Metastatic burden in lungs of MDA231 - LM2 SUM159 - LM1 - bearing mice treated with paclitaxel as single therapy or in combination with JNKi . Each value represents mean ± . MDA231 - LM2 - bearing mice : n = 6 mice per group . SUM159 - LM1 - bearing mice : vehicle , JNKi PAX n = 6 mice per group ; PAX + JNKi n = 5 . Ten fields were quantified per lung section . FOV , field of view , LS , lung section . PAX , paclitaxel . Representative histological of metastatic foci ( arrows ) in lungs of MDA231 - LM2 - implanted mice described in panel C , stained for human vimentin expression in differentially treated groups . Scale bar , 100 μm .

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Metastatic burden in lungs of MDA231-LM2 and SUM159-LM1 tumor-bearing mice treated with paclitaxel as single therapy or in combination with JNKi. Each value represents the mean ± SEM. MDA231-LM2 tumor-bearing mice: n = 6 mice per group. SUM159-LM1 tumor-bearing mice: vehicle, JNKi and PAX n = 6 mice per group; PAX+ JNKi n = 5. Ten fields were quantified per lung section. FOV, field of view, LS, lung section. PAX, paclitaxel. Representative histological images of metastatic foci (arrows) in lungs of the MDA231-LM2-implanted mice described in panel C, stained for human vimentin expression in differentially treated groups. Scale bar, 100 μm.


Apoptosis was determined by of TUNEL - positive per field of view ( FOV ) in MDA231 - LM2 tumors ( from described in ( C ) ) treated with JNKi / or PAX . Values are means from 6 mice per condition ± .

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Apoptosis was determined by quantification of TUNEL-positive cells per field of view (FOV) in MDA231-LM2 tumors (from the animals described in (C)) treated with JNKi and/or PAX. Values are means from 6 mice per condition ± SEM.


GSEA showing enrichment of JNK signature in samples from breast cancer from TOP trial ( GSE16446 ) , stratified according to pathological response to neoadjuvant chemotherapy . NES , normalized enrichment score . pCR , pathological complete response .

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GSEA showing enrichment of the JNK signature in tumor samples from breast cancer patients from the TOP trial (GSE16446), stratified according to pathological response to neoadjuvant chemotherapy. NES, normalized enrichment score. pCR, pathological complete response.


Kaplan - Meier analyses of chemotherapy - treated breast cancer associating JNK signature ( JNK - S ) with metastasis - free survival ( TOP trial data set , JNK - S low n = 54 ; JNK - S high n = 53 ( G ) ) or overall survival ( METABRIC data set , JNK - S low n = 135 ; JNK - S high n = 135 ( H ) ) . HR , hazard . P values were determined by log - rank .

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Kaplan-Meier analyses of chemotherapy-treated breast cancer patients associating JNK signature (JNK-S) with metastasis-free survival (TOP trial data set, JNK-S low n = 54 patients; JNK-S high n = 53 patients (G)) or overall survival (METABRIC data set, JNK-S low n = 135 patients; JNK-S high n = 135 patients (H)). HR, hazard ratio. P values were determined by log-rank test.


( A ) Intracellular detection of NOD2 ( bold in histogram plot ) was performed by flow cytometry using freshly isolated alveolar macrophages selected from a gate set on large granular bronchoalveolar ( R1 , dot plot ) . One representative experiment out of three experiments is presented .

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(A) Intracellular detection of NOD2 (bold line in the histogram plot) was performed by flow cytometry using freshly isolated alveolar macrophages selected from a gate set on large granular bronchoalveolar cells (R1, dot plot). One representative experiment out of three experiments is presented.


( B ) were stimulated with 10 μg / mL of MDP for 24 h , NOD2 IκBα were measured in fractions by western blot . fold increase relative to unstimulated normalized to tubulin of one representative experiment out of three is reported .

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(B) Cells were stimulated with 10 μg/mL of MDP for 24 h, and NOD2 and IκBα proteins were measured in the cytosolic fractions by western blot analysis. The fold increase relative to unstimulated cells and normalized to tubulin of one representative experiment out of three is reported.


( C ) upregulation of NOD2 gene expression was assessed by quantitative PCR . results are depicted as mean fold change in NOD2 gene expression relative to unstimulated ( n = 11 ) .

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(C) The upregulation of NOD2 gene expression was assessed by quantitative PCR. The results are depicted as the mean fold change in NOD2 gene expression relative to unstimulated cells (n = 11).


( D ) Subcellular localization of NOD2 was ascertained in unstimulated ( left , × 80 , 000 ) MDP ‐ stimulated ( right , × 63 , 000 ) using an anti ‐ NOD2 antibody detected with a secondary antibody coupled to 5 ‐ nm gold particles , indicated with arrowheads . Bar represents 150 nm . were obtained from one experiment .

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(D) Subcellular localization of NOD2 was ascertained in unstimulated (left, ×80,000) and MDP‐stimulated cells (right, ×63,000) using an anti‐NOD2 antibody and detected with a secondary antibody coupled to 5‐nm gold particles, indicated with arrowheads. Bar represents 150 nm. Micrographs were obtained from one experiment.


NOD2 ligation in alveolar macrophages induces pro ‐ inflammatory cytokine release . were incubated for 24 h in presence of 10 μg / mL of MDP or 100 ng / mL of LPS . Production of IL ‐ 1β , IL ‐ 6 , TNF ‐ α , IL ‐ 10 was measured in culture supernatants using Milliplex technology . Depicted are box plots with median values quartiles for each cytokine . data are representative of two independent experiments ( n = 11 ) ; * p 0 . 05 * * p 0 . 01 using two ‐ Wilcoxon signed ‐ rank .

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NOD2 ligation in alveolar macrophages induces pro‐inflammatory cytokine release. Cells were incubated for 24 h in the presence of 10 μg/mL of MDP or 100 ng/mL of LPS. Production of IL‐1β, IL‐6, TNF‐α, and IL‐10 was measured in culture supernatants using Milliplex technology. Depicted are box plots with median values and quartiles for each cytokine. The data are representative of two independent experiments (n = 11); *p 0.05 and **p 0.01 using the two‐tailed Wilcoxon signed‐rank test.


LL37 IRGM gene expression is upregulated following NOD2 activation ( A ‐ D ) . Alveolar macrophages were incubated in presence of 10 μg / mL of MDP or 100 ng / mL of LPS for 24 h . Upregulation of ( A ) LL37 ( D ) IRGM gene expression was assessed after specific ligand recognition by quantitative PCR using Taqman system ΔΔCT method for relative . fold change in gene expression relative to unstimulated is depicted . Data are representative of two independent experiments ( n = 7 - 12 ) . Bold lines indicate median values .

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LL37 and IRGM gene expression is upregulated following NOD2 activation (A‐D). Alveolar macrophages were incubated in the presence of 10 μg/mL of MDP or 100 ng/mL of LPS for 24 h. Upregulation of (A) LL37 and (D) IRGM gene expression was assessed after specific ligand recognition by quantitative PCR using the Taqman system and the ΔΔCT method for relative quantification. The fold change in gene expression relative to unstimulated cells is depicted. Data are representative of two independent experiments (n = 7-12). Bold lines indicate median values.


( B , E ) LL37 IRGM were measured in fractions by western blot .

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(B, E) LL37 and IRGM protein levels were measured in cytosolic fractions by western blot analysis.


( C , F ) Data are expressed as fold increases relative to unstimulated normalized to tubulin calculated by densitometry are shown as means ± . One representative experiment out of three experiments is presented .

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(C, F) Data are expressed as the protein fold increases relative to unstimulated cells and normalized to tubulin calculated by densitometry and are shown as means ± SEM. One representative experiment out of three experiments is presented.


( A ) Intracellular burden was measured by quantifying CFU , intracellular growth index was calculated after 1 4 days post infection relative to phagocytosed at day 0 . Data are shown as medians quartiles are representative of n = 7 experiments .

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(A) Intracellular bacterial burden was measured by quantifying CFU, and the intracellular growth index was calculated after 1 and 4 days post infection relative to phagocytosed bacteria at day 0. Data are shown as medians and quartiles and are representative of n = 7 experiments.


production of ( B ) TNF ‐ α , ( C ) IL ‐ 6 ( D ) IL ‐ 1β was measured in culture supernatants using Milliplex technology . Box plots depicting median values quartiles are representative of n = 9 experiments .

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The production of (B) TNF‐α, (C) IL‐6 and (D) IL‐1β was measured in culture supernatants using Milliplex technology. Box plots depicting median values and quartiles are representative of n = 9 experiments.


( E ) LL37 ( F ) IRGM gene expression was assessed by quantitative PCR using Taqman system ΔΔCT method for relative . Fold changes in gene expression relative to unstimulated are reported . Bold lines indicate median values of n = 9 . * p 0 . 05 * * p 0 . 01 using two ‐ Wilcoxon signed ‐ rank .

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(E) LL37 and (F) IRGM gene expression was assessed by quantitative PCR using the Taqman system and the ΔΔCT method for relative quantification. Fold changes in gene expression relative to unstimulated cells are reported. Bold lines indicate median values of n = 9. *p 0.05 and **p 0.01 using the two‐tailed Wilcoxon signed‐rank test.


( G ) LL37 IRGM was assessed by western blot . One representative experiment out of three experiments is presented .

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(G) LL37 and IRGM protein was assessed by western blot. One representative experiment out of three experiments is presented.


( H ) amount of normalized to tubulin relative to infected ‐ only was calculated by densitometry . Data are depicted as means ± are representative of n = 3 experiments .

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(H) The amount of protein normalized to tubulin relative to infected‐only cells was calculated by densitometry. Data are depicted as the means ± SEM and are representative of n = 3 experiments.


( A , B , D , E , G , H ) subcellular localization of autophagy was ascertained in untreated MDP ‐ treated using ( A , B ) anti ‐ IRGM , ( D , E ) anti ‐ LC3 , ( G , H ) anti ‐ ATG16L1 antibodies detected with a secondary antibody coupled to 5 ‐ nm gold particles indicated with arrowheads ; bar represents 150 nm .

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(A, B, D, E, G, H) The subcellular localization of autophagy proteins was ascertained in untreated and MDP‐treated cells using (A, B) anti‐IRGM, (D, E) anti‐LC3, and (G, H) anti‐ATG16L1 antibodies and detected with a secondary antibody coupled to 5‐nm gold particles indicated with arrowheads; bar represents 150 nm.


( C , F , I ) Gold particles co ‐ localizing with were manually counted in ten macrophages of each condition . Means ± are depicted differences between treatments are indicated , * p 0 . 05 using a two ‐ paired t ‐ . ( B ) Transmission electron microscopy ( ) magnification × 50 , 000 ; of are magnification × 80 , 000 . Data were generated from one experiment .

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(C, F, I) Gold particles co‐localizing with bacteria were manually counted in ten macrophages of each condition. Means ± SEM are depicted and differences between treatments are indicated, *p 0.05 using a two‐tailed paired t‐test. (B) Transmission electron microscopy (TEM) magnification ×50,000; the rest of the micrographs are TEM magnification ×80,000. Data were generated from one experiment.


Macrophages treated with MDP after M . tuberculosis infection develop autophagy vacuoles . Alveolar macrophages were infected with M . tuberculosis H37Rv at an MOI of 5 for 1 h . Nonphagocytosed were washed away , macrophages were incubated for an additional hour . were then treated with 10 μg / mL of MDP for 24 h . Autophagy indicators ( arrowheads ) such as ( A ) vesicles containing organelles ( magnification × 20 , 000 ) ( B ) onion skin ‐ like lamellar multivesicular bodies ( magnification × 16 , 000 ) were identified by ultrastructural . .

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Macrophages treated with MDP after M. tuberculosis infection develop autophagy vacuoles. Alveolar macrophages were infected with M. tuberculosis H37Rv at an MOI of 5 for 1 h. Nonphagocytosed bacteria were washed away, and the macrophages were incubated for an additional hour. The cells were then treated with 10 μg/mL of MDP for 24 h. Autophagy indicators (arrowheads) such as (A) vesicles containing organelles (TEM magnification ×20,000) and (B) onion skin‐like lamellar multivesicular bodies (TEM magnification ×16,000) were identified by ultrastructural analysis..


( C , D ) Autophagosomes containing mycobacteria ( Mtb ) are indicated with arrows ( C , magnification × 40 , 000 ; D , magnification × 12 , 000 ) . were obtained from one experiment . Bar represents 300 nm .

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(C, D) Autophagosomes containing mycobacteria (Mtb) are indicated with arrows (C, TEM magnification ×40,000; D, TEM magnification ×12,000). Micrographs were obtained from one experiment. Bar represents 300 nm.


( a ) Chemical structure of PIK - III . ( b ) indicated kinases were incubated with different doses of PIK - III IC50 values were determined .

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(a) Chemical structure of PIK-III. (b) The indicated kinases were incubated with different doses of PIK-III and IC50 values were determined.


( c ) U2OS expressing GFP - FYVE were incubated with indicated compounds for 2 h , fixed , stained with Hoechst 33342 imaged by automated epifluorescence microscopy . GFP - positive per were calculated used to determine IC50 values for each compound . Data points represent values from three wells of one experiment .

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(c) U2OS cells expressing GFP-FYVE were incubated with the indicated compounds for 2 h, fixed, stained with Hoechst 33342 and imaged by automated epifluorescence microscopy. GFP-positive puncta per cell were calculated and used to determine IC50 values for each compound. Data points represent values from three wells of one experiment.


( d ) U2OS expressing GFP - FOXO3A were incubated with indicated compounds for 2 h , fixed , stained with Hoechst 33342 imaged by automated epifluorescence microscopy . of to GFP was calculated used to determine IC50 values for each compound . Data points represent values from three wells of one experiment .

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(d) U2OS cells expressing GFP-FOXO3A were incubated with the indicated compounds for 2 h, fixed, stained with Hoechst 33342 and imaged by automated epifluorescence microscopy. The ratio of nuclear to cellular GFP fluorescence intensity was calculated and used to determine IC50 values for each compound. Data points represent values from three wells of one experiment.


( a ) General domain structure of human VPS34 kinase comprising helical domain ( yellow ) kinase domain ( green ) . ATP - binding pocket active site lie between - terminal lobe carboxy - terminal lobe of kinase domain . ( b ) Close - up view of VPS34 active site with PIK - III bound in ATP - binding pocket . PIK - III forms two hydrogen - bond interactions between compound ' s donor / acceptor moiety backbone amide carbonyl oxygen of residue Ile 685 . A prominent hydrophobic pocket , bounded in part by side chains of Phe 612 , Pro 618 Phe 684 accommodates cyclopropyl functional group . An extended solvent - mediated hydrogen - bonding network bridges interactions between PIK - III aminopyrimidine moiety side chains of Asp 671 Asp 644 . ( c ) binding properties surface of VPS34 active site is superimposed on solvent - accessible surface . Green surfaces are regions with hydrophobic binding properties ; note hydrophobic nature of active site floor deep hydrophobic pocket in which cyclopropyl moiety fits . Blue red surfaces represent hydrogen bond donor acceptor regions , respectively . PIK - III is shown bound in active site . ( d ) Superposition of PIK - III - bound VPS34 pocket surface with a mesh ( ) mapping human PI ( 3 ) Kα active site . displacement of PI ( 3 ) Kα hydrophobic pocket away from hinge ( relative to that of VPS34 ) is readily apparent .

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(a) General domain structure of human VPS34 kinase comprising the helical domain (yellow) and the kinase domain (green). The ATP-binding pocket and active site lie between the amino-terminal lobe and the carboxy-terminal lobe of the kinase domain. (b) Close-up view of the VPS34 active site with PIK-III bound in the ATP-binding pocket. PIK-III forms two hydrogen-bond interactions between the compound's donor/acceptor moiety and the backbone amide and carbonyl oxygen of residue Ile 685. A prominent hydrophobic pocket, bounded in part by the side chains of Phe 612, Pro 618 and Phe 684 accommodates the cyclopropyl functional group. An extended solvent-mediated hydrogen-bonding network bridges interactions between the PIK-III aminopyrimidine moiety and the side chains of Asp 671 and Asp 644. (c) The binding properties surface of the VPS34 active site is superimposed on the solvent-accessible surface. Green surfaces are regions with hydrophobic binding properties; note the hydrophobic nature of the active site floor and the deep hydrophobic pocket in which the cyclopropyl moiety fits. Blue and red surfaces represent hydrogen bond donor and acceptor regions, respectively. PIK-III is shown bound in the active site. (d) Superposition of the PIK-III-bound VPS34 pocket surface with a mesh (white) mapping the human PI(3)Kα active site. The displacement of the PI(3)Kα hydrophobic pocket away from the hinge (relative to that of VPS34) is readily apparent.


( a ) H4 expressing mCherry - GFP - LC3 were treated overnight with indicated compounds , fixed , stained with Hoechst 33342 imaged by automated acquisition . Representative of an overlay of GFP ( green ) , mCherry ( red ) Hoechst ( blue ) are shown with a scale bar equivalent to 10 μm . Bars represent mean GFP - mCherry - positive LC3 per data points represent values from 18 imaged fields in two wells from one experiment .

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(a) H4 cells expressing mCherry-GFP-LC3 were treated overnight with the indicated compounds, fixed, stained with Hoechst 33342 and imaged by automated acquisition. Representative images of an overlay of GFP (green), mCherry (red) and Hoechst (blue) are shown with a scale bar equivalent to 10 μm. Bars represent the mean GFP- and mCherry-positive LC3 puncta per cell and data points represent values from 18 imaged fields in two wells from one experiment.


b ) HeLa expressing GFP - Parkin were treated with PIK - III for 12 h followed by addition of CCCP for 12 h , fixed , stained for endogenous Tom20 imaged . Representative of an overlay of Tom20 ( red ) Hoechst ( blue ) are shown with a scale bar equivalent to 50 μm . Bars represent mean Tom20 per data points represent values from four wells from one experiment .

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b) HeLa cells expressing GFP-Parkin were treated with PIK-III for 12 h followed by the addition of CCCP for 12 h, fixed, stained for endogenous Tom20 and imaged. Representative images of an overlay of Tom20 (red) and Hoechst (blue) are shown with a scale bar equivalent to 50 μm. Bars represent the mean cellular Tom20 staining intensity per cell and data points represent values from four wells from one experiment.


( c ) H4 expressing GFP - p62 were treated overnight with indicated compounds , fixed , stained with Hoechst 33342 imaged . Representative of an overlay of GFP ( green ) Hoechst ( blue ) are shown with a scale bar equivalent to 10 μm . Bars represent mean GFP - positive per data points represent values from 18 imaged fields in 2 wells from one experiment .

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(c) H4 cells expressing GFP-p62 were treated overnight with the indicated compounds, fixed, stained with Hoechst 33342 and imaged. Representative images of an overlay of GFP (green) and Hoechst (blue) are shown with a scale bar equivalent to 10 μm. Bars represent the mean GFP-positive puncta area per cell and data points represent values from 18 imaged fields in 2 wells from one experiment.


( d ) Steady - state of autophagy markers visualized by western blotting in H4 GFP - p62 treated overnight with 0 . 5 μM AZD8055 or 5 μM PIK - III .

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(d) Steady-state levels of autophagy markers visualized by western blotting in H4 GFP-p62 cells treated overnight with 0.5 μM AZD8055 or 5 μM PIK-III.


( e ) Steady - state of p62 , LC3 GAPDH visualized by western blotting in PSN - 1 treated for 48 h with indicated doses of PIK - III . ( f ) Steady - state of p62 , LC3 GAPDH visualized by western blotting in Panc10 . 05 treated for 48 h with indicated doses of PIK - III .

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(e) Steady-state levels of p62, LC3 and GAPDH visualized by western blotting in PSN-1 cells treated for 48 h with the indicated doses of PIK-III. (f) Steady-state levels of p62, LC3 and GAPDH visualized by western blotting in Panc10.05 cells treated for 48 h with the indicated doses of PIK-III.


( g ) RKO expressing inducible HA - ATG4B - C74A were treated with doxycycline ( DOX induction ) for five days then incubated in DOX - free medium for 24 h ( DOX washout ) . Bafilomycin A1 was added with or without PIK - III at beginning of washout period ( lanes 5 6 ) . Steady - state of ATG4B , LC3 GAPDH were visualized by western blotting . Uncropped of blots are shown in Supplementary Fig . 7 .

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(g) RKO cells expressing inducible HA-ATG4B-C74A were treated with doxycycline (DOX induction) for five days and then incubated in DOX-free medium for 24 h (DOX washout). Bafilomycin A1 was added with or without PIK-III at the beginning of the washout period (lanes 5 and 6). Steady-state levels of ATG4B, LC3 and GAPDH were visualized by western blotting. Uncropped images of blots are shown in Supplementary Fig. 7.


. ( b ) represented by unique ubiquitylation sites with > 1 . 5 log2 ( fold change ) in both replicates after diglycyl - peptide enrichment were searched for > 1 . 5 log2 ( fold change ) at proteome level in both replicates . resulting 15 are listed to right in descending order of average fold change in flow - through replicate analyses . Two independent biological replicates are shown in crossplots .

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. (b) Proteins represented by unique ubiquitylation sites with >1.5 log2 (fold change) in both replicates after diglycyl-peptide enrichment were searched for >1.5 log2 (fold change) at the proteome level in both replicates. The resulting 15 proteins are listed to the right in descending order of the average protein fold change in the flow-through replicate analyses. Two independent biological replicates are shown in crossplots.


( c ) Steady - state of NCOA4 in DLD1 treated with bafilomycin A1 or PIK - III for 20 h .

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(c) Steady-state levels of NCOA4 in DLD1 cells treated with bafilomycin A1 or PIK-III for 20 h.


( d ) of NCOA4 p62 in ATG7 wild - type ( + / + ) , heterozygous ( + / − ) or homozygous knockout ( − / − ) DLD1 clones 1 2 were

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(d) The levels of NCOA4 and p62 in ATG7 wild-type (+/+), heterozygous (+/−) or homozygous knockout (−/−) DLD1 clones 1 and 2 were determined


e ) DLD1 cultured in rich medium ( Fed ) were deprived of serum ( Starve ) for indicated times ( hours , h ) of NCOA4 , p62 , LC3 GAPDH were determined by western blotting .

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e) DLD1 cells cultured in rich medium (Fed) were deprived of serum and amino acids (Starve) for the indicated times (hours, h) and levels of NCOA4, p62, LC3 and GAPDH were determined by western blotting.


( f ) Confocal microscopy was used to evaluate distribution of endogenous NCOA4 , LC3 , LAMP2 EEA1 in treated overnight with bafilomycin A1 or PIK - III . Regions outlined with dashed lines are magnified to right of each panel . Scale bars in full panels on left , zoomed panels on right correspond to 10 2 μm respectively . Uncropped of blots are shown in Supplementary Fig . 7 .

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(f) Confocal microscopy was used to evaluate the cellular distribution of endogenous NCOA4, LC3, LAMP2 and EEA1 in cells treated overnight with bafilomycin A1 or PIK-III. Regions outlined with white dashed lines are magnified to the right of each panel. Scale bars in full panels on left, and zoomed panels on right correspond to 10 and 2 μm respectively. Uncropped images of blots are shown in Supplementary Fig. 7.


( a ) Endogenous immunoprecipitation of NCOA4 with ferritin heavy ( FTH1 ) ( FTL ) chains from DLD1 lysates .

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(a) Endogenous immunoprecipitation of NCOA4 with ferritin heavy (FTH1) and light (FTL) chains from DLD1 cell lysates.


b ) Steady - state of FTH1 FTL expressed in DLD1 growing in rich medium or starved for times indicated ( hours , h ) .

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b) Steady-state levels of FTH1 and FTL expressed in DLD1 cells growing in rich medium or starved for the times indicated (hours, h).


( c ) DLD1 were cultured in presence or absence of bafilomycin A1overnight , fixed processed for confocal immunofluorescence microscopy to determine distribution of endogenous FTH1 or FTL with LC3 , LAMP2 or EEA1 . Regions outlined with dashed lines are magnified to right of each panel . Scale bars in full zoomed panels correspond to 10 2 μm respectively .

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(c) DLD1 cells were cultured in the presence or absence of bafilomycin A1overnight, fixed and processed for confocal immunofluorescence microscopy to determine the cellular distribution of endogenous FTH1 or FTL with LC3, LAMP2 or EEA1. Regions outlined with white dashed lines are magnified to the right of each panel. Scale bars in full and zoomed panels correspond to 10 and 2 μm respectively.


( d ) DLD1 parental or NCOA4 − / − ( clones 1 2 ; KO - 1 , KO - 2 ) were starved for 6 h of NCOA4 , FTH1 , FTL GAPDH determined .

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(d) DLD1 parental or NCOA4−/− cells (clones 1 and 2; KO-1, KO-2) were starved for 6 h and the levels of NCOA4, FTH1, FTL and GAPDH determined.


( e ) DLD1 parental or NCOA4 − / − were cultured in presence or absence of bafilomycin A1overnight , fixed processed for confocal immunofluorescence microscopy to determine distribution of endogenous ferritin ( FTH1 - FTL ) LAMP2 . Regions outlined with dashed lines are magnified to right of each panel . Scale bars of full zoomed panels correspond to 10 2 μm respectively . Uncropped of blots are shown in Supplementary Fig . 7 .

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(e) DLD1 parental or NCOA4−/− cells were cultured in the presence or absence of bafilomycin A1overnight, fixed and processed for confocal immunofluorescence microscopy to determine the cellular distribution of endogenous ferritin (FTH1-FTL) and LAMP2. Regions outlined with white dashed lines are magnified to the right of each panel. Scale bars of full and zoomed panels correspond to 10 and 2 μm respectively. Uncropped images of blots are shown in Supplementary Fig. 7.


( a ) extracts from DLD1 treated with DMSO or bafilomycin A1 overnight were resolved using SEC each fraction was analysed for presence of NCOA4 FTH1 by western blot . elute in high - to low - molecular - fractions from 18 to 33 , respectively . Dashed lines are for visual comparison only .

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(a) Cell extracts from DLD1 cells treated with DMSO or bafilomycin A1 overnight were resolved using SEC and each fraction was analysed for the presence of NCOA4 and FTH1 by western blot. Proteins elute in high- to low-molecular-weight fractions from 18 to 33, respectively. Dashed lines are for visual comparison only.


( b ) DLD1 parental or NCOA4 − / − extracts were resolved using SEC each fraction was analysed for presence of NCOA4 , FTH1 FTL by western blot .

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(b) DLD1 parental or NCOA4−/− cell extracts were resolved using SEC and each fraction was analysed for the presence of NCOA4, FTH1 and FTL by western blot.


( c ) NCOA4 − / − were transfected with control or SBP - NCOA4 cDNA for 36 h incubated overnight in presence or absence of indicated inhibitors , lysed occurrence of faster - migrating FTH1 band was determined .

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(c) NCOA4−/− cells were transfected with control or SBP-NCOA4 cDNA for 36 h and incubated overnight in the presence or absence of the indicated inhibitors, lysed and the occurrence of the faster-migrating FTH1 band was determined.


( d ) Endogenous NCOA4 immunoprecipitations from DLD1 parental , FTH1 − / − or FTL − / − ( clones 1 2 for each genotype ) extracts . presence of FTH1 FTL was detected by western blot . Uncropped of blots are shown in Supplementary Fig . 7 .

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(d) Endogenous NCOA4 immunoprecipitations from DLD1 parental, FTH1−/− or FTL−/− (clones 1 and 2 for each genotype) protein extracts. The presence of FTH1 and FTL was detected by western blot. Uncropped images of blots are shown in Supplementary Fig. 7.


( a ) DLD1 were incubated in presence or absence of DFX ( 30 μM ) PIK - III ( 5 μM ) of FTH1 FTL were analysed using western blotting .

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(a) DLD1 cells were incubated in the presence or absence of DFX (30 μM) and PIK-III (5 μM) and the levels of FTH1 and FTL were analysed using western blotting.


. ( b ) DLD parental or NCOA4 − / − were incubated in presence or absence of DFX of FTH FTL were determined .

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. (b) DLD parental or NCOA4−/− cells were incubated in the presence or absence of DFX and the levels of FTH and FTL were determined.


( c ) Ncoa4 − / − mouse embryonic were analysed for Ncoa4 Fth1 by western blotting . Note complete absence of lower Fth1 band consistent with earlier findings .

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(c) Ncoa4−/− mouse embryonic stem cells were analysed for Ncoa4 and Fth1 by western blotting. Note the complete absence of the lower Fth1 band consistent with earlier findings.


( d ) Representative haemotoxylin eosin of spleens from control or Ncoa4 − / − mice is shown brown pigmented regions apparent in Ncoa4 − / − sections are indicated ( arrows ) .

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(d) Representative haemotoxylin and eosin staining of spleens from control or Ncoa4−/− mice is shown and brown pigmented regions apparent in Ncoa4−/− sections are indicated (white arrows).


( e ) presence of iron in spleens from control or Ncoa4 − / − mice was determined using Perls ' Prussian blue . Representative ( left panels ) or magnifications ( right panels ) are shown . Scale bars correspond to 100 μm 20 μm in full magnified panels respectively . ( f ) Quantitative image of data shown in e was performed as described in Methods . Each data point represents relative iron in a single mouse spleen section horizontal lines are means of each group ( n = 6 mice per group ; ∗ P 0 . 0006 for unpaired t - ) . Uncropped of blots are shown in Supplementary Fig . 7 .

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(e) The presence of iron in spleens from control or Ncoa4−/− mice was determined using Perls' Prussian blue staining. Representative micrographs (left panels) or magnifications (right panels) are shown. Scale bars correspond to 100 μm and 20 μm in full and magnified panels respectively. (f) Quantitative image analysis of the data shown in e was performed as described in the Methods. Each data point represents relative iron levels in a single mouse spleen section and horizontal lines are means of each group (n = 6 mice per group; ∗P 0.0006 for unpaired t-test). Uncropped images of blots are shown in Supplementary Fig. 7.


B , C Inhibitory effect of mycolactone on expression of CD38 ( Type II ) CD138 , IL - 6R , CD40 ( Type I ) transmembrane ( B ) . Stimulatory effect of mycolactone on expression of BCMA , a Type III transmembrane ( C ) . Mean Intensities ( MFIs ) were measured in live 48 h after addition of mycolactone . Data are Mean MFIs ± SD from technical duplicates of 2 independent experiments , relative to vehicle - treated controls ( Ctrl ) .

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B, C Inhibitory effect of mycolactone on cell expression of CD38 (Type II) and CD138, IL-6R, and CD40 (Type I) transmembrane proteins (B). Stimulatory effect of mycolactone on cell expression of BCMA, a Type III transmembrane protein (C). Mean Fluorescence Intensities (MFIs) were measured in live cells 48 h after addition of mycolactone. Data are Mean MFIs ± SD from technical duplicates of 2 independent experiments, relative to vehicle-treated controls (Ctrl).


C Synergy between mycolactone BZ in MM . 1S , JIM3 KMS - 11 lines , when treated as in ( B ) . Data are Mean Loewe scores ± SD shown as heatmaps . N = 6 ( cumulative data of 3 independent experiments with technical duplicates ) for MM . 1S KMS - 11 , N = 4 ( cumulative data of 2 independent experiments with technical duplicates ) for JIM3 . Statistical significance was assessed by Student ' s t - : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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C Synergy between mycolactone and BZ in the MM.1S, JIM3 and KMS-11 cell lines, when treated as in (B). Data are Mean Loewe scores ± SD shown as heatmaps. N = 6 (cumulative data of 3 independent experiments with technical duplicates) for MM.1S and KMS-11, and N = 4 (cumulative data of 2 independent experiments with technical duplicates) for JIM3. Statistical significance was assessed by Student's t-test: *p<0.05; **p<0.01; ***p<0.001.


D , E MM . 1S were treated as in ( B ) but using carfilzomib instead of BZ ( D ) . Synergy scores ( E ) were calculated as in ( C ) . Data are Mean Loewe scores of technical duplicates , shown as heatmaps .

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D,E MM.1S cell were treated as in (B) but using carfilzomib instead of BZ (D). Synergy scores (E) were calculated as in (C). Data are Mean Loewe scores of technical duplicates, shown as heatmaps.


In MM . 1S treated with mycolactone / or BZ at indicated concentrations , or vehicle as control for 6 h : ( D ) CHOP GADD34 mRNA were quantified by qPCR Data information : Shown mRNA data are Mean fold changes ( 2 - ∆ ∆ CT ) ± SD , relative to untreated controls ( cumulative data of at least 2 independent experiments with technical duplicates or triplicates ) , pairwise compared by nested one - Way ANOVA with Tukey ' s multiple comparison , exact p - values indicated . Thapsigargin ( Tg , 2 µM , 6 h ) , Tunicamycin ( Tu , 2 µM , 6 h ) were used as positive controls .

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In MM.1S cells treated with mycolactone and/or BZ at the indicated concentrations, or vehicle as control for 6 h: (D) CHOP and GADD34 mRNA levels were quantified by qPCR Data information: Shown mRNA data are Mean fold changes (2-∆∆CT) ± SD, relative to untreated controls (cumulative data of at least 2 independent experiments with technical duplicates or triplicates), pairwise compared by nested one-Way ANOVA with Tukey's multiple comparison test, exact p-values indicated. Thapsigargin (Tg, 2 µM, 6 h), Tunicamycin (Tu, 2 µM, 6 h) were used as positive controls.


In MM . 1S treated with mycolactone / or BZ at indicated concentrations , or vehicle as control for 6 h : ( E ) ATF6 GAPDH in lysates were assessed by Western blot with molecular markers ( kDa ) indicated on right ; Bands corresponding to glycosylated ( G - ATF6 ) , non - glycosylated ( NG - ATF6 ) ATF6 are indicated by arrows ; Data information : DTT ( 4 mM , 2 h ) were used as positive controls . shown data are representative of 2 independent experiments with similar results .

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In MM.1S cells treated with mycolactone and/or BZ at the indicated concentrations, or vehicle as control for 6 h: (E) ATF6 and GAPDH protein levels in cell lysates were assessed by Western blot with molecular weight markers (kDa) indicated on the right; Bands corresponding to glycosylated (G-ATF6), non-glycosylated (NG-ATF6) and cleaved ATF6 are indicated by arrows; Data information: DTT (4 mM, 2 h) were used as positive controls. shown data are representative of 2 independent experiments with similar results.


In MM . 1S treated with mycolactone / or BZ at indicated concentrations , or vehicle as control for 6 h : ( F ) Total was isolated used as a template for RT - PCR of XBP - 1 ( upper panel ) which was then digested with Pst1 separated on a 2 % agarose gel ( lower panel ) with molecular markers ( bp ) indicated on left ; spliced XBP1 ( sXBP1 ) mRNA were also quantified by qPCR normalized to total ( spliced + unspliced ) XBP1 mRNA , ( G ) BiP mRNA level were quantified by qPCR . Data information : Shown mRNA data are Mean fold changes ( 2 - ∆ ∆ CT ) ± SD , relative to untreated controls ( cumulative data of at least 2 independent experiments with technical duplicates or triplicates ) , pairwise compared by nested one - Way ANOVA with Tukey ' s multiple comparison , exact p - values indicated . Thapsigargin ( Tg , 2 µM , 6 h ) , Tunicamycin ( Tu , 2 µM , 6 h ) were used as positive controls . shown data are representative of 2 independent experiments with similar results .

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In MM.1S cells treated with mycolactone and/or BZ at the indicated concentrations, or vehicle as control for 6 h: (F) Total RNA was isolated and used as a template for RT-PCR of XBP-1 (upper panel) which was then digested with Pst1 and separated on a 2% agarose gel (lower panel) with molecular weight markers (bp) indicated on the left; spliced XBP1 (sXBP1) mRNA levels were also quantified by qPCR and normalized to total (spliced + unspliced) XBP1 mRNA levels, (G) BiP mRNA level were quantified by qPCR. Data information: Shown mRNA data are Mean fold changes (2-∆∆CT) ± SD, relative to untreated controls (cumulative data of at least 2 independent experiments with technical duplicates or triplicates), pairwise compared by nested one-Way ANOVA with Tukey's multiple comparison test, exact p-values indicated. Thapsigargin (Tg, 2 µM, 6 h), Tunicamycin (Tu, 2 µM, 6 h) were used as positive controls. shown data are representative of 2 independent experiments with similar results.


C MM . 1S BzR were treated for 24 h with mycolactone / or BZ at indicated concentrations , or vehicle as control . Data are Mean % ± SD of live , apoptotic dead relative to total . N = 6 ( cumulative data of 3 independent experiments with technical duplicates ) .

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C MM.1S BzR cells were treated for 24 h with mycolactone and/or BZ at the indicated concentrations, or vehicle as control. Data are Mean % ± SD of live, apoptotic and dead cells relative to total cells. N = 6 (cumulative data of 3 independent experiments with technical duplicates).


E ATF4 , CHOP , sXBP1 BiP mRNA were quantified by qPCR in MM . 1s BzR treated as in ( C ) . sXBP1 mRNA were normalized to total ( spliced + unspliced ) XBP1 mRNA level . Data are Mean fold changes ( 2 - ∆ ∆ CT ) ± SD , relative to untreated controls . N = 5 ( cumulative data of 2 independent experiments with technical duplicates triplicates ) , pairwise compared by nested one - Way ANOVA with Tukey ' s multiple comparison , exact p - values indicated . Thapsigargin ( Tg , 2 µM , 6h ) was used as a positive control .

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

E ATF4, CHOP, sXBP1 and BiP mRNA levels were quantified by qPCR in MM.1s BzR cells treated as in (C). sXBP1 mRNA levels were normalized to total (spliced + unspliced) XBP1 mRNA level. Data are Mean RNA fold changes (2-∆∆CT) ± SD, relative to untreated controls. N = 5 (cumulative data of 2 independent experiments with technical duplicates and triplicates), pairwise compared by nested one-Way ANOVA with Tukey's multiple comparison test, exact p-values indicated. Thapsigargin (Tg, 2 µM, 6h) was used as a positive control.


C v - abl pro - B were treated with mycolactone / or BZ at indicated concentrations for 6 h . CHOP mRNA were quantified by qPCR . Data are Mean fold changes ( 2 - ∆ ∆ CT ) ± SD relative to untreated controls . N = 6 ( cumulative data of 3 independent experiments with technical duplicates ) , pairwise compared by nested one - Way ANOVA with Tukey ' s multiple comparison , exact p - values indicated . Data information : Thapsigargin ( Tg , 2 µM , 6h ) was used as a positive control .

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C v-abl pro-B cells were treated with mycolactone and/or BZ at the indicated concentrations for 6 h. CHOP mRNA levels were quantified by qPCR. Data are Mean RNA fold changes (2-∆∆CT) ± SD relative to untreated controls. N = 6 (cumulative data of 3 independent experiments with technical duplicates), pairwise compared by nested one-Way ANOVA with Tukey's multiple comparison test, exact p-values indicated. Data information: Thapsigargin (Tg, 2 µM, 6h) was used as a positive control.


D v - abl pro - B were treated for 6 h with mycolactone / or BZ at indicated concentrations . ATF4 were assessed in lysates by Western blot ( top panel ) quantified relative to GADPH ( lower panel ) . Data information : Data shown are representative of experiments performed with two independent v - abl clones , with similar results . Thapsigargin ( Tg , 2 µM , 6h ) was used as a positive control .

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

D v-abl pro-B cells were treated for 6 h with mycolactone and/or BZ at the indicated concentrations. ATF4 protein levels were assessed in cell lysates by Western blot (top panel) and quantified relative to GADPH levels (lower panel). Data information: Data shown are representative of experiments performed with two independent v-abl cell clones, with similar results. Thapsigargin (Tg, 2 µM, 6h) was used as a positive control.


Mononuclear from bone marrow aspirates of newly diagnosed ( # 2 , # 5 , # 9 # 13 ) or relapsed ( # 7 # 12 ) MM were treated with mycolactone / or BZ at indicated concentrations , or vehicle as control , for 18 h . Then , MM were identified by with anti - CD38 anti - CD138 antibodies A Following treatment , induction of apoptosis / necrosis was measured by exposure of annexin V PI incorporation . Data are Mean % of live from technical duplicates , relative to controls .

B-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-DISEASE O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-DISEASE O O O O O B-GENEPROD I-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

Mononuclear cells from bone marrow aspirates of newly diagnosed (#2, #5, #9 and #13) or relapsed (#7 and #12) MM patients were treated with mycolactone and/or BZ at the indicated concentrations, or vehicle as control, for 18 h. Then, MM cells were identified by staining with anti-CD38 and anti-CD138 antibodies A Following treatment, induction of apoptosis/necrosis was measured by exposure of annexin V and PI incorporation. Data are Mean % of live cells from technical duplicates, relative to controls.


Mononuclear from bone marrow aspirates of newly diagnosed ( # 2 , # 5 , # 9 # 13 ) or relapsed ( # 7 # 12 ) MM were treated with mycolactone / or BZ at indicated concentrations , or vehicle as control , for 18 h . B Synergy between mycolactone BZ in treated tumors . Data are Mean Loewe scores from technical duplicates , shown as heatmaps .

B-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

Mononuclear cells from bone marrow aspirates of newly diagnosed (#2, #5, #9 and #13) or relapsed (#7 and #12) MM patients were treated with mycolactone and/or BZ at the indicated concentrations, or vehicle as control, for 18 h. B Synergy between mycolactone and BZ in treated tumors. Data are Mean Loewe scores from technical duplicates, shown as heatmaps.


A C57BL / 6 mice ( N = 2 / group ) were injected intraperitoneally with BZ ( 0 . 5 mg . Kg - 1 ) , BZ ( 0 . 5 mg . Kg - 1 ) + mycolactone ( 0 . 3 mg . Kg - 1 ) , DMSO as vehicle controls every 3 . 5 days , or left untreated . Blood samples were analyzed 5 , 15 , 20 25 days after first injection percentages of CD3 + CD19 + determined by flow cytometry . Results are expressed as % of positive among total mononuclear , each dot representing Mean % from 2 mice .

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

A C57BL/6 mice (N=2/group) were injected intraperitoneally with BZ (0.5 mg.Kg-1), BZ (0.5 mg.Kg-1) + mycolactone (0.3 mg.Kg-1), DMSO as vehicle controls every 3.5 days, or left untreated. Blood samples were analyzed 5, 15, 20 and 25 days after the first injection and percentages of CD3+ and CD19+ cells determined by flow cytometry. Results are expressed as % of positive cells among total mononuclear cells, each dot representing the Mean % from 2 mice.


B NSG mice ( N = 9 / group ) were injected subcutaneously with 3 . 106 MM . 1S at day 0 . Seven days later , mice were injected with DMSO , BZ ( 0 . 5 mg . Kg - 1 ) / or mycolactone ( 0 . 3 mg . Kg - 1 ) every 3 . 5 days intraperitoneally growth was followed by daily measurement of diameter . Data are Mean diameters ± SD represent cumulative data from 3 independent experiments with 3 mice / group . Difference between groups were analyzed with Tukey ' s multiple comparison using Two - way Anova , with mixed effect model for each time points : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ( exact p - values indicated ) . Only significant differences are shown .

O O B-ORGANISM O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B NSG mice (N=9/group) were injected subcutaneously with 3.106 MM.1S cells at day 0. Seven days later, mice were injected with DMSO, BZ (0.5 mg.Kg-1) and/or mycolactone (0.3 mg.Kg-1) every 3.5 days intraperitoneally and tumor growth was followed by daily measurement of the tumor diameter. Data are Mean tumor diameters ± SD and represent cumulative data from 3 independent experiments with 3 mice/group. Difference between groups were analyzed with Tukey's multiple comparison test using Two-way Anova, with mixed effect model for each time points: *p<0.05; **p<0.01; ***p<0.001 (exact p-values indicated). Only significant differences are shown.


a , Seckel contain normal centrosome numbers mitotic spindle poles . Interphase centrosomes of Seckel have no CPAP immunoreactivity but recruit similar of centrosomal ( ii iii ) .

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a, Seckel cells contain normal centrosome numbers and mitotic spindle poles. Interphase centrosomes of Seckel cells have no CPAP immunoreactivity but recruit similar levels of centrosomal proteins (ii and iii).


b , Seckel display overly long cilia . Arl13b labels cilium . Scale bar 1 μm . of cilia , * * * p < 0 . 001 ; t - ; Scatter plot showing mean with ± SD ( iii ) . displaying normal ( 0 - 5 μm ) , long ( 5 - 10 μm ) extra long ( > 10 μm ) cilia . * * * p < 0 . 001 ; Ordinary Two - way ANOVA ( iv ) . Average cilia of WT Seckel ( v ) . n = 3 independent experiments , 228 for WT 297 for Seckel . Error bars are s . e . m . * * * p < 0 . 001 , t - .

O O O O O O B-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O 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, Seckel cells display overly long cilia. Arl13b labels cilium. Scale bar 1 μm. Quantification of cilia length, *** p<0.001; t-test; Scatter plot showing mean with ±SD (iii). Cells displaying normal (0-5 μm), long (5-10 μm) and extra long (>10 μm) cilia. *** p<0.001; Ordinary Two-way ANOVA (iv). Average cilia length of WT and Seckel cells (v). n = 3 independent experiments, 228 cells for WT and 297 cells for Seckel. Error bars are s. e. m. *** p<0.001, t-test.


c , Electron microscopy ( EM ) of Seckel WT fibroblasts . In contrast to WT , Seckel basal has disorganized microtubule of doublet triplets ( red arrows red circles in schematics ) . d , Longitudinal serial - sectioning EM of WT ( top ) Seckel fibroblasts ( bottom ) . In contrast to WT basal bodies that have structurally normal distal sub - distal appendages ( yellow arrows red triangles of DA SDA in schematics ) , Seckel basal displays either lack or misplaced appendages . n > 15 basal bodies . e , Seckel show reduced EdU incorporation after 72 hrs . Error bars are s . e . m . * * p < 0 . 01 , ttest ; n = 4 independent experiments , 727 for WT 844 for Seckel .

O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

c, Electron microscopy (EM) of Seckel and WT fibroblasts. In contrast to WT, Seckel basal body has disorganized microtubule of doublet and triplets (red arrows and red circles in schematics).d, Longitudinal serial-sectioning EM of WT (top) Seckel fibroblasts (bottom). In contrast to WT basal bodies that have structurally normal distal and sub-distal appendages (yellow arrows and red triangles of DA and SDA in schematics), Seckel basal body displays either lack or misplaced appendages. n> 15 basal bodies.e, Seckel cells show reduced EdU incorporation after 72 hrs. Error bars are s. e. m. ** p<0.01, ttest; n = 4 independent experiments, 727 cells for WT and 844 cells for Seckel.


a . Experimental plan . Both Seckel WT were synchronized at G0 by serum starvation for 96 hrs . This treatment causes Seckel WT to have equivalent cilia allowing us to estimate rate of cilia disassembly ( see below Appendix Fig . S2a ) . b , Dynamic CPAP localization during cilium disassembly cycle re - entry . Cep152 remain unchanged . Arl13b ( green ) or acetylated α - tubulin ( magenta ) marks cilium . n > 100 . Bar diagrams at right shows semi quantitative measurements . Error bars are ± SD .

O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O

a. Experimental plan. Both Seckel and WT cells were synchronized at G0 by serum starvation for 96 hrs. This treatment causes Seckel and WT cells to have equivalent cilia length allowing us to estimate the rate of cilia disassembly (see below and Appendix Fig. S2a).b, Dynamic CPAP localization during cilium disassembly and cell cycle re-entry. Cep152 levels remain unchanged. Arl13b (green) or acetylated α-tubulin (magenta) marks the cilium. n> 100 cells. Bar diagrams at right shows semi quantitative intensity measurements. Error bars are ± SD.


c , Seckel show neither CPAP dynamics nor cilium disassembly upon serum stimulation . Cep152 remain constant . n > 100 . Bar diagrams at right shows semi quantitative measurements . Error bars are ± SD .

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c, Seckel cells show neither CPAP dynamics nor cilium disassembly upon serum stimulation. Cep152 levels remain constant. n> 100 cells. Bar diagrams at right shows semi quantitative intensity measurements. Error bars are ± SD.


d , Cilium disassembly dynamics in WT Seckel . Relative cilium disassembly rate shows retarded cilium disassembly in Seckel ( Error bars are s . e . m . ) ( ii ) . distribution of cilia in WT Seckel fibroblasts . Scatter plot shows mean with ± SD ( i ) .

O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O O O O O O O O O O O

d, Cilium disassembly dynamics in WT and Seckel cells. Relative cilium disassembly rate shows retarded cilium disassembly in Seckel cells (Error bars are s. e. m.) (ii). Quantification and distribution of cilia length in WT and Seckel fibroblasts. Scatter plot shows mean with ± SD (i).


e , Seckel with retarded cilium disassembly show delayed G1 - S transition assessed by nuclearCyclin A immunoreactivity . n = 3 independent experiments , n > 200 for WT Seckel . Error bars are s . e . m .

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e, Seckel cells with retarded cilium disassembly show delayed G1-S transition assessed by nuclearCyclin A immunoreactivity. n = 3 independent experiments, n> 200 cells for WT and Seckel. Error bars are s.e.m.


f , Seckel show delayed mitotic entry . Note that an unresorbed Arl13b - positive ciliary remnant ( ciliary , green ) is present with one of centrosomes ( Paridaen et al , 2013 ) . Bar diagram shows mitotic index of WT Seckel 24 hrs after serum stimulation . n = 3 independent experiments , n > 300 for WT Seckel . Error bars are s . e . m . Scale bar 1 μm .

O O O O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f, Seckel cells show delayed mitotic entry. Note that an unresorbed Arl13b-positive ciliary remnant (ciliary membrane, green) is present with one of the centrosomes(Paridaen et al, 2013). Bar diagram shows mitotic index of WT and Seckel cells 24 hrs after serum stimulation. n = 3 independent experiments, n> 300 cells for WT and Seckel. Error bars are s.e.m. Scale bar 1 μm.


a , CPAP co - immunopurifies with CDC components from HeLa extracts . Centrosomal Cap350 ODF2 do not co - purify with CPAP .

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

a, CPAP co-immunopurifies with CDC components from HeLa extracts. Centrosomal proteins Cap350 and ODF2 do not co-purify with CPAP.


b , of CDC components ( CPAP , Aurora A , OFD1 , Nde - 1 ) are differentially regulated in response to serum starvation stimulation in WT Seckel fibroblasts . Specifically , they are elevated 1h after serum stimulation , thus enabling cilium disassembly . Elevated CDC in WT correlate with an elevated level of CPAP as shown in Fig . 2 as well as with expression of cyclin A , a G1 - S marker . GAPDH was used as a loading control indicating an equivalent amount of WT Seckel fibroblast total were loaded .

O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-TISSUE O O O O O B-CELL_TYPE O O O O O O O O B-TISSUE O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE O O O O

b, Levels of CDC components (CPAP, Aurora A, OFD1, and Nde-1) are differentially regulated in response to serum starvation and stimulation in WT and Seckel fibroblasts. Specifically, they are elevated 1h after serum stimulation, thus enabling cilium disassembly. Elevated CDC levels in WT cells correlate with an elevated level of CPAP as shown in Fig. 2 as well as with the expression of cyclin A, a G1-S marker. GAPDH was used as a loading control indicating an equivalent amount of WT and Seckel fibroblast total proteins were loaded.


c . WT CPAP ( WT - IP ) but not Seckel variant CPAP ( Seckel IP ) co - immunopurifies with CDC components from asynchronously grown WT Seckelfibroblastscell extracts . IP samples were adjusted so that equivalent amounts of immunoprecipitatedCPAP were used in samples . Seckel ( 5x ) WT ( 1x ) fibroblast extracts were used as input controls to show efficacy of various antibodies . Note that CPAP abundance in Seckel is ~ 5 times lower than that of WT . CentrosomalproteinsCap350 ODF2 do not co - purify with CPAP indicating specificity of CPAP interaction with CDC components .

O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O B-GENEPROD O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O B-SUBCELLULAR O O

c. WT CPAP (WT-cells IP) but not Seckel variant CPAP (Seckel cells IP) co-immunopurifies with CDC components from asynchronously grown WT and Seckelfibroblastscell extracts. The IP samples were adjusted so that equivalent amounts of immunoprecipitatedCPAP were used in samples. Seckel (5x) and WT (1x) fibroblast cell extracts were used as input controls to show the efficacy of the various antibodies. Note that CPAP abundance in Seckel cells is ~5 times lower than that of WT cells. CentrosomalproteinsCap350 and ODF2 do not co-purify with CPAP indicating the specificity of CPAP interaction with CDC components.


d , GST pull down ( i ) immunopurification assays ( ii ) of CPAP ' s CC5 domain demonstrate its sufficiency in mediating CPAP - CDC interactions . CC5 fragment is expressed as a GFP fusion in HeLa immunopurified using anti - GFP antibodies . HeLa extracts were used as input controls . CAP350 does not co - purify with CPAP . Both Cap350 ODF2 are not pulled down by CPAP ' s CC5 domain .

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

d, GST pull down (i) and immunopurification assays (ii) of CPAP's CC5 domain demonstrate its sufficiency in mediating CPAP-CDC protein interactions. The CC5 fragment is expressed as a GFP fusion protein in HeLa cells and immunopurified using anti-GFP antibodies. HeLa extracts were used as input controls. CAP350 does not co-purify with CPAP. Both Cap350 and ODF2 are not pulled down by CPAP's CC5 domain.


a , Seckel NPCs fail to efficiently recruit CDC components . Fraction of non - ciliated ciliated WT Seckel NPCs . Ciliated are further quantified grouped on whether they are positive or negative for Aurora A CPAP at their basal bodies . Scale bar 0 . 5 μm ( i - ii ) . Proportion of ciliated is higher in Seckel NPCs ( * * p < 0 . 01 ) . Compared to WT , a higher proportion of Seckel NPCs lack Aurora A CPAP in their basal bodies ( * p < 0 . 05 for Auroa A * * * p < 0 . 001 for CPAP ) . Ordinary Two - way ANOVA followed by Sidak ' s multiple comparisons . n = 3 independent experiments . 127 for WT 97 for Seckel . Error bars are s . e . m . ( iii ) Seckel consistently contain Nde - 1 Ofd1 with a significantly lesser . t ; error bars are s . e . m . ; n = 3 independent experiments . 110 for WT 100 for Seckel .

O O O B-CELL_TYPE O O O O B-SUBCELLULAR O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

a, Seckel NPCs fail to efficiently recruit CDC components. Fraction of non-ciliated and ciliated WT and Seckel NPCs. Ciliated cells are further quantified and grouped based on whether they are positive or negative for Aurora A and CPAP at their basal bodies. Scale bar 0.5 μm (i-ii). Proportion of ciliated cells is higher in Seckel NPCs (**p<0.01). Compared to WT, a higher proportion of Seckel NPCs lack Aurora A and CPAP in their basal bodies (*p<0.05 for Auroa A and ***p<0.001 for CPAP). Ordinary Two-way ANOVA followed by Sidak's multiple comparisons test. n = 3 independent experiments. 127 cells for WT and 97 cells for Seckel. Error bars are s. e. m. (iii) Seckel cells consistently contain Nde-1 and Ofd1 with a significantly lesser intensity. t test; error bars are s.e.m.; n = 3 independent experiments. 110 cells for WT and 100 cells for Seckel.


b , Proportion of ciliated is higher in Seckel NPCs . Error bars are s . e . m . * * p < 0 . 01 , t - . n = 3 independent experiments . 127 for WT 97 for Seckel .

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b, Proportion of ciliated cells is higher in Seckel NPCs. Error bars are s. e. m. ** p<0.01, t-test. n = 3 independent experiments. 127 cells for WT and 97 cells for Seckel.


c , Seckel NPCs exhibit longer cilia . Error bars are ± s . e . m . * * * p < 0 . 001 , t - . NPCs were derived from two independent iPS clones . n = 3 independent experiments . 146 for WT , 135 for Seckel ( clone 1 ) 202 ( clone 2 ) . d , SeckelNPCs show reduced proliferation delayed G1 - S transition . Error bars are ± s . e . m . * * * p < 0 . 001 ( EdU ) , * p < 0 . 05 ( Cyclin - A ) , t - . For cyclin A , n = 3 independent experiments . 270 for WT 232 for Seckel . For EdU , 700 for WT 1630 for Seckel .

O O O B-CELL_TYPE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O

c, Seckel NPCs exhibit longer cilia. Error bars are ± s. e. m. *** p<0.001, t-test. NPCs were derived from two independent iPS clones. n = 3 independent experiments. 146 cells for WT, 135 cells for Seckel (clone 1) and 202 (clone 2).d, SeckelNPCs show reduced proliferation and delayed G1-S transition. Error bars are ± s. e. m. *** p<0.001 (EdU), * p<0.05 (Cyclin-A), t-test. For cyclin A, n = 3 independent experiments. 270 cells for WT and 232 cells for Seckel cells. For EdU, 700 cells for WT and 1630 cells for Seckel.


e , Olomoucine triggers differentiation of WT NPCs into TUJ1 - positive neurons under non - differentiating conditions . Error bars are s . e . m . * * * p < 0 . 001 , Ordinary Two - way ANOVA followed by Tukey ' s multiple comparisons . n = 3 independent experiments . 298 for untreated , 151 for DMSO 146 for olomoucine , 25 μM .

O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O

e, Olomoucine triggers differentiation of WT NPCs into TUJ1-positive neurons under non-differentiating conditions. Error bars are s. e. m. *** p<0.001, Ordinary Two-way ANOVA followed by Tukey's multiple comparisons test. n = 3 independent experiments. 298 cells for untreated, 151 cells for DMSO and 146 cells for olomoucine, 25 μM.


f , Seckel NPCs differentiated into TUJ1 - positive neurons under non - differentiating conditions . Error bars show ± s . e . m . * * * p < 0 . 001 , t - . n = 3 independent experiments . 633 for WT 275 for Seckel . Scale bar 5 μm .

O O O B-CELL_TYPE O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O 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, Seckel NPCs differentiated into TUJ1-positive neurons under non-differentiating conditions. Error bars show ±s. e. m. *** p<0.001, t-test. n = 3 independent experiments. 633 cells for WT and 275 cells for Seckel. Scale bar 5 μm.


g , Expression of CPAP - GFP or CPAP - CC5 - GFP in Seckel NPCs slightly increases nestin - positive ( i ) , decreases neuronal differentiation ( for CC5 * p < 0 . 05 ) ( ii ) , decreases ciliary ( for CPAP * * * p < 0 . 001 for CC5 * * p < 0 . 01 ) ( iii ) , elevates mitotic index ( for CC5 * p < 0 . 05 ) ( iv ) decreases number of ciliated ( for CPAP * p < 0 . 05 for CC5 * * p < 0 . 01 ) ( v ) compared to control ( mock electroporation ) . Error bars show s . e . m . One - way ANOVA followed by Tukey ' s multiple comparisons . n = 3 independent experiments with > 350 ( nestinstaining , differentiation mitotic index ) , > 70 ( cilium ) .

O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O

g, Expression of CPAP-GFP or CPAP-CC5-GFP in Seckel NPCs slightly increases nestin-positive cells (i), decreases neuronal differentiation (for CC5 *p<0.05) (ii), decreases ciliary length (for CPAP ***p<0.001 and for CC5 **p<0.01) (iii), elevates mitotic index (for CC5 *p<0.05) (iv) and decreases the number of ciliated cells (for CPAP *p<0.05 and for CC5 **p<0.01) (v) compared to control (mock electroporation). Error bars show s. e. m. One-way ANOVA followed by Tukey's multiple comparisons test. n = 3 independent experiments with >350 (nestinstaining, differentiation and mitotic index), and >70 cells (cilium length) cells.


a - b , siRNA - mediated down regulation of CDC components in WT NPCs causes longer cilia ( Ofd1 * * * p < 0 . 001 , Nde1 * * p < 0 . 01 ) , retarded cilia disassembly as shown by increased numbers of ciliated ( Ofd1 * * p < 0 . 01 , Nde1 * p < 0 . 05 ) differentiation of NPCs into TUJ1 positive ( Ofd1 * * p < 0 . 001 , Nde1 * p < 0 . 01 ) . In controls , a non - targeting siRNA ( scrambled ) is used . Western blots at right validate siRNAs - mediated Nde1 OFD1 depletion . Error bars are s . e . m . Ordinary Two - way ANOVA followed by Sidak ' s multiple comparisons . n = 3 independent experiments . 275 for scrambled , 423 for Ofd1 siRNA 255 for Nde1 siRNA .

O O O O O O O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O

a-b, siRNA-mediated down regulation of CDC components in WT NPCs causes longer cilia (Ofd1 ***p<0.001, Nde1 **p<0.01), retarded cilia disassembly as shown by increased numbers of ciliated cells (Ofd1 **p<0.01, Nde1 *p<0.05) and differentiation of NPCs into TUJ1 positive cells (Ofd1 **p<0.001, Nde1 *p<0.01). In controls, a non-targeting siRNA (scrambled) is used. Western blots at right validate siRNAs-mediated Nde1 and OFD1 depletion. Error bars are s. e. m. Ordinary Two-way ANOVA followed by Sidak's multiple comparisons test. n = 3 independent experiments. 275 cells for scrambled, 423 cells for Ofd1 siRNA and 255 cells for Nde1 siRNA.


O O O O O O O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O


c , VX680 ( 0 . 5 μM , a that will specifically allow neuronal differentiation ) was used to inhibit Aurora A activity . Bar diagrams below show quantifications . Compared to DMSO - treated control NPCs , a higher proportion of VX680 treated NPCs are ciliated ( * * * p < 0 . 001 ) , exhibiting longer cilia ( * * * p < 0 . 001 ) show Tuj1 positive neurons ( * * * p < 0 . 001 ) . Error bars are s . e . m . For ciliated cilium : n = 3 independent experiments . 164 for DMSO - treated 77 for VX680 - treated . For NPC differentiation : n = 4 independent experiments . 286 for DMSO - treated 118 for VX680 - treated . Scale bar 1 μm .

O O B-SMALL_MOLECULE O O O O O O O O O O O B-CELL_TYPE O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O

c, VX680 (0.5 μM, a concentration that will specifically allow neuronal differentiation) was used to inhibit Aurora A activity. Bar diagrams below show quantifications. Compared to DMSO-treated control NPCs, a higher proportion of VX680 treated NPCs are ciliated (***p<0.001), exhibiting longer cilia (***p<0.001) and show Tuj1 positive neurons (***p<0.001). Error bars are s. e. m. For ciliated cells and cilium length: n = 3 independent experiments. 164 cells for DMSO-treated and 77 cells for VX680-treated. For NPC differentiation: n = 4 independent experiments. 286 cells for DMSO-treated and 118 cells for VX680-treated. Scale bar 1 μm.


d , cycle progression of siRNA or VX680 treated WT NPCs compared to control assessed by EdU incorporation for 24 hours . Inhibition of Ofd1 ( * p < 0 . 05 ) , Nde1 ( * * p < 0 . 01 ) Aurora A ( * * * p < 0 . 001 ) caused significantly reduced incorporation of EdU .

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

d, Cell cycle progression of siRNA or VX680 treated WT NPCs compared to control assessed by EdU incorporation for 24 hours. Inhibition of Ofd1 (*p<0.05), Nde1 (**p<0.01) and Aurora A (***p<0.001) caused significantly reduced incorporation of EdU.


e - g , Western blots show that CPAP targeting siRNA treatment depletes endogenous CPAP . Both low - high - exposures are given ( e ) . Increased cilia NPC differentiation upon expression of RNAi - resistant CPAP E1235V compared to WT CPAP ( f - g ) . Error bars are s . e . m . * * * p < 0 . 001 ( cilia ) , * p < 0 . 05 ( differentiation ) , t - . For cilia : n = 4 independent experiments . 25 for CPAP , 47 for CPAP E1235V . For differentiation : n = 4 independent experiments . 145 for CPAP 81 for CPAP E1235V . Scale bar 1 μm .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR B-CELL_TYPE O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O

e-g, Western blots show that CPAP targeting siRNA treatment depletes endogenous CPAP. Both low -and high -exposures are given (e). Increased cilia length and NPC differentiation upon expression of RNAi-resistant CPAP E1235V compared to WT CPAP (f-g). Error bars are s. e. m. *** p<0.001 (cilia length), * p<0.05 (differentiation), t-test. For cilia length: n = 4 independent experiments. 25 cells for CPAP, 47 cells for CPAP E1235V. For differentiation: n = 4 independent experiments. 145 for CPAP and 81 for CPAP E1235V. Scale bar 1 μm.


a , iPS - derived 14 - day old organoid displaying morphological structures of brain - like tissues , parenthesis . Scale bar 500 μm . ventricular zone ( VZ ) with radial glial stained with nestin ( magenta ) neurons stained with TUJ1 is shown at higher magnification . Scale bar 20 μm .

O O B-CELL_TYPE O O O O O O O O O O O B-TISSUE O O O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O

a, iPS-derived 14-day old organoid displaying complex morphological structures of brain-like tissues, parenthesis. Scale bar 500 μm. The ventricular zone (VZ) with radial glial cells stained with nestin (magenta) and neurons stained with TUJ1 is shown at higher magnification. Scale bar 20 μm.


b , Reduced Nestin / Pax - 6 - positive progenitors increased TUJ1 - positive neurons in Seckel organoids . In ventricular zones of WT organoids , there are significantly more Pax6 positive than TUJ1 positive , which is not case in Seckel organoids ( * p < 0 . 05 ) . TUJ1 / Pax6 in graph on right shows significantly more TUJ1 positive vs . Pax6 in Seckel organoid ventricular zones ( * p < 0 . 05 ) . Error bars are s . e . m . Ordinary Two - way ANOVA followed by Sidak ' s multiple comparisons . n = 10 WT organoids ( 2520 ) . Seckeln = 10 organoids ( 2024 ) . Scale bar 20 μm .

O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-CELL_TYPE O O O O O B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

b, Reduced Nestin/Pax-6-positive progenitors and increased TUJ1-positive neurons in Seckel organoids. In ventricular zones of WT organoids, there are significantly more Pax6 positive cells than TUJ1 positive cells, which is not the case in Seckel organoids (*p<0.05). TUJ1/Pax6 ratio in graph on the right shows significantly more TUJ1 positive cells vs. Pax6 cells in Seckel organoid ventricular zones (* p<0.05). Error bars are s. e. m. Ordinary Two-way ANOVA followed by Sidak's multiple comparisons test. n = 10 WT organoids (2520 cells). Seckeln = 10 organoids (2024 cells). Scale bar 20 μm.


c , ( i ) Seckel organoid tissues contain increased numbers of cilia with an obvious observation of enlarged lateral ventricular lumen ( * p < 0 . 05 ) . Graph at far right shows an increased lumen as well as thinner ambient VZ in Seckel organoids . ( * p < 0 . 05 ) . Error bars are s . e . m . t - . n = 6 for WT organoid 6 for Seckel organoid . Scale bar 20 μm . ( ii ) Seckel organoid display longer cilia . Error bars are s . e . m . * * * p < 0 . 001 , t - . n = 3 for WT organoids 4 for Seckel organoids . Scale bar 1 μm .

O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O B-TISSUE O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O 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, (i) Seckel organoid tissues contain increased numbers of cilia with an obvious observation of enlarged lateral ventricular lumen (* p<0.05). Graph at far right shows an increased lumen area as well as thinner ambient VZ in Seckel organoids. (*p<0.05). Error bars are s. e. m. t-test. n=6 for WT organoid and 6 for Seckel organoid. Scale bar 20 μm. (ii) Seckel organoid cells display longer cilia. Error bars are s. e. m. *** p<0.001, t-test. n=3 for WT organoids and 4 for Seckel organoids. Scale bar 1 μm.


d , Seckel organoids electroporated with constructs expressing GFP alone ( i ) , GFP with CC5 - GFP ( ii ) GFP with CPAP - GFP ( iii ) . Middle panel shows with anti - GFP antibodies ( red ) to show transfected . Note presence of increased Pax6 - positive ( magenta ) NPCs in CC5 CPAP electroporated tissues compared to GFP control . Likewise , there is a larger neural epithelium with multiple RGs ( arrowheads ) . Bar diagrams at right shows rescue effects of CPAP CC5 electroporation compared with GFP alone ( iv ) lower TUJ1 / Pax6 ( * * p < 0 . 01 * * * p < 0 . 001 respectively ) ( v ) smaller lumen diameter ( * * p < 0 . 01 ) ( vi ) shorter cilia ( * * p < 0 . 01 * * * p < 0 . 001 respectively ) . Error bars are s . e . m . One - way ANOVA followed by Dunnett ' s multiple comparisons . n = 3 organoids with 225 for GFP , 537 for CPAP 569 for CC5 . At least 10 ventricles from 3 independent organoids were used to measure CPAP diameter . t . Error bars are s . e . m . Scale bar 20 μm . e , Rescue effect of CC5 CPAPelectroporation on cleavage planes of phospho - vimentin - positive RGs in Seckel organoids . Compared to GFP control , electroporation of CC5 or GFP increases proportion of horizontally orientated SeckelRGs ( for CC5 * * * p < 0 . 001 ) decreases portion of obliquely ( for CC5 * p < 0 . 05 ) or vertically oriented SeckelGFP ( for CPAP CC5 * * p < 0 . 01 ) . Error bars are s . e . m . Two - way ANOVA followed by Tukey ' s multiple comparisons . n = 4 organoids . 100 for GFP control , 112 for CPAP 99 for CC5 .

O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-CELL_TYPE O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-TISSUE I-TISSUE O O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O

d, Seckel organoids electroporated with constructs expressing GFP alone (i), GFP with CC5-GFP and (ii) GFP with CPAP-GFP (iii). Middle panel shows staining with anti-GFP antibodies (red) to show the transfected area. Note the presence of increased Pax6-positive (magenta) NPCs in CC5 and CPAP electroporated tissues compared to GFP control. Likewise, there is a larger neural epithelium with multiple RGs (arrowheads). Bar diagrams at right shows rescue effects of CPAP and CC5 electroporation compared with GFP alone (iv) lower TUJ1/Pax6 ratio (**p<0.01 and ***p<0.001 respectively) (v) smaller lumen diameter (**p<0.01) and (vi) shorter cilia length (**p<0.01 and ***p<0.001 respectively). Error bars are s. e. m. One-way ANOVA followed by Dunnett's multiple comparisons test. n = 3 organoids with 225 cells for GFP, 537 cells for CPAP and 569 for CC5. At least 10 ventricles from 3 independent organoids were used to measure the CPAP diameter. t test. Error bars are s.e.m. Scale bar 20 μm.e, Rescue effect of CC5 and CPAPelectroporation on cleavage planes of phospho-vimentin-positive RGs in Seckel organoids. Compared to GFP control, electroporation of CC5 or GFP increases the proportion of horizontally orientated SeckelRGs (for CC5 ***p<0.001) and decreases the portion of obliquely (for CC5 *p<0.05) or vertically oriented SeckelGFP (for CPAP and CC5 **p<0.01). Error bars are s.e.m. Two-way ANOVA followed by Tukey's multiple comparisons test. n = 4 organoids. 100 cells for GFP control, 112 cells for CPAP and 99 cells for CC5.


B Competitive inhibition of mitochondrial import by clogger inhibits growths . Yeast expressing clogger ( cytochrome b2 - DHFR cytochrome b2 ∆ 19 - DHFR ) or DHFR for comparison under control of galactose - inducible promoter were grown to mid - log phase on lactate medium . Ten - fold serial dilutions were dropped on lactate ( no induction ) or lactate with 0 . 5 % galactose medium ( induction ) . MTS , matrix - targeting signal .

O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-ORGANISM O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

B Competitive inhibition of mitochondrial protein import by clogger proteins inhibits cell growths. Yeast cells expressing clogger proteins (cytochrome b2-DHFR and cytochrome b2∆19-DHFR) or cytosolic DHFR for comparison under control of the galactose-inducible promoter were grown to mid-log phase on lactate medium. Ten-fold serial dilutions were dropped on lactate (no induction) or lactate with 0.5% galactose medium (induction). MTS, matrix-targeting signal.


E of proteasome Pre6 were detected upon expression of DHFR or clogger by Western blotting quantified from three replicates . were grown in lactate medium clogger expression was induced for 4 . 5 h with 0 . 5 % galactose before samples were harvested lysed . Data are displayed as mean ± standard deviations from n = 3 independent biological replicates .

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

E Levels of the proteasome protein Pre6 were detected upon expression of cytosolic DHFR or clogger by Western blotting and quantified from three replicates. Cells were grown in lactate medium and clogger expression was induced for 4.5 h with 0.5% galactose before samples were harvested and lysed. Data are displayed as mean ± standard deviations from n = 3 independent biological replicates.


A clogger was expressed in wild type ( WT ) ∆ rpn4 ( ∆ ) for 4 . 5 h with medium containing 0 . 5 % galactose . Medium was exchanged for a non - inducing lactate medium . Precursor ( pre ) mature ( m ) forms of b2 ∆ - DHFR clogger were visualized by Western blotting using a DHFR - specific antibody .

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

A The clogger was expressed in wild type (WT) and ∆rpn4 (∆) cells for 4.5 h with medium containing 0.5% galactose. Medium was exchanged for a non-inducing lactate medium. Precursor (pre) and mature (m) forms of the b2∆-DHFR clogger were visualized by Western blotting using a DHFR-specific antibody.


G Signals of Hsp104 Hsp42 before after clogger induction in wild type ∆ rpn4 . soluble DHFR was expressed for control . strains were grown in lactate medium clogger expression was induced with 0 . 5 % galactose for 6 h .

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

G Signals of Hsp104 and Hsp42 before and after clogger induction in wild type and ∆rpn4 cells. The cytosolic soluble DHFR protein was expressed for control. The strains were grown in lactate medium and clogger expression was induced with 0.5% galactose for 6 h.


A DHFR ( control ) or clogger b2 - DHFR were expressed in wild type ∆ rpn4 for 4 . 5 h . distribution of constitutively expressed Hsp104 - GFP was visualized by microscopy . Diameter of Hsp104 - GFP - bound were quantified . . Data are displayed as mean ± standard deviations from n = 36 independent biological replicates . Significance was assessed using two - sided , paired Student ' s t - . P values are indicated as asterisks * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 . See also Fig . EV4A for of number of per .

O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Cytosolic DHFR (control) or the clogger protein b2-DHFR were expressed in wild type and ∆rpn4 cells for 4.5 h. The distribution of constitutively expressed Hsp104-GFP was visualized by fluorescence microscopy. Diameter of Hsp104-GFP-bound aggregates were quantified. . Data are displayed as mean ± standard deviations from n = 36 independent biological replicates. Significance was assessed using two-sided, paired Student's t-test. P values are indicated as asterisks ** p ≤ 0.01, **** p ≤ 0.0001. See also Fig. EV4A for quantification of the number of aggregates per cell.


B Hsp104 - GFP was purified on nanotrap sepharose in wild type that were grown at indicated temperatures . SN , supernatant representing non - bound fraction . signals for Ssa1 Sod1 are shown for control .

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

B Hsp104-GFP was purified on nanotrap sepharose in wild type cells that were grown at the indicated temperatures. SN, supernatant representing the non-bound fraction. The signals for Ssa1 and Sod1 are shown for control.