SUM159 - LM1 cells expressing either control shRNA ( control ) , or one of two different TNC shRNAs ( shTNC 1 and 2 ) , were injected intravenously into NSG mice . ( J ) Metastatic colonization was quantified by lung bioluminescence . Control and shTNC ( 1 ) n = 8 mice per group , shTNC ( 2 ) n = 7 mice . P value was determined by a two - tailed Mann - Whitney test . ( K ) Representative images of metastatic foci ( top ) and ex vivo lung bioluminescence ( bottom ) . Arrows denote vimentin - expressing cancer cells in metastases .

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SUM159-LM1 cells expressing either control shRNA (control), or one of two different TNC shRNAs (shTNC 1 and 2), were injected intravenously into NSG mice. (J) Metastatic colonization was quantified by lung bioluminescence. Control and shTNC (1) n = 8 mice per group, shTNC (2) n = 7 mice. P value was determined by a two-tailed Mann-Whitney test. (K) Representative images of metastatic foci (top) and ex vivo lung bioluminescence (bottom). Arrows denote vimentin-expressing cancer cells in metastases.


Kaplan - Meier analysis of breast cancer patients ( combined GSE2603 and GSE5327 datasets ) showing a link between high SPP1 and TNC expression and poor lung metastasis - free survival . Patient samples , SPP1 and TNC low n = 70 ; SPP1 and TNC high n = 70 . HR , Hazard ratio .

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Kaplan-Meier analysis of breast cancer patients (combined GSE2603 and GSE5327 datasets) showing a link between high SPP1 and TNC expression and poor lung metastasis-free survival. Patient samples, SPP1 and TNC low n = 70; SPP1 and TNC high n = 70. HR, Hazard ratio.


Gene expression profile of MDA231 - LM2 cells treated with paclitaxel ( PAX ) for 48 hours . Highlighted are examples of JNK signature genes that are induced by PAX treatment .

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Gene expression profile of MDA231-LM2 cells treated with paclitaxel (PAX) for 48 hours. Highlighted are examples of JNK signature genes that are induced by PAX treatment.


GSEA normalized enrichment scores of four independent mammary stem cell signatures that arise in response to paclitaxel treatment of MDA231 - LM2 cells .

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GSEA normalized enrichment scores of four independent mammary stem cell signatures that arise in response to paclitaxel treatment of MDA231-LM2 cells.


Immunofluorescence analysis of p - c - Jun + cancer cells in lung metastases from mice injected intravenously with MDA231 - LM2 cells and treated with PAX at week 3 post injection . Lungs were harvested 72h after PAX treatment . Cancer cells express GFP as a marker . DAPI was used for nuclear staining . Scale bar , 50 µm .

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Immunofluorescence analysis of p-c-Jun+ cancer cells in lung metastases from mice injected intravenously with MDA231-LM2 cells and treated with PAX at week 3 post injection. Lungs were harvested 72h after PAX treatment. Cancer cells express GFP as a marker. DAPI was used for nuclear staining. Scale bar, 50 µm.


Quantification of cancer cells expressing p - c - Jun in panel D . Values are mean from three mice ± SEM . Nine metastatic foci were analyzed per mouse .

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Quantification of cancer cells expressing p-c-Jun in panel D. Values are mean from three mice ± SEM. Nine metastatic foci were analyzed per mouse.


Western blot analyzing phosphorylated JNK ( p - JNK ) , JNK , phosphorylated c - Jun ( p - c - Jun ) and c - Jun in MDA231 - LM2 cells treated with PAX for 48 hours . Tubulin was used as a loading control .

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Western blot analyzing phosphorylated JNK (p-JNK), JNK, phosphorylated c-Jun (p-c-Jun) and c-Jun in MDA231-LM2 cells treated with PAX for 48 hours. Tubulin was used as a loading control.


SPP1 and TNC expression in SUM159 - LM1 cells treated with increasing concentrations of PAX qPCR triplicates ± SD .

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SPP1 and TNC expression in SUM159-LM1 cells treated with increasing concentrations of PAX qPCR triplicates ± SD.


SPP1 and TNC expression in SUM159 - LM1 cells treated with increasing concentrations of doxorubicin ( DOXO ) . qPCR triplicates ± SD .

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SPP1 and TNC expression in SUM159-LM1 cells treated with increasing concentrations of doxorubicin (DOXO). qPCR triplicates ± SD.


SPP1 and TNC expression in SUM159 - LM1 cancer cells treated with 4 μM 5 - Fluorouracil or 4 μM methotrexate . Cells were treated for 48 hours . qPCR triplicates ± SD .

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SPP1 and TNC expression in SUM159-LM1 cancer cells treated with 4 μM 5-Fluorouracil or 4 μM methotrexate. Cells were treated for 48 hours. qPCR triplicates ± SD.


SPP1 and TNC expression in SUM159 - LM1 cells treated with paclitaxel and JNKi . qPCR triplicates ± SD . SPP1 and TNC repression by JNKi , P < 0 . 05 for both vehicle and PAX - treated cells .

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SPP1 and TNC expression in SUM159-LM1 cells treated with paclitaxel and JNKi. qPCR triplicates ± SD. SPP1 and TNC repression by JNKi, P < 0.05 for both vehicle and PAX-treated cells.


Growth curves of mammary tumors in vehicle - or PAX - treated Spp1 + / - mice implanted with the indicated cells expressing control shRNA , and Spp1 - / - mice implanted with the indicated cells expressing SPP1 shRNA . MDA231 - LM2 : control , PAX and SPP1 def . + PAX n = 16 tumors per group , SPP1 def . n = 14 tumors . SUM159 - LM1 : control , PAX and SPP1 def . + PAX n = 16 tumors per group , SPP1 def . n = 15 tumors .

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Growth curves of mammary tumors in vehicle- or PAX-treated Spp1+/- mice implanted with the indicated cells expressing control shRNA, and Spp1-/- mice implanted with the indicated cells expressing SPP1 shRNA. MDA231-LM2: control, PAX and SPP1 def. + PAX n = 16 tumors per group, SPP1 def. n = 14 tumors. SUM159-LM1: control, PAX and SPP1 def. + PAX n = 16 tumors per group, SPP1 def. n = 15 tumors.


Quantification of lung metastasis from the animals described in panels A and B . The number of metastatic foci per field of view ( FOV ) or per lung section ( LS ) was determined . MDA231 - LM2 tumor - bearing mice : control , PAX and SPP1 def . + PAX n = 8 mice per group , SPP1 def . n = 7 mice . SUM159 - LM1 tumor - bearing mice : control , PAX and SPP1 def . + PAX n = 8 mice per group , SPP1 def . n = 7 mice .

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Quantification of lung metastasis from the animals described in panels A and B. The number of metastatic foci per field of view (FOV) or per lung section (LS) was determined. MDA231-LM2 tumor-bearing mice: control, PAX and SPP1 def. + PAX n = 8 mice per group, SPP1 def. n = 7 mice. SUM159-LM1 tumor-bearing mice: control, PAX and SPP1 def. + PAX n = 8 mice per group, SPP1 def. n = 7 mice.


Tumor weight in mice at day 40 after implantation of MDA231 - LM2 breast cancer cells in a control or SPP1 deficient setting undergoing combination treatment with doxorubicin ( Adriamycin ) and cyclophosphamide ( AC regimen ) . Control , AC and SPP1 def . n = 16 tumors per group , SPP1 def . + AC n = 14 tumors .

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Tumor weight in mice at day 40 after implantation of MDA231-LM2 breast cancer cells in a control or SPP1 deficient setting undergoing combination treatment with doxorubicin (Adriamycin) and cyclophosphamide (AC regimen). Control, AC and SPP1 def. n = 16 tumors per group, SPP1 def. + AC n = 14 tumors.


Lung metastasis was quantified in mice that were implanted with MDA231 - LM2 tumors and subjected to AC chemotherapy . Each value represents the mean ± SEM ; n = 5 mice per group . Ten random fields per lung section were analyzed for percentage of metastatic area . Representative histological images of metastatic nodules in lungs of mice from the experiment described in panel E . Metastases were determined by expression of human vimentin ( arrows ) . Scale bar , 100 µm .

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Lung metastasis was quantified in mice that were implanted with MDA231-LM2 tumors and subjected to AC chemotherapy. Each value represents the mean ± SEM; n = 5 mice per group. Ten random fields per lung section were analyzed for percentage of metastatic area. Representative histological images of metastatic nodules in lungs of mice from the experiment described in panel E. Metastases were determined by expression of human vimentin (arrows). Scale bar, 100 µm.


Mammary tumor growth curves in PAX - treated NSG mice bearing control or TNC knockdown MDA231 - LM2 tumors . Values are means ± SEM , n = 16 tumors per group . * * * * P < 0 . 0001 . Lung metastases quantified in tumor bearing

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Mammary tumor growth curves in PAX-treated NSG mice bearing control or TNC knockdown MDA231-LM2 tumors. Values are means ± SEM, n = 16 tumors per group. **** P < 0.0001. Lung metastases quantified in tumor bearing mice


Representative histology of human vimentin - positive lung metastases ( arrows )

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Representative histology of human vimentin-positive lung metastases (arrows)


Mammary tumor growth curves in NSG mice implanted with MDA231 - LM2 or SUM159 - LM1 cells and treated with paclitaxel as single agent or in combination with JNKi . Each value represents the mean ± SEM . MDA231 - LM2 : n = 12 tumors per group . SUM159 - LM1 : vehicle , JNKi and PAX n = 12 tumors per group ; PAX + JNKi n = 10 tumors . * * P < 0 . 01 , * * * P < 0 . 001 .

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Mammary tumor growth curves in NSG mice implanted with MDA231-LM2 or SUM159-LM1 cells and treated with paclitaxel as single agent or in combination with JNKi. Each value represents the mean ± SEM. MDA231-LM2: n = 12 tumors per group. SUM159-LM1: vehicle, JNKi and PAX n = 12 tumors per group; PAX + JNKi n = 10 tumors. **P < 0.01, ***P < 0.001.


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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 ) 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 ) .

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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 ) 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 .

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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 . 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 .

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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 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 .

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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 and IRGM protein levels were measured in cytosolic fractions by western blot analysis .

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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 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 .

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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 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 .

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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.


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 .

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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 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 .

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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 and IRGM protein 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 ) 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 .

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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 ) 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 .

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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 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 .

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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 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 . .

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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 , TEM magnification × 40 , 000 ; D , TEM magnification × 12 , 000 ) . Micrographs 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 ) The indicated kinases were incubated with different doses of PIK - III and 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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

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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 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 .

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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 ) and light ( FTL ) chains from DLD1 cell 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 levels of FTH1 and FTL expressed in DLD1 cells growing in rich medium or starved for the 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 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 .

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

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

(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 ) 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 .

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

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

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

(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 − / − 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 .

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

(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 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 .

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

(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 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 .

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

(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 − / − cells were incubated in the presence or absence of DFX and the levels of FTH and FTL were determined .

O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O

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

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

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

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

(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 ) 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 .

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

(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 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 ) .

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

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 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 .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O 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 O O O O O O O O O O O O O O

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 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 .

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

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 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 .

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

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 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 .

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

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 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 ) .

O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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 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 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 .

O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O 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 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 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 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 .

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

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 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 .

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

B-CELL_TYPE I-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 O O B-DISEASE B-ORGANISM O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-DISEASE O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-DISEASE O O O O O B-GENEPROD I-GENEPROD O B-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. 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 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 .

B-CELL_TYPE I-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 O O B-DISEASE B-ORGANISM O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-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 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 .

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 O O O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O 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 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 .

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 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 O B-SMALL_MOLECULE 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 O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O 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 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 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 ) .

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

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 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 .

O O O O O O O B-SUBCELLULAR O B-GENEPROD 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 O O O O O O O O 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 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 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 .

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

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

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 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 .

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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 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 ) .

O O B-SUBCELLULAR O O 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 O O O B-SUBCELLULAR O O 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 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 .

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

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 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 .

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

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 proteins Cap350 and 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 O B-GENEPROD O B-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 , 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 .

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

O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O B-GENEPROD O 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 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 O B-CELL_TYPE 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 B-SUBCELLULAR O B-GENEPROD O O O O O O B-GENEPROD O 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 ) 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 .

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

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

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 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 .

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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 - 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 .

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 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 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 B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 O O O O B-EXP_ASSAY O O O 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 test . n = 3 independent experiments . 298 cells for untreated , 151 cells for DMSO and 146 cells for olomoucine , 25 μM .

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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 - test . n = 3 independent experiments . 633 cells for WT and 275 cells for Seckel . Scale bar 5 μm .

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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 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 .

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