Pxyl‐HCVAH‐NocNΔ10) on nutrient agar plates at 30 and 48°C in the absence and presence of 0.5% w/v xylose, as indicated.


C - F Cell morphology of strains DWA350 and DWA307 following growth in LB at 30 ° C ( C and D ) and at 42 ° C in LB + 0 . 5 % w / v xylose ( E and F ) . Arrowheads indicate minicells . Cell membranes were stained with FM5 - 95 . Insets show the corresponding phase contrast light microscopy images . Scale bar , 5 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 O O O O O O O O O O O O O O O O O

C-F Cell morphology of strains DWA350 and DWA307 following growth in LB at 30°C (C and D) and at 42°C in LB + 0.5% w/v xylose (E and F). Arrowheads indicate minicells. Cell membranes were stained with FM5-95. Insets show the corresponding phase contrast light microscopy images. Scale bar, 5 μm.


G , H Cellular localisation of HCVAH ‐ NocNΔ10 ‐ YFP ( G ) in strain DWA193 ( Δnoc , Pxyl ‐ HCVAH ‐ nocNΔ10 ‐ yfp ) and overlay showing DAPI ‐ stained DNA ( H ) . The strain was grown at 30 ° C in CH medium . Inset shows the corresponding phase contrast light microscopy image . Scale bar , 5 μm .

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G, H Cellular localisation of HCVAH‐NocNΔ10‐YFP (G) in strain DWA193 (Δnoc, Pxyl‐HCVAH‐nocNΔ10‐yfp) and overlay showing DAPI‐stained DNA (H). The strain was grown at 30°C in CH medium. Inset shows the corresponding phase contrast light microscopy image. Scale bar, 5 μm.


A - F Localisation of ParB ‐ box mutants . Cellular localisation of ( A ) Noc ‐ mYFP ( DWA206 ) , ( B ) NocQ68R ‐ mYFP ( DWA285 ) , ( C ) NocG86S ‐ mYFP ( DWA286 ) , ( D ) NocR88A ‐ mYFP ( DWA545 ) , ( E ) NocR89A ‐ mYFP ( DWA546 ) and ( F ) NocR91A ‐ mYFP ( DWA547 ) . Insets show the corresponding phase contrast images . Scale bar , 5 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 O O O O O O 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-F Localisation of ParB‐box mutants. Cellular localisation of (A) Noc‐mYFP (DWA206), (B) NocQ68R‐mYFP (DWA285), (C) NocG86S‐mYFP (DWA286), (D) NocR88A‐mYFP (DWA545), (E) NocR89A‐mYFP (DWA546) and (F) NocR91A‐mYFP (DWA547). Insets show the corresponding phase contrast images. Scale bar, 5 μm.


G Ability of ParB ‐ box mutants to rescue the growth defect of Δnoc ΔminCD . Strains DWA564 ( Pxyl ‐ noc ‐ myfp ) , 590 ( Pxyl ‐ nocQ68R ‐ myfp ) , 568 ( Pxyl ‐ nocG86S ‐ myfp ) , 598 ( Pxyl ‐ nocR88A ‐ myfp ) , 600 ( Pxyl ‐ nocR89A ‐ myfp ) and 602 ( Pxyl ‐ nocR91A ‐ myfp ) were streaked on plates containing 0 . 5 % w / v xylose and incubated for 18 h at either 30 or 39 ° C , as indicated , before being photographed .

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G Ability of ParB‐box mutants to rescue the growth defect of Δnoc ΔminCD. Strains DWA564 (Pxyl‐noc‐myfp), 590 (Pxyl‐nocQ68R‐myfp), 568 (Pxyl‐nocG86S‐myfp), 598 (Pxyl‐nocR88A‐myfp), 600 (Pxyl‐nocR89A‐myfp) and 602 (Pxyl‐nocR91A‐myfp) were streaked on plates containing 0.5% w/v xylose and incubated for 18 h at either 30 or 39°C, as indicated, before being photographed.


H - O ParB ‐ box mutants are dominant ‐ negative . Cells of strains DWA362 ( ΔminCD , Pspac ( hy ) ‐ noc ) , 363 ( ΔminCD , Pspac ( hy ) ‐ nocNΔ10 ) , 364 ( ΔminCD , Pspac ( hy ) ‐ nocQ68R ) and 365 ( ΔminCD , Pspac ( hy ) ‐ nocG86S ) were examined after growth for 2 h at 42 ° C with either no additions ( NA ) ( H - K ) or in the presence of 1 mM IPTG ( L - O ) , as indicated . Cell membranes were stained with FM5 ‐ 95 . Insets show the corresponding phase contrast images . Scale bar , 5 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H-O ParB‐box mutants are dominant‐negative. Cells of strains DWA362 (ΔminCD, Pspac(hy)‐noc), 363 (ΔminCD, Pspac(hy)‐nocNΔ10), 364 (ΔminCD, Pspac(hy)‐nocQ68R) and 365 (ΔminCD, Pspac(hy)‐nocG86S) were examined after growth for 2 h at 42°C with either no additions (NA) (H-K) or in the presence of 1 mM IPTG (L-O), as indicated. Cell membranes were stained with FM5‐95. Insets show the corresponding phase contrast images. Scale bar, 5 μm.


A - C Representative images of TetR ‐ mCherry in cells lacking ( A ) or containing ( B and C ) the NBS plasmid pDWA117 . Strains DWA427 ( A ) and 429 ( B and C ) ( both strains contain Pspac ‐ noc ) were examined after growth for 2 h in the absence ( A and B ) and presence ( C ) of 1 mM IPTG . DNA was stained with DAPI .

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A-C Representative images of TetR‐mCherry in cells lacking (A) or containing (B and C) the NBS plasmid pDWA117. Strains DWA427 (A) and 429 (B and C) (both strains contain Pspac‐noc) were examined after growth for 2 h in the absence (A and B) and presence (C) of 1 mM IPTG. DNA was stained with DAPI.


D - G Representative images showing the localisation of ( D ) Noc ‐ mYFP ( DWA519 ) , ( E ) NocNΔ10 ‐ mYFP ( DWA522 ) , ( F ) NocQ68R ‐ mYFP ( DWA520 ) and ( G ) NocG86S ‐ mYFP ( DWA521 ) , in the presence of the NBS plasmid pSG4929 .

D-G Representative images showing the localisation of (D) Noc‐mYFP (DWA519), (E) NocNΔ10‐mYFP (DWA522), (F) NocQ68R‐mYFP (DWA520) and (G) NocG86S‐mYFP (DWA521), in the presence of the NBS plasmid pSG4929.


A , B Effects of Noc overproduction on cell division and nucleoid morphology in E . coli . Cells of strain DWA261 carrying pDWA37 ( PA1 / 04 / 03 ‐ noc ) were examined after growth in LB with either no additions ( A ) or after induction for 1 h with 1 mM IPTG ( B ) .

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A, B Effects of Noc overproduction on cell division and nucleoid morphology in E. coli. Cells of strain DWA261 carrying pDWA37 (PA1/04/03‐noc) were examined after growth in LB with either no additions (A) or after induction for 1 h with 1 mM IPTG (B).


C - E Effects of overproduction of Noc variants on nucleoid morphology . Cells of strains DWA266 ( PA1 / 04 / 03 ‐ nocNΔ10 ) ( C ) , 270 ( PA1 / 04 / 03 ‐ HCVAH ‐ nocNΔ10 ) ( D ) and 267 ( PA1 / 04 / 03 ‐ nocCΔ50 ) ( E ) carrying plasmids for the overproduction of the indicated mutants ( see cartoons underneath panels ) were grown in LB and examined after growth for 1 h in the presence of 1 mM IPTG . Cell membranes and DNA were stained with FM5 ‐ 95 and DAPI , respectively . Scale bar , 5 μm .

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C-E Effects of overproduction of Noc variants on nucleoid morphology. Cells of strains DWA266 (PA1/04/03‐nocNΔ10) (C), 270 (PA1/04/03‐HCVAH‐nocNΔ10) (D) and 267 (PA1/04/03‐nocCΔ50) (E) carrying plasmids for the overproduction of the indicated mutants (see cartoons underneath panels) were grown in LB and examined after growth for 1 h in the presence of 1 mM IPTG. Cell membranes and DNA were stained with FM5‐95 and DAPI, respectively. Scale bar, 5 μm.


A , B Effects of Spo0J and Noc30 ‐ Spo0J overproduction on cell division and nucleoid morphology in E . coli . Cells of strains DWA271 ( PA1 / 04 / 03 ‐ spo0J ) ( A ) and 272 ( PA1 / 04 / 03 ‐ noc ‐ spo0J ) ( B ) were grown in LB in the presence of 1 mM IPTG , to induce the expression of either Spo0J ( A ) or the Noc ‐ Spo0J hybrid ( B ) , and were examined 1 h post ‐ induction . Cell membranes and DNA were stained with FM5 - 95 and DAPI , respectively . Scale bar , 5 μm .

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A, B Effects of Spo0J and Noc30‐Spo0J overproduction on cell division and nucleoid morphology in E. coli. Cells of strains DWA271 (PA1/04/03‐spo0J) (A) and 272 (PA1/04/03‐noc‐spo0J) (B) were grown in LB in the presence of 1 mM IPTG, to induce the expression of either Spo0J (A) or the Noc‐Spo0J hybrid (B), and were examined 1 h post‐induction. Cell membranes and DNA were stained with FM5-95 and DAPI, respectively. Scale bar, 5 μm.


( a ) Western blot analysis of CALM , actin , GAPDH and LC3 - II in several cell lines ( HeLa , HEK , CAD and murine embryonic fibroblast ( MEF ) ) where CALM was knocked down using shRNA or siRNA , as indicated , or knockout with rescue experiment . In all experiments in this paper , we used a scramble siRNA or a luciferase shRNA as controls . The cells were starved in Hanks balanced salt solution ( HBSS ) and treated with Baf A1 as indicated . ( BC , basal conditions ; SE , short exposure ; LE , longer exposure ) . Quantification of LC3 - II / actin or GAPDH ratio is shown in Supplementary Fig . 1A . * Not specific .

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

(a) Western blot analysis of CALM, actin, GAPDH and LC3-II in several cell lines (HeLa, HEK, CAD and murine embryonic fibroblast (MEF)) where CALM was knocked down using shRNA or siRNA, as indicated, or knockout with rescue experiment. In all experiments in this paper, we used a scramble siRNA or a luciferase shRNA as controls. The cells were starved in Hanks balanced salt solution (HBSS) and treated with Baf A1 as indicated. (BC, basal conditions; SE, short exposure; LE, longer exposure). Quantification of LC3-II/actin or GAPDH ratio is shown in Supplementary Fig. 1A. *Not specific.


( b ) LC3 dot counting in CALM knockdown cells . CALM was knocked down in HeLa cells expressing GFP - LC3 and with or without an siRNA - resistant form of wild - type CALM ( rescue ) , as indicated . The cells were starved in HBSS for 4 h , fixed and subjected to fluorescence microscopy to score the number of LC3 dots per cell . The knockdown efficiency and the level of the siRNA - resistant form of CALM are shown on the left on the western blotting . The number of LC3 dots per cell ( shown as mean ± s . d . ) is shown on the graph for each condition ( n ≥ 300 cells per condition ; BC , basal conditions ) . The ratio of the number of LC3 dots per cell between starvation and basal conditions is shown on the right ( * P0 . 01 ; NS , not significant , two - tailed t - test ) . Pictures obtained from automated microscope are shown . Scale bars , 20 μm .

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(b) LC3 dot counting in CALM knockdown cells. CALM was knocked down in HeLa cells expressing GFP-LC3 and with or without an siRNA-resistant form of wild-type CALM (rescue), as indicated. The cells were starved in HBSS for 4 h, fixed and subjected to fluorescence microscopy to score the number of LC3 dots per cell. The knockdown efficiency and the level of the siRNA-resistant form of CALM are shown on the left on the western blotting. The number of LC3 dots per cell (shown as mean ±s.d.) is shown on the graph for each condition (n≥300 cells per condition; BC, basal conditions). The ratio of the number of LC3 dots per cell between starvation and basal conditions is shown on the right (*P0.01; NS, not significant, two-tailed t-test). Pictures obtained from automated microscope are shown. Scale bars, 20 μm.


( a ) p62 vesicle formation in CALM knockdown HeLa cells . Confocal pictures are shown . # CALM - downregulated cells where p62 vesicles accumulate . Data are representative of three independent experiments and shown as mean ± s . e . m . ( n ≥ 500 cells ; * P0 . 01 ; two - tailed t - test ) . Scale bars , 5 μm .

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(a) p62 vesicle formation in CALM knockdown HeLa cells. Confocal pictures are shown. #CALM-downregulated cells where p62 vesicles accumulate. Data are representative of three independent experiments and shown as mean ±s.e.m. (n≥500 cells; *P0.01; two-tailed t-test). Scale bars, 5 μm.


( b ) Western blot analysis of p62 , actin and GAPDH in HeLa cells and HEK cells ( basal conditions , BC , or without serum for 1 h ) where CALM was knocked down using shRNA or siRNA , as indicated . Data are mean ± s . d ( n = 3 experiments for HeLa cells and HEK cells ; * P0 . 05 ; two - tailed t - test ) .

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(b) Western blot analysis of p62, actin and GAPDH in HeLa cells and HEK cells (basal conditions, BC, or without serum for 1 h) where CALM was knocked down using shRNA or siRNA, as indicated. Data are mean ±s.d (n=3 experiments for HeLa cells and HEK cells; *P0.05; two-tailed t-test).


( c ) Percentage of huntingtinQ74 - expressing cells with aggregates in CALM knockdown HeLa cells . Data depict one representative experiments performed in triplicate , out of three independent experiments and shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ) .

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(c) Percentage of huntingtinQ74-expressing cells with aggregates in CALM knockdown HeLa cells. Data depict one representative experiments performed in triplicate, out of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).


( d ) Tau - positive tangle formation in CALM knockdown cells . HeLa cells transiently expressing DsRed - tau 4R were treated with Baf A1 for 4 h as indicated . Cells were fixed and analysed by confocal microscopy after immnunostaining for phosphorylated tau using PHF1 antibody ( green ) . Data represent the number of cells with phosphorylated tau - positive tangles as mean ± s . e . m . ( n = 3 experiments ; * P0 . 01 ; two - tailed t - test ) . Scale bars , 5 μm .

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(d) Tau-positive tangle formation in CALM knockdown cells. HeLa cells transiently expressing DsRed-tau 4R were treated with Baf A1 for 4 h as indicated. Cells were fixed and analysed by confocal microscopy after immnunostaining for phosphorylated tau using PHF1 antibody (green). Data represent the number of cells with phosphorylated tau-positive tangles as mean ±s.e.m. (n=3 experiments; *P0.01; two-tailed t-test). Scale bars, 5 μm.


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(b) Western blot analysis of p62, actin and GAPDH in HeLa cells and HEK cells (basal conditions, BC, or without serum for 1 h) where CALM was knocked down using shRNA or siRNA, as indicated. Data are mean ±s.d (n=3 experiments for HeLa cells and HEK cells; *P0.05; two-tailed t-test).


( a ) Formation of endogenous ATG12 vesicles in CALM knockdown HeLa cells in basal ( BC ) and serum starvation conditions . Confocal pictures are shown with arrows indicating ATG12 vesicles in starvation conditions . Data are from one representative experiment , out of three independent experiments . Data shown as mean ± s . e . m . ( n ≥ 500 cells ; * P0 . 01 ; two - tailed t - test ) . Scale bars , 5 μm .

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(a) Formation of endogenous ATG12 vesicles in CALM knockdown HeLa cells in basal (BC) and serum starvation conditions. Confocal pictures are shown with arrows indicating ATG12 vesicles in starvation conditions. Data are from one representative experiment, out of three independent experiments. Data shown as mean ±s.e.m. (n≥500 cells; *P0.01; two-tailed t-test). Scale bars, 5 μm.


( b ) Co - localization between GFP - ATG16L1 and internalized cholera toxin subunit B conjugated with Alexa555 ( CTX ; 20 min ) in CALM knockdown HeLa cells . Confocal pictures are presented with magnified areas showing the co - localization between ATG16L1 and CTX in greater detail . Scale bars , 5 μm . The Pearson ' s coefficient between ATG16L1 and cholera toxin is shown . Data are representative of three independent experiments and shown as mean ± s . d . ( n ≥ 20 cells ; * P0 . 05 ; two - tailed t - test ) .

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(b) Co-localization between GFP-ATG16L1 and internalized cholera toxin subunit B conjugated with Alexa555 (CTX; 20 min) in CALM knockdown HeLa cells. Confocal pictures are presented with magnified areas showing the co-localization between ATG16L1 and CTX in greater detail. Scale bars, 5 μm. The Pearson's coefficient between ATG16L1 and cholera toxin is shown. Data are representative of three independent experiments and shown as mean ±s.d. (n≥20 cells; *P0.05; two-tailed t-test).


( c ) Size of endogenous ATG12 vesicles in CALM knockdown HeLa cells in starvation conditions . Confocal pictures are shown with arrows indicating ATG12 vesicles and # indicating CALM knockdown cells . Data are representative of three independent experiments and shown as mean ± s . d . ( n ≥ 100 vesicles ; * P0 . 05 ; two - tailed t - test ) . ( AU , arbitrary unit ) . Scale bars , 5 μm .

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(c) Size of endogenous ATG12 vesicles in CALM knockdown HeLa cells in starvation conditions. Confocal pictures are shown with arrows indicating ATG12 vesicles and # indicating CALM knockdown cells. Data are representative of three independent experiments and shown as mean ±s.d. (n≥100 vesicles; *P0.05; two-tailed t-test). (AU, arbitrary unit). Scale bars, 5 μm.


( d ) Live - cell imaging of ATG16L1 - GFP in CALM knockdown HeLa cells . ATG16L1 pictures from various time points of a 5 - min movie are shown in inverted grey style . Arrows indicate ATG16L1 vesicles . The number of fusion events per 5 min is shown . Data are representative of five movies and shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ) . Scale bars , 5 μm .

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(d) Live-cell imaging of ATG16L1-GFP in CALM knockdown HeLa cells. ATG16L1 pictures from various time points of a 5-min movie are shown in inverted grey style. Arrows indicate ATG16L1 vesicles. The number of fusion events per 5 min is shown. Data are representative of five movies and shown as mean ±s.d. (*P0.05; two-tailed t-test). Scale bars, 5 μm.


( e ) In vitro fusion assay of post - nuclear supernatant from HeLa cells expressing either GFP - ATG16L1 or mStrawberry - ATG16L1 in control and CALM knockdown conditions . Confocal pictures are shown where ATG16L1 - mStrawberry signal is shown in purple to enable better visualization . Fused vesicles appear in white . The ATP - negative condition , which prevents SNARE - dependent fusion , is also shown as a control for the reaction . Magnified areas are shown to allow visualization of the vesicles . The percentage of fused vesicles is represented . n = numbers of vesicles scored per field ( a minimum of five fields were analysed per condition ) . Data are representative of two independent experiments and shown as mean ± s . d . ( n ≥ 100 vesicles ) . Scale bars , 5 μm . ( * P0 . 05 ; two - tailed t - test ) .

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

(e) In vitro fusion assay of post-nuclear supernatant from HeLa cells expressing either GFP-ATG16L1 or mStrawberry-ATG16L1 in control and CALM knockdown conditions. Confocal pictures are shown where ATG16L1-mStrawberry signal is shown in purple to enable better visualization. Fused vesicles appear in white. The ATP-negative condition, which prevents SNARE-dependent fusion, is also shown as a control for the reaction. Magnified areas are shown to allow visualization of the vesicles. The percentage of fused vesicles is represented. n=numbers of vesicles scored per field (a minimum of five fields were analysed per condition). Data are representative of two independent experiments and shown as mean ±s.d. (n≥100 vesicles). Scale bars, 5 μm. (*P0.05; two-tailed t-test).


( a ) LC3 levels in CALM knockdown cells . CALM was knocked down in cells expressing or not expressing an siRNA - resistant form of CALM wild type ( wt ) or CALM 219 mutant , as indicated . The cells were lysed and subjected to western blotting . The knockdown efficiency , LC3 - II levels and the level of the siRNA - resistant form of CALM are shown on the western blotting .

(a) LC3 levels in CALM knockdown cells. CALM was knocked down in cells expressing or not expressing an siRNA-resistant form of CALM wild type (wt) or CALM 219 mutant, as indicated. The cells were lysed and subjected to western blotting. The knockdown efficiency, LC3-II levels and the level of the siRNA-resistant form of CALM are shown on the western blotting.


( b ) LC3 dots counting in CALM knockdown cells . CALM was knocked down in cells expressing GFP - LC3 with or without an siRNA - resistant form of CALM wild type ( wt ) or CALM mutant ( 219 mutant ) as indicated . The cells were kept in full medium or starved in Hanks balanced salt solution ( HBSS ) for 4 h , fixed and subjected to microscopy to score the number of LC3 dots per cell . The ratio of the number of LC3 dots per cell ( shown as mean ± s . d . ) between starvation and basal conditions is shown on the graph ( * P0 . 01 ; NS , not significant , two - tailed t - test ; n ≥ 300 cells per condition ; BC , basal conditions ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) LC3 dots counting in CALM knockdown cells. CALM was knocked down in cells expressing GFP-LC3 with or without an siRNA-resistant form of CALM wild type (wt) or CALM mutant (219 mutant) as indicated. The cells were kept in full medium or starved in Hanks balanced salt solution (HBSS) for 4 h, fixed and subjected to microscopy to score the number of LC3 dots per cell. The ratio of the number of LC3 dots per cell (shown as mean ±s.d.) between starvation and basal conditions is shown on the graph (*P0.01; NS, not significant, two-tailed t-test; n≥300 cells per condition; BC, basal conditions).


( c ) Co - localization between ATG9A and VAMP3 - HA or GFP - LC3 and VAMP8 - HA in control and CALM knockdown HeLa cells stably expressing VAMP3 - HA or VAMP8 - HA . Confocal pictures are shown with magnified areas showing co - localization between ATG9A and VAMP3 or GFP - LC3 and VAMP8 in control cells and no co - localization in CALM knockdown cells . Quantification of ATG9A - VAMP3 or GFP - LC3 - VAMP8 co - localization is shown on the graph as a Pearson ' s coefficient ( data are mean ± s . d . ; * P0 . 05 ; two - tailed t - test ) . Scale bars , 5 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-localization between ATG9A and VAMP3-HA or GFP-LC3 and VAMP8-HA in control and CALM knockdown HeLa cells stably expressing VAMP3-HA or VAMP8-HA. Confocal pictures are shown with magnified areas showing co-localization between ATG9A and VAMP3 or GFP-LC3 and VAMP8 in control cells and no co-localization in CALM knockdown cells. Quantification of ATG9A-VAMP3 or GFP-LC3-VAMP8 co-localization is shown on the graph as a Pearson's coefficient (data are mean ±s.d.; *P0.05; two-tailed t-test). Scale bars, 5 μm.


( a ) Co - localization between ATG12 and VAMP2 - HA in control and CALM knockdown HeLa cells stably expressing VAMP2 - HA . Confocal pictures are shown with magnified areas on the right showing co - localization between ATG12 and VAMP2 in control cells and no co - localization in CALM knockdown cells . Quantification of ATG12 - VAMP2 co - localization is shown on the graph as the Pearson ' s coefficient ( data are mean ± s . d . ; * P0 . 05 ; two - tailed t - test ) . Scale bars , 5 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Co-localization between ATG12 and VAMP2-HA in control and CALM knockdown HeLa cells stably expressing VAMP2-HA. Confocal pictures are shown with magnified areas on the right showing co-localization between ATG12 and VAMP2 in control cells and no co-localization in CALM knockdown cells. Quantification of ATG12-VAMP2 co-localization is shown on the graph as the Pearson's coefficient (data are mean ±s.d.;*P0.05; two-tailed t-test). Scale bars, 5 μm.


( b ) Western blot analysis of VAMP2 , actin and LC3 - II in HeLa cells where VAMP2 was knocked down , as indicated . The cells were starved in Hanks balanced salt solution ( HBSS ) and treated with Baf A1 as indicated . ( SE , short exposure ; LE , longer exposure . ) Quantification of LC3 - II / actin ratio is shown . Data are representative of three independent experiments and shown as mean ± s . d . ( * P0 . 05 ; NS , not significant , two - tailed t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-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

(b) Western blot analysis of VAMP2, actin and LC3-II in HeLa cells where VAMP2 was knocked down, as indicated. The cells were starved in Hanks balanced salt solution (HBSS) and treated with Baf A1 as indicated. (SE, short exposure; LE, longer exposure.) Quantification of LC3-II/actin ratio is shown. Data are representative of three independent experiments and shown as mean ±s.d. (*P0.05; NS, not significant, two-tailed t-test).


( c ) LC3 dot counting in CALM and VAMP2 knockdown cells . CALM or VAMP2 were knocked down in cells expressing GFP - LC3 . The cells were kept in full medium or starved in HBSS for 4 h , fixed and subjected to automated fluorescence microscopy to score the number of LC3 dots per cell . The number of LC3 dots per cell ( shown as mean ± s . d . ) is shown on the graph for each condition ( n ≥ 300 cells per condition ; BC , basal conditions ) . ( * P0 . 01 ; NS , not significant , two - tailed t - test ) .

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

(c) LC3 dot counting in CALM and VAMP2 knockdown cells. CALM or VAMP2 were knocked down in cells expressing GFP-LC3. The cells were kept in full medium or starved in HBSS for 4 h, fixed and subjected to automated fluorescence microscopy to score the number of LC3 dots per cell. The number of LC3 dots per cell (shown as mean ±s.d.) is shown on the graph for each condition (n≥300 cells per condition; BC, basal conditions). (*P0.01; NS, not significant, two-tailed t-test).


( d ) Number of p62 dots per cell in VAMP2 knockdown . HeLa cells where VAMP2 was knocked down were fixed and subjected to microscopy after labelling endogenous p62 using specific antibody . The data represent the number of p62 dots per cell shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ; n ≥ 300 cells per condition ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Number of p62 dots per cell in VAMP2 knockdown. HeLa cells where VAMP2 was knocked down were fixed and subjected to microscopy after labelling endogenous p62 using specific antibody. The data represent the number of p62 dots per cell shown as mean ±s.d. (*P0.05; two-tailed t-test; n≥300 cells per condition).


( e ) Percentage of huntingtinQ74 - expressing cells with aggregates in VAMP2 knockdown HeLa cells . Data are from one representative experiments performed in triplicate , out of three independent experiments and shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ) .

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

(e) Percentage of huntingtinQ74-expressing cells with aggregates in VAMP2 knockdown HeLa cells. Data are from one representative experiments performed in triplicate, out of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).


( f ) Western blot analysis of VAMP2 , actin and LC3 - II in HeLa cells stably expressing VAMP2 - HA wild type or VAMP2 - HA mutated in the CALM - binding site at different levels ( wild - type clone 6 , wt Cl6 : low level ; wild - type clone 11 , wt Cl11 : high level ; mutant clone 11 , AA Cl11 : low level ; mutant clone 12 , AA Cl12 : high level ) . The cells were treated with Baf A1 as indicated . ( SE , short exposure ; LE , longer exposure ) . Quantification of LC3 - II / actin ratio is shown . Data are representative of three independent experiments and shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-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

(f) Western blot analysis of VAMP2, actin and LC3-II in HeLa cells stably expressing VAMP2-HA wild type or VAMP2-HA mutated in the CALM-binding site at different levels (wild-type clone 6, wt Cl6: low level; wild-type clone 11, wt Cl11: high level; mutant clone 11, AA Cl11: low level; mutant clone 12, AA Cl12: high level). The cells were treated with Baf A1 as indicated. (SE, short exposure; LE, longer exposure). Quantification of LC3-II/actin ratio is shown. Data are representative of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).


( a ) Size of endogenous ATG12 vesicles in HeLa cells stably expressing either wild - type or mutant VAMP2 . Confocal pictures are presented with magnified areas showing ATG12 vesicles . Data are representative of three independent experiments and shown as mean ± s . d . ( n ≥ 100 vesicles ; * P0 . 05 ; two tail t - test ) . Scale bars , 5 μm .

(a) Size of endogenous ATG12 vesicles in HeLa cells stably expressing either wild-type or mutant VAMP2. Confocal pictures are presented with magnified areas showing ATG12 vesicles. Data are representative of three independent experiments and shown as mean ±s.d. (n≥100 vesicles; *P0.05; two tail t-test). Scale bars, 5 μm.


( b ) Co - localization between ATG12 and LC3 in HeLa cells stably expressing either wild - type clone 11 or mutant clone 12 VAMP2 . Confocal pictures are presented with magnified areas showing ATG12 - LC3 co - localization . The Pearson ' s coefficient between ATG12 and LC3 is shown . Data are representative of three independent experiments and shown as mean ± s . d . ( n ≥ 100 vesicles ; * P0 . 05 ; two - tailed t - test ) . Scale bars , 5 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 O O O O O O O O O O O O O O O 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) Co-localization between ATG12 and LC3 in HeLa cells stably expressing either wild-type clone 11 or mutant clone 12 VAMP2. Confocal pictures are presented with magnified areas showing ATG12-LC3 co-localization. The Pearson's coefficient between ATG12 and LC3 is shown. Data are representative of three independent experiments and shown as mean ±s.d. (n≥100 vesicles; *P0.05; two-tailed t-test). Scale bars, 5 μm.


( c ) Size and number of endogenous ATG12 vesicles in VAMP2 - knockdown HeLa cells in starvation condition . Confocal pictures are shown with arrows indicating ATG12 vesicles . Data are representative of three independent experiments and shown as mean ± s . d . for the size of ATG12 vesicle . ( n ≥ 100 vesicles ; * P0 . 05 ; based two - tailed t - test ) . Scale bars , 5 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 O O O O O O O O O O O O O O O O O O O O O O O

(c) Size and number of endogenous ATG12 vesicles in VAMP2-knockdown HeLa cells in starvation condition. Confocal pictures are shown with arrows indicating ATG12 vesicles. Data are representative of three independent experiments and shown as mean ±s.d. for the size of ATG12 vesicle. (n≥100 vesicles; *P0.05; based two-tailed t-test). Scale bars, 5 μm.


( d ) Live - cell imaging of ATG16L1 - GFP in VAMP2 - knockdown HeLa cells . Confocal pictures from various time points of a 5 - min movie are shown in inverted greyscale . Arrows indicate ATG16L1null . The number of fusion events per vesicles is shown . Data are representative of five movies and shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ) . Scale bars , 5 μm .

(d) Live-cell imaging of ATG16L1-GFP in VAMP2-knockdown HeLa cells. Confocal pictures from various time points of a 5-min movie are shown in inverted greyscale. Arrows indicate ATG16L1null. The number of fusion events per vesicles is shown. Data are representative of five movies and shown as mean ±s.d. (*P0.05; two-tailed t-test). Scale bars, 5 μm.


( e ) In vitro fusion assay of post - nuclear supernatants from control and VAMP2 knockdown HeLa cells expressing either GFP - ATG16L1 or mStrawberry - ATG16L1 . Confocal pictures are shown where ATG16L1 - mStrawberry signal is shown in purple to enable better visualization . Fused vesicles appear in white . The ATP - negative condition , which prevents SNARE - dependent fusion , is also shown as a control of reaction . Magnified areas are shown to allow visualization of the vesicles . The percentage of fused vesicles ( white ) is represented . Data are representative of two independent experiments and shown as mean ± s . d . ( n ≥ 100 vesicles ) . Scale bars , 5 μm . ( * P0 . 05 ; two - tailed t - test ) . n = numbers of vesicles scored per field .

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

(e) In vitro fusion assay of post-nuclear supernatants from control and VAMP2 knockdown HeLa cells expressing either GFP-ATG16L1 or mStrawberry-ATG16L1. Confocal pictures are shown where ATG16L1-mStrawberry signal is shown in purple to enable better visualization. Fused vesicles appear in white. The ATP-negative condition, which prevents SNARE-dependent fusion, is also shown as a control of reaction. Magnified areas are shown to allow visualization of the vesicles. The percentage of fused vesicles (white) is represented. Data are representative of two independent experiments and shown as mean ±s.d. (n≥100 vesicles). Scale bars, 5 μm. (*P0.05; two-tailed t-test). n=numbers of vesicles scored per field.


( a ) Western blotting showing downregulation of lap ( i ) or increase in Atg8a - II ( ii ) expression level in adult fly heads on lap downregulation using the UAS - RNAi lines lapGD12732 or lapKK105767 , or the heterozygous allele lap1 . Quantification of lap / actin and Atg8a - II / actin is shown . Genotypes : Control w ; elav - GAL4 / + ; for the RNAi lines : w ; elav - GAL4 / lapKK105767 and w ; elav - GAL4 / + ; lapGD12732 / + ; for the lap1 allele : w ; elav - GAL4 / + ; lap1 / + .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blotting showing downregulation of lap (i) or increase in Atg8a-II (ii) expression level in adult fly heads on lap downregulation using the UAS-RNAi lines lapGD12732 or lapKK105767, or the heterozygous allele lap1. Quantification of lap/actin and Atg8a-II/actin is shown. Genotypes: Control w; elav-GAL4/+; for the RNAi lines: w; elav-GAL4/ lapKK105767 and w; elav-GAL4/+; lapGD12732/+; for the lap1 allele: w; elav-GAL4/+; lap1/+.


( b ) Western blotting showing the accumulation of tau in Drosophila adult flyheads on lap downregulation using the UAS - RNAi lines lapGD12732 or lapKK105767 , or the heterozygous allele lap1 . Quantification of tau / lapGD12732 is shown . Genotypes : Control w ; elav - GAL4 / + ; for tau - WT : w ; elav - GAL4 / + ; UAS - tau - WT / + ; for RNAi lines : w ; elav - GAL4 / lapKK105767 ; UAS - tau - WT / + and w ; elav - GAL4 / + ; lapGD12732 / tau - WT ; for the lap1 allele : w ; elav - GAL4 / + ; lap1 / + or w ; elav - GAL4 / + ; lap1 / UAS - tau - WT .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blotting showing the accumulation of tau in Drosophila adult flyheads on lap downregulation using the UAS-RNAi lines lapGD12732 or lapKK105767, or the heterozygous allele lap1. Quantification of tau/lapGD12732 is shown. Genotypes: Control w; elav-GAL4/+; for tau-WT: w; elav-GAL4/+; UAS-tau-WT/+; for RNAi lines: w; elav-GAL4/ lapKK105767; UAS-tau-WT/+ and w; elav-GAL4/+; lapGD12732/tau-WT; for the lap1 allele: w; elav-GAL4/+; lap1/+ or w; elav-GAL4/+; lap1/UAS-tau-WT.


( ai ) Western blot analysis of tubulin , LC3 - I and LC3 - II in zebrafish larvae where ATG7 was downregulated , as indicated . ( ii ) Western blot analysis of CALM - HA , actin and LC3 - II in zebrafishlarvae where CALM - HA was expressed , as indicated . The larvae were treated with ammonium chloride ( NH4Cl ) , as indicated . ( SE , short exposure ; LE , longer exposure ) . Quantification of LC3 - II / actin ratio is shown . Data are representative of three independent experiments and shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ) .

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

(ai) Western blot analysis of tubulin, LC3-I and LC3-II in zebrafish larvae where ATG7 was downregulated, as indicated. (ii) Western blot analysis of CALM-HA, actin and LC3-II in zebrafishlarvae where CALM-HA was expressed, as indicated. The larvae were treated with ammonium chloride (NH4Cl), as indicated. (SE, short exposure; LE, longer exposure). Quantification of LC3-II/actin ratio is shown. Data are representative of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).