( E ) HCC70 parental , PI3Kβi - Res or AKTi - Res cells were treated with vehicle , AKTi ( MK2206 250 nM ) or gefitinib ( 3 μM ) , alone or in the indicated combinations . The cell lysates were probed with the indicated antibodies .

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(E) HCC70 parental, PI3Kβi-Res or AKTi-Res cells were treated with vehicle, AKTi (MK2206 250 nM) or gefitinib (3 μM), alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.


( F ) HCC70 MK res ( acquired resistance models to MK2206 ) were treated with serial dilutions of gefitinib , alone or in combination with MK2206 ( 810 nM ) , as indicated , and viability measured after 4 days of treatment . Average ± SD of triplicates and representative of two independent experiments .

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(F) HCC70 MK res (acquired resistance models to MK2206) were treated with serial dilutions of gefitinib, alone or in combination with MK2206 (810 nM), as indicated, and viability measured after 4 days of treatment. Average ± SD of triplicates and representative of two independent experiments.


( G ) HCC70 AZD res ( acquired resistance models to AZD8186 ) were treated with serial dilutions of gefitinib , alone or in combination with AZD8186 ( 270 nM ) , as indicated , and viability measured after 4 days . Average ± SD of triplicates and representative of two independent experiments .

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(G) HCC70 AZD res (acquired resistance models to AZD8186) were treated with serial dilutions of gefitinib, alone or in combination with AZD8186 (270 nM), as indicated, and viability measured after 4 days. Average ± SD of triplicates and representative of two independent experiments.


( H , I ) p110β co - immunoprecipitates with EGFR in MDA - MB - 468 ( H ) and in HCC70 ( I ) . Cells were pre - treated with different inhibitors , including vehicle , pan - PI3Ki GDC0941 ( 1 μM for MDA - MB - 468 or 0 . 5 μM for HCC70 ) , EGFRi ( gefitinib 3 μM ) , PI3Kβi ( AZD8186 250 nM ) or a combination of EGFRi and PI3Kβi . Cell lysates were incubated with IgG control or anti - EGFR antibody and the immuno - complexes or the total lysates were immune - blotted with the indicated antibodies .

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(H,I) p110β co-immunoprecipitates with EGFR in MDA-MB-468 (H) and in HCC70 (I). Cells were pre-treated with different inhibitors, including vehicle, pan-PI3Ki GDC0941 (1 μM for MDA-MB-468 or 0.5 μM for HCC70), EGFRi (gefitinib 3 μM), PI3Kβi (AZD8186 250 nM) or a combination of EGFRi and PI3Kβi. Cell lysates were incubated with IgG control or anti-EGFR antibody and the immuno-complexes or the total lysates were immune-blotted with the indicated antibodies.


( J ) EGF - induced increase in phospho - AKT is dependent on p110β kinase activity . MDA - MB - 468 were starved in 0 % FBS and pre - treated with vehicle or PI3Kβi ( AZD8186 250 nM ) for 1h . Cell lysates were probed with the indicated antibodies . Phospho - AKT and pan - AKT bands were quantified by the use of ImageLite software : the ratio of phospho - AKT to pan - AKT normalised to the control ( left hand lane ) is shown .

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(J) EGF-induced increase in phospho-AKT is dependent on p110β kinase activity. MDA-MB-468 were starved in 0% FBS and pre-treated with vehicle or PI3Kβi (AZD8186 250 nM) for 1h. Cell lysates were probed with the indicated antibodies. Phospho-AKT and pan-AKT bands were quantified by the use of ImageLite software: the ratio of phospho-AKT to pan-AKT normalised to the control (left hand lane) is shown.


( A Results of CRISPR - Cas9 screening in combination with AZD8186 100nM ( A ) The dot - plots show for each gene knocked - out by sgRNAs the fold change ( log2 ) between treated conditions ( AZD8186 and vehicle in the fluorescence signal ( anti - phosphoS6 immunofluorescence ) versus the p - value of the difference calculated by two sided t - test . The plots represent means of biological triplicates . Genes for which it was calculated a p < 0 . 0001 and Log2 ( fold change ) > 0 . 25 are reported and highlighted in green ; genes having a 0 . 0001 0 . 25 are reported and shown in red . Gefitinib combined with AZD8186 represents the positive control of the experiment ( A ) . < 0 . 05 >

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(A Results of CRISPR-Cas9 screening in combination with AZD8186 100nM (A) The dot-plots show for each gene knocked-out by sgRNAs the fold change (log2) between treated conditions (AZD8186 and vehicle in the fluorescence signal (anti-phosphoS6 immunofluorescence) versus the p-value of the difference calculated by two sided t-test. The plots represent means of biological triplicates. Genes for which it was calculated a p<0.0001 and Log2 (fold change)>0.25 are reported and highlighted in green; genes having a 0.0001 0.25 are reported and shown in red. Gefitinib combined with AZD8186 represents the positive control of the experiment (A). <0.05>


B ) Results of CRISPR - Cas9 screening in combination with GDC0941 400nM ( B ) . The dot - plots show for each gene knocked - out by sgRNAs the fold change ( log2 ) between treated conditions GDC0941 , and vehicle in the fluorescence signal ( anti - phosphoS6 immunofluorescence ) versus the p - value of the difference calculated by two sided t - test . The plots represent means of biological triplicates . Genes for which it was calculated a p < 0 . 0001 and Log2 ( fold change ) > 0 . 25 are reported and highlighted in green ; genes having a 0 . 0001 0 . 25 are reported and shown in red . < 0 . 05 >

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B) Results of CRISPR-Cas9 screening in combination with GDC0941 400nM (B). The dot-plots show for each gene knocked-out by sgRNAs the fold change (log2) between treated conditions GDC0941, and vehicle in the fluorescence signal (anti-phosphoS6 immunofluorescence) versus the p-value of the difference calculated by two sided t-test. The plots represent means of biological triplicates. Genes for which it was calculated a p<0.0001 and Log2 (fold change)>0.25 are reported and highlighted in green; genes having a 0.0001 0.25 are reported and shown in red. <0.05>


( C ) Box & Whisker plot showing the fold change in fluorescence signal between vehicle and GDC0941 - treated conditions for MDA - MB - 468 cells transduced with non - target Control , GNB2 or GNG5 sgRNAs ( N = 2 or 3 ) . Data presented in a box and whisker plot with the central band indicating the median , the upper and lower extremes of the box or hinge being the third and first quartiles respectively and the whiskers extending to the most extreme data values within 1 . 5 times the inter - quartile range . Statistical significance of unpaired t - test * * * * P < 0 . 0001 .

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(C) Box & Whisker plot showing the fold change in fluorescence signal between vehicle and GDC0941-treated conditions for MDA-MB-468 cells transduced with non-target Control, GNB2 or GNG5 sgRNAs (N=2 or 3). Data presented in a box and whisker plot with the central band indicating the median, the upper and lower extremes of the box or hinge being the third and first quartiles respectively and the whiskers extending to the most extreme data values within 1.5 times the inter-quartile range. Statistical significance of unpaired t-test ****P<0.0001.


( D ) Biochemical analysis of GNB2 KO cells . Cell lysates of MDA - MB - 468 parental cells and three MDA - MB - 468 GNB2 KO clones were probed with the indicated antibodies . Quantification of the bands was performed by ImageLite software .

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(D) Biochemical analysis of GNB2 KO cells. Cell lysates of MDA-MB-468 parental cells and three MDA-MB-468 GNB2 KO clones were probed with the indicated antibodies. Quantification of the bands was performed by ImageLite software.


( A ) MDA - MB - 468 parental cells and three MDA - MB - 468 GNB2 KO clones were treated with vehicle , GDC0941 1 μM , gefitinib 3 μM or lapatinib 1 μM for 24 hours . The cell lysates were probed with the indicated antibodies .

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(A) MDA-MB-468 parental cells and three MDA-MB-468 GNB2 KO clones were treated with vehicle, GDC0941 1 μM, gefitinib 3 μM or lapatinib 1 μM for 24 hours. The cell lysates were probed with the indicated antibodies.


( B - D ) Viability assays of MDA - MB - 468 parental cells and three MDA - MB - 468 GNB2 KO clones treated with serial dilutions of the indicated drugs for six days . Mean ± SD of triplicates and representative of two or three independent experiments .

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(B-D) Viability assays of MDA-MB-468 parental cells and three MDA-MB-468 GNB2 KO clones treated with serial dilutions of the indicated drugs for six days. Mean ± SD of triplicates and representative of two or three independent experiments.


( E ) Patient samples from METABRIC dataset classified as TNBCs ( N = 299 ) were assigned to the groups " PTEN - low " when falling in the lower quartile for PTEN expression , " PTEN - mut " when harbouring a non - synonymous mutation on PTEN gene , " PTEN - WT " in all other cases . Comparison of the expression of GNB2 between the PTEN low or mut and the PTEN - WT groups . Box & Whisker plot ( Median / IQR / 1 . 5 * IQR Whiskers ) . P value calculated by unpaired t - test .

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(E) Patient samples from METABRIC dataset classified as TNBCs (N=299) were assigned to the groups "PTEN-low" when falling in the lower quartile for PTEN expression, "PTEN-mut" when harbouring a non-synonymous mutation on PTEN gene, "PTEN-WT" in all other cases. Comparison of the expression of GNB2 between the PTEN low or mut and the PTEN-WT groups. Box & Whisker plot (Median/IQR/1.5*IQR Whiskers). P value calculated by unpaired t-test.


( A ) Schematic of the results from the drug screening with compounds targeting GPCR signaling . MDA - MB - 468 cells were treated with the compounds of the GPCR - targeted library at three different concentrations ( 0 . 1 , 1 or 10 μM ) in combination with vehicle , GDC0941 450 nM or lapatinib 1 μM . The pS6 signal was measured by IF after 24 hours of treatment and normalized to DAPI . Z scores of the normalized fluorescence values for each drug measured in the presence of vehicle , GDC0941 or lapatinib are reported on the y axis or on the left or right x axis of the dot plot , respectively . Readings acquired following treatment with the three different concentrations of the GPCR - targeted drugs are reported in 3 different colours . Dots corresponding to vorapaxar treatments at the three different concentrations are highlighted in the plot . The values reported are mean of a biological triplicate of the experiment .

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(A) Schematic of the results from the drug screening with compounds targeting GPCR signaling. MDA-MB-468 cells were treated with the compounds of the GPCR-targeted library at three different concentrations (0.1, 1 or 10 μM) in combination with vehicle, GDC0941 450 nM or lapatinib 1 μM. The pS6 signal was measured by IF after 24 hours of treatment and normalized to DAPI. Z scores of the normalized fluorescence values for each drug measured in the presence of vehicle, GDC0941 or lapatinib are reported on the y axis or on the left or right x axis of the dot plot, respectively. Readings acquired following treatment with the three different concentrations of the GPCR-targeted drugs are reported in 3 different colours. Dots corresponding to vorapaxar treatments at the three different concentrations are highlighted in the plot. The values reported are mean of a biological triplicate of the experiment.


( B ) MDA - MB - 468 were treated for 24 h with vehicle , vorapaxar ( 10 μM ) , GDC0941 ( 1 μM ) or lapatinib ( 1 μM ) alone or in the indicated combinations . The cell lysates were probed with the indicated antibodies .

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(B) MDA-MB-468 were treated for 24 h with vehicle, vorapaxar (10 μM), GDC0941 (1 μM) or lapatinib (1 μM) alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.


( C ) MDA - MB - 468 parental cells or GNB2 KO clones were starved and treated with scramble or PAR1 activating peptide for 5 minutes , alone or in combination with vorapaxar . The cell lysates were probed with the indicated antibodies . Phospho - AKT , pan - AKT , phospho - ERK1 / 2 and pan - ERK1 / 2 bands were quantified by the use of ImageLite software : the ratio of phospho - AKT to pan - AKT and phospho - ERK1 / 2 to pan - ERK1 / 2 normalised to the control peptide - treated conditions for WT and GNB2 KO cells ( first and forth lanes , respectively ) is shown .

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(C) MDA-MB-468 parental cells or GNB2 KO clones were starved and treated with scramble or PAR1 activating peptide for 5 minutes, alone or in combination with vorapaxar. The cell lysates were probed with the indicated antibodies. Phospho-AKT, pan-AKT, phospho-ERK1/2 and pan-ERK1/2 bands were quantified by the use of ImageLite software: the ratio of phospho-AKT to pan-AKT and phospho-ERK1/2 to pan-ERK1/2 normalised to the control peptide-treated conditions for WT and GNB2 KO cells (first and forth lanes, respectively) is shown.


( D ) p - S6 / loading control signal from western blots experiments in which a panel of six TNBC PTEN - null cell lines and HCC70 cells that acquired resistance to AZD8186 or MK2206 were treated with vehicle , vorapaxar ( 10 μM ) , GDC0941 ( 1 μM ) or lapatinib ( 1 μM ) alone or in the indicated combinations . The values were normalised to vehicle treatment for all cell lines . Mean of 2 independent experiments ± SD . P values calculated by two - tailed paired student t - test * P = 0 . 0235 and * * P = 0 . 002 . Cell lines used in the experiments were : MDA - MB - 468 , HCC70 , HCC1937 , HCC38 , HCC1395 , BT - 549 , HCC70 AZD8186 - resistant and HCC70 MK2206 - resistant .

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(D) p-S6/loading control signal from western blots experiments in which a panel of six TNBC PTEN-null cell lines and HCC70 cells that acquired resistance to AZD8186 or MK2206 were treated with vehicle, vorapaxar (10 μM), GDC0941 (1 μM) or lapatinib (1 μM) alone or in the indicated combinations. The values were normalised to vehicle treatment for all cell lines. Mean of 2 independent experiments ± SD. P values calculated by two-tailed paired student t-test *P=0.0235 and **P=0.002. Cell lines used in the experiments were: MDA-MB-468, HCC70, HCC1937, HCC38, HCC1395, BT-549, HCC70 AZD8186-resistant and HCC70 MK2206-resistant.


( E ) Long - term proliferation assay of MDA - MB - 468 cells treated with vorapaxar ( 5 or 2 . 5 μM ) , GDC0941 ( 1 μM ) or lapatinib ( 1 μM ) , alone or in the indicated combinations . Cells were treated for two weeks and stained by crystal violet . One representative experiment of three is shown .

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(E) Long-term proliferation assay of MDA-MB-468 cells treated with vorapaxar (5 or 2.5 μM), GDC0941 (1 μM) or lapatinib (1 μM), alone or in the indicated combinations. Cells were treated for two weeks and stained by crystal violet. One representative experiment of three is shown.


A Robust immunofluorescence detection of ubiquitination on LAMP1 - positive compartments with pan - ubiquitin FK2 or K48 chain - specific antibodies . HeLa cells were treated with 250 µM LLOMe or EtOH ( untreated ) for 3 h , fixed and stained with indicated antibodies . Confocal microscopy was followed by automated image analysis . Graphs represent data from three independent experiments with ≥ 50 cells per condition ( mean ± SD ) . Scale bar : 20 µm .

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A Robust immunofluorescence detection of ubiquitination on LAMP1-positive compartments with pan-ubiquitin FK2 or K48 chain-specific antibodies. HeLa cells were treated with 250 µM LLOMe or EtOH (untreated) for 3 h, fixed and stained with indicated antibodies. Confocal microscopy was followed by automated image analysis. Graphs represent data from three independent experiments with ≥ 50 cells per condition (mean ± SD). Scale bar: 20 µm.


C Primary screen using pools of four single siRNAs targeting 37 human E2 enzymes . HeLa cells were seeded in 384 - well plates , transfected with a siRNA library in quadruplicates for 72 h . Cells were LLOMe - treated and processed for imaging Plates were imaged automatically with a spinning disk confocal microscope . Automated image analysis was done using the Acapella software and the percentage of ubiquitinated lysosomes was determined . Robust z - scores were calculated for LLOMe - treated samples for each depletion against the values of the whole plate . Samples exceeding the threshold of - 2 of robust z - score ( highlighted in red ) were considered for the secondary screen .

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C Primary screen using pools of four single siRNAs targeting 37 human E2 enzymes. HeLa cells were seeded in 384-well plates, transfected with a siRNA library in quadruplicates for 72 h. Cells were LLOMe-treated and processed for imaging Plates were imaged automatically with a spinning disk confocal microscope. Automated image analysis was done using the Acapella software and the percentage of ubiquitinated lysosomes was determined. Robust z-scores were calculated for LLOMe-treated samples for each depletion against the values of the whole plate. Samples exceeding the threshold of -2 of robust z-score (highlighted in red) were considered for the secondary screen.


D Selected candidates were re - screened with the same work pipeline but with the four single siRNAs of pools of each candidate from the primary screen . Candidates exceeding the threshold in both stainings are highlighted in red .

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D Selected candidates were re-screened with the same work pipeline but with the four single siRNAs of pools of each candidate from the primary screen. Candidates exceeding the threshold in both stainings are highlighted in red.


E Damage - induced translocation of UBE2QL1 to lysosomes . The three best candidates ( from D ) were transiently expressed with N - terminal HA - tag in HeLa cells . Cells were treated with LLOMe or EtOH ( untreated ) for 3 h , fixed and stained for HA and LAMP1 as indicated and imaged by confocal laser scanning microscopy . Scale bar : 10 µm . F Automated quantification of ( E ) . Shown are Pearson correlation coefficients ( P . C . C ) of HA and LAMP1 signals . Graph represents data from three independent experiments with ≥ 30 cells per condition ( mean ± SD ) . * * * * P < 0 . 0001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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E Damage-induced translocation of UBE2QL1 to lysosomes. The three best candidates (from D) were transiently expressed with N-terminal HA-tag in HeLa cells. Cells were treated with LLOMe or EtOH (untreated) for 3 h, fixed and stained for HA and LAMP1 as indicated and imaged by confocal laser scanning microscopy. Scale bar: 10 µm. F Automated quantification of (E). Shown are Pearson correlation coefficients (P.C.C) of HA and LAMP1 signals. Graph represents data from three independent experiments with ≥ 30 cells per condition (mean ± SD). ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).


G Western blot detection of endogenous UBE2QL1 and depletion efficiency . Cells were transfected with control siRNA ( Ctrl ) , two UBE2QL1 siRNAs from the screen ( # 2 or # 4 ) or an additional siRNA ( # 5 ) , and lysates probed with an antibody to UBE2QL1 as indicated . GAPDH was probed as loading control .

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G Western blot detection of endogenous UBE2QL1 and depletion efficiency. Cells were transfected with control siRNA (Ctrl), two UBE2QL1 siRNAs from the screen (#2 or #4) or an additional siRNA (#5), and lysates probed with an antibody to UBE2QL1 as indicated. GAPDH was probed as loading control.


A HeLa cells were transfected with different UBE2QL1 siRNAs for 60 h , and LLOMe or control - treated ( untreated ) for 3 h . Cells were fixed and immuno - stained for LAMP1 along with antibodies specific for K63 or K48 - linked ubiquitin chains as indicated and imaged with a laser scanning confocal microscope . Note the strong reduction of damage - induced K48 ubiquitination and decreased intensity of the K63 signal . Scale bar : 20 µm . B , C Automated image quantification of ( A ) . Shown are percentages of K48 or K63 - positive LAMP1 vesicles per cell . Graphs represent data from three independent experiments with ≥ 70 cells per condition ( mean ± SD ) . * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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A HeLa cells were transfected with different UBE2QL1 siRNAs for 60 h, and LLOMe or control-treated (untreated) for 3 h. Cells were fixed and immuno-stained for LAMP1 along with antibodies specific for K63 or K48-linked ubiquitin chains as indicated and imaged with a laser scanning confocal microscope. Note the strong reduction of damage-induced K48 ubiquitination and decreased intensity of the K63 signal. Scale bar: 20 µm. B, C Automated image quantification of (A). Shown are percentages of K48 or K63-positive LAMP1 vesicles per cell. Graphs represent data from three independent experiments with ≥ 70 cells per condition (mean ± SD). **P<0.01; ***P<0.001; ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).


A HeLa cells expressing UBE2QL1 - HA were fixed at indicated time points after LLOMe treatment and processed for confocal microscopy . Cells were stained with antibodies specific for LAMP1 ( not shown ) , and combinations of HA with K48 chains , K63 chains or p62 as indicated . Note the delayed co - emergence of K48 chains and UBE2QL1 vesicles , compared to the earlier appearance of K63 chains and p62 . Arrows indicate colocalizing or non - colocalizing vesicles . Scale bars : 10 µm , 2 µm for inlays . B Automated quantification of ( A ) . Shown are the Pearson correlation coefficients ( P . C . C . ) representing colocalization of UBE2QL1 - HA , K48 chains , K63 chains or p62 with LAMP1 - positive vesicles . Graphs represent data from three independent experiments with ≥ 30 cells per condition ( mean ± SD ) . C Automated quantification of ( A ) . P . C . C . ' s representing colocalization of K48 chains , K63 chains or p62 with UBE2QL1 - HA in cells treated with LLOMe or vehicle alone ( untreated ) for 180 min . Graphs represent data from three independent experiments with ≥ 30 cells per condition ( mean ± SD ) .

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A HeLa cells expressing UBE2QL1-HA were fixed at indicated time points after LLOMe treatment and processed for confocal microscopy. Cells were stained with antibodies specific for LAMP1 (not shown), and combinations of HA with K48 chains, K63 chains or p62 as indicated. Note the delayed co-emergence of K48 chains and UBE2QL1 vesicles, compared to the earlier appearance of K63 chains and p62. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 10 µm, 2 µm for inlays. B Automated quantification of (A). Shown are the Pearson correlation coefficients (P.C.C.) representing colocalization of UBE2QL1-HA, K48 chains, K63 chains or p62 with LAMP1-positive vesicles. Graphs represent data from three independent experiments with ≥ 30 cells per condition (mean ± SD). C Automated quantification of (A). P.C.C.'s representing colocalization of K48 chains, K63 chains or p62 with UBE2QL1-HA in cells treated with LLOMe or vehicle alone (untreated) for 180 min. Graphs represent data from three independent experiments with ≥ 30 cells per condition (mean ± SD).


Immuno - electron microscopy of LLOMe - treated HeLa cells . Cells in ( A ) overexpress mCherry - Gal3 and a dominant - negative C160S mutant of GFP - YOD1 , and were immunostained for p62 ( A ) p62 ( 10 nm gold particles ) localizes as a cap ( arrowheads ) to the outer limiting membrane of lysosomes ( L ) . Scale bars : 200 nm .

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

Immuno-electron microscopy of LLOMe-treated HeLa cells. Cells in (A) overexpress mCherry-Gal3 and a dominant-negative C160S mutant of GFP-YOD1, and were immunostained for p62 (A) p62 (10 nm gold particles) localizes as a cap (arrowheads) to the outer limiting membrane of lysosomes (L). Scale bars: 200 nm.


Immuno - electron microscopy of LLOMe - treated HeLa cells . Cells in ( B ) overexpress mCherry - Gal3 and a dominant - negative C160S mutant of GFP - YOD1 , and were immunostained for p62 and K48 . ( B ) K48 ubiquitin chains ( 15 nm gold particles , arrows ) are found in the lumen of lysosomes , that also contain p62 ( 10 nm gold particles , arrowheads ) at their outer membrane . Scale bars : 200 nm .

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Immuno-electron microscopy of LLOMe-treated HeLa cells. Cells in (B) overexpress mCherry-Gal3 and a dominant-negative C160S mutant of GFP-YOD1, and were immunostained for p62 and K48. (B) K48 ubiquitin chains (15 nm gold particles, arrows) are found in the lumen of lysosomes, that also contain p62 (10 nm gold particles, arrowheads) at their outer membrane. Scale bars: 200 nm.


Immuno - electron microscopy of LLOMe - treated HeLa cells . ( C ) Overexpressed UBE2QL1 - GFP ( 10 nm gold particles ) is found in late endosomes ( LE , Inset ) and lysosomes . M = mitochondrion . Scale bars : 200 nm .

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Immuno-electron microscopy of LLOMe-treated HeLa cells. (C) Overexpressed UBE2QL1-GFP (10 nm gold particles) is found in late endosomes (LE, Inset) and lysosomes. M = mitochondrion. Scale bars: 200 nm.


D , E Comparative UBE2QL1 proximity mapping before and after lysosome damage using SILAC mass spectrometry . HeLa cells expressing a UBE2QL1 - APEX2 fusion were EtOH ( light - labelled , L ) - or LLOMe ( heavy - labelled , H ) - treated and pulsed with biotin phenol ( 30 min ) and H2O2 ( 1 min ) . Proteins detected in replicative streptavidin purifications are depicted in a Venn diagram ( D ) and displayed high correlation coefficients ( E ) .

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

D, E Comparative UBE2QL1 proximity mapping before and after lysosome damage using SILAC mass spectrometry. HeLa cells expressing a UBE2QL1-APEX2 fusion were EtOH (light-labelled, L)- or LLOMe (heavy-labelled, H)-treated and pulsed with biotin phenol (30 min) and H2O2 (1 min). Proteins detected in replicative streptavidin purifications are depicted in a Venn diagram (D) and displayed high correlation coefficients (E).


, F Comparative UBE2QL1 proximity mapping before and after lysosome damage using SILAC mass spectrometry . HeLa cells expressing a UBE2QL1 - APEX2 fusion were EtOH ( light - labelled , L ) - or LLOMe ( heavy - labelled , H ) - treated and pulsed with biotin phenol ( 30 min ) and H2O2 ( 1 min ) . ( F ) Results from the four experiments are summarized in a volcano plot . Horizontal dotted line represents the significance threshold ( p > 0 . 05 ) . The vertical lines indicate the fold change cutoff ( log2 ( H / L ) ≥ 1 . 5 ) . Red dots indicate proteins above the significance and fold - change thresholds . Protein names are color - coded in blue ( endosome / lysosome - associated ) , green ( endolysosomal damage / autophagy - associated ) and orange ( VCP / p97 and its cofactors ) .

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

, F Comparative UBE2QL1 proximity mapping before and after lysosome damage using SILAC mass spectrometry. HeLa cells expressing a UBE2QL1-APEX2 fusion were EtOH (light-labelled, L)- or LLOMe (heavy-labelled, H)-treated and pulsed with biotin phenol (30 min) and H2O2 (1 min). (F) Results from the four experiments are summarized in a volcano plot. Horizontal dotted line represents the significance threshold (p > 0.05). The vertical lines indicate the fold change cutoff (log2 (H/L) ≥ 1.5). Red dots indicate proteins above the significance and fold-change thresholds. Protein names are color-coded in blue (endosome/lysosome-associated), green (endolysosomal damage/autophagy-associated) and orange (VCP/p97 and its cofactors).


A HeLa cells stably expressing p97 - GFP were control or UBE2QL1 - depleted for 48 h and expression of p97 - GFP was induced for the last 24 h . Cells were LLOMe or control treated for 1 h , fixed after a chase of 2 h , and stained with antibodies to K48 ubiquitin chains and LAMP1 as indicated . Note the reduction of the K48 and p97 - GFP signals on damaged lysosomes in UBE2QL1 - depleted cells . Arrows indicate colocalizing or non - colocalizing vesicles . Scale bars : 20 µm , 10 µm for inlays .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O 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 HeLa cells stably expressing p97-GFP were control or UBE2QL1-depleted for 48 h and expression of p97-GFP was induced for the last 24 h. Cells were LLOMe or control treated for 1 h, fixed after a chase of 2 h, and stained with antibodies to K48 ubiquitin chains and LAMP1 as indicated. Note the reduction of the K48 and p97-GFP signals on damaged lysosomes in UBE2QL1-depleted cells. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 10 µm for inlays.


B Automated quantification Shown are fold increases of Pearson correlation coefficients ( P . C . C . ) of p97 - GFP and LAMP1 signals , normalized to untreated siCtrl . Data are from four independent experiments with ≥ 30 cells per condition . Data information : data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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B Automated quantification Shown are fold increases of Pearson correlation coefficients (P.C.C.) of p97-GFP and LAMP1 signals, normalized to untreated siCtrl. Data are from four independent experiments with ≥ 30 cells per condition. Data information: data are presented as mean ± SD. *P<0.05; ***P<0.001 (One-way-ANOVA with Bonferroni's multiple comparison test).


C HeLa cells were control or UBE2QL1 - depleted for 60 h , fixed after 1 h of LLOMe or control treatment and stained for LAMP1 and p62 and analyzed by confocal microscopy . Arrows indicate colocalizing or non - colocalizing vesicles . Scale bars : 20 µm , 5 µm for inlays .

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C HeLa cells were control or UBE2QL1-depleted for 60 h, fixed after 1 h of LLOMe or control treatment and stained for LAMP1 and p62 and analyzed by confocal microscopy. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 5 µm for inlays.


D Quantification Shown are fold increases of Pearson correlation coefficients ( P . C . C . ) of p62 on LAMP1 signals , normalized to untreated siCtrl . Graph represents data from three independent experiments with ≥ 30 cells per condition . Data information : data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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D Quantification Shown are fold increases of Pearson correlation coefficients (P.C.C.) of p62 on LAMP1 signals, normalized to untreated siCtrl. Graph represents data from three independent experiments with ≥ 30 cells per condition. Data information: data are presented as mean ± SD. *P<0.05; ***P<0.001 (One-way-ANOVA with Bonferroni's multiple comparison test).


E HeLa cells were treated but fixed after additional 2 h of chase . Samples were stained for Galectin - 3 ( Gal3 ) to detect damaged lysosomes , and LC3B as marker for autophagosomes . Note the reduction of LC3B colocalization with Gal3 indicating impaired autophagosome formation . Arrows indicate colocalizing or non - colocalizing vesicles . Scale bars : 20 µm , 5 µm for inlays .

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E HeLa cells were treated but fixed after additional 2 h of chase. Samples were stained for Galectin-3 (Gal3) to detect damaged lysosomes, and LC3B as marker for autophagosomes. Note the reduction of LC3B colocalization with Gal3 indicating impaired autophagosome formation. Arrows indicate colocalizing or non-colocalizing vesicles. Scale bars: 20 µm, 5 µm for inlays.


F Quantification Shown are fold increases of Pearson correlation coefficients ( P . C . C . ) of LC3B and Gal3 signals , normalized to untreated siCtrl , representing LC3B colocalization with Gal3 . Graph represents data from three independent experiments with ≥ 50 cells per condition . Data information : data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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F Quantification Shown are fold increases of Pearson correlation coefficients (P.C.C.) of LC3B and Gal3 signals, normalized to untreated siCtrl, representing LC3B colocalization with Gal3. Graph represents data from three independent experiments with ≥ 50 cells per condition. Data information: data are presented as mean ± SD. *P<0.05; ***P<0.001 (One-way-ANOVA with Bonferroni's multiple comparison test).


G Western blot analysis of lysates of cells transfected with UBE2QL1 or control siRNA for 72 h , treated with 200 nM Bafilomycin A1 for 5 h or 250 µM LLOMe for 3 h as indicated and probed with an antibody specific for LC3A / B . GAPDH was probed as loading control .

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G Western blot analysis of lysates of cells transfected with UBE2QL1 or control siRNA for 72 h, treated with 200 nM Bafilomycin A1 for 5 h or 250 µM LLOMe for 3 h as indicated and probed with an antibody specific for LC3A/B. GAPDH was probed as loading control.


A Gal3 puncta assay for damaged lysosomes . HeLa cells were depleted of UBE2QL1 with two siRNAs for 60 h and treated with LLOMe or EtOH alone ( untreated ) for 1 h . Cells were fixed at indicated times after washout , stained with DAPI and Gal3 and LAMP1 antibodies , and processed for confocal microscopy . Note increased number of Gal3 puncta in untreated UBE2QL1 - depleted cells and their persistence 10 h after LLOMe - induced damage . Scale bars : 10 µm , 2 µm for inlays . Arrows indicate Gal3 and LAMP1 colocalizing vesicles .

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

A Gal3 puncta assay for damaged lysosomes. HeLa cells were depleted of UBE2QL1 with two siRNAs for 60 h and treated with LLOMe or EtOH alone (untreated) for 1 h. Cells were fixed at indicated times after washout, stained with DAPI and Gal3 and LAMP1 antibodies, and processed for confocal microscopy. Note increased number of Gal3 puncta in untreated UBE2QL1-depleted cells and their persistence 10 h after LLOMe-induced damage. Scale bars: 10 µm, 2 µm for inlays. Arrows indicate Gal3 and LAMP1 colocalizing vesicles.


B Automated quantification The percentage of cells with more than three Gal3 puncta is shown . Graph represents data from three independent experiments with ≥ 50 cells per condition ( mean ± SD ) . * P < 0 . 05 ; * * P < 0 . 01 ( One - way - ANOVA with Dunnett ' s multiple comparison test ) .

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B Automated quantification The percentage of cells with more than three Gal3 puncta is shown. Graph represents data from three independent experiments with ≥ 50 cells per condition (mean ± SD). *P<0.05; **P<0.01 (One-way-ANOVA with Dunnett's multiple comparison test).


C Survival assay . Control or UBE2QL1 - depleted HeLa cells ( 48 h ) were treated with increasing concentrations of LLOMe as indicated . Cell viability was measured with the MTS assay . Graph represents data from one experiment including three replicates ( mean ± SD ) . * P < 0 . 05 ; * * * * P < 0 . 0001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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C Survival assay. Control or UBE2QL1-depleted HeLa cells (48 h) were treated with increasing concentrations of LLOMe as indicated. Cell viability was measured with the MTS assay. Graph represents data from one experiment including three replicates (mean ± SD). *P<0.05; ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).


A Control or UBE2QL1 - depleted ( for 60 h ) HeLa cells were stained with DAPI and an antibody against TFEB and automatically analyzed by confocal microscopy . Note nuclear translocation of TFEB upon loss of UBE2QL1 . Scale bar : 20µm .

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A Control or UBE2QL1-depleted (for 60 h) HeLa cells were stained with DAPI and an antibody against TFEB and automatically analyzed by confocal microscopy. Note nuclear translocation of TFEB upon loss of UBE2QL1. Scale bar: 20µm.


B Automated quantification The intensity ratio of the cytoplasmic and nuclear TFEB signals was determined in both conditions . Graph represents data from four independent experiments with ≥ 100 cells per condition ( mean ± SD ) . * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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

B Automated quantification The intensity ratio of the cytoplasmic and nuclear TFEB signals was determined in both conditions. Graph represents data from four independent experiments with ≥ 100 cells per condition (mean ± SD). ***P<0.001; ****P<0.0001 (One-way-ANOVA with Bonferroni's multiple comparison test).


C Cells were stained for LAMP1 and mTOR . Note the increase in lysosomes and lysosomal dissociation of mTOR in UBE2QL1 depleted cells . Scale bar : 20 µm .

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C Cells were stained for LAMP1 and mTOR. Note the increase in lysosomes and lysosomal dissociation of mTOR in UBE2QL1 depleted cells. Scale bar: 20 µm.


D Automated quantification The colocalization of mTOR and LAMP1 is shown as Pearson correlation coefficients ( P . C . C . ) of the signals . Graph represents data from three independent experiments with ≥ 70 cells per condition ( mean ± SD ) . * P < 0 . 05 ( One - way - ANOVA with Bonferroni ' s multiple comparison test ) .

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D Automated quantification The colocalization of mTOR and LAMP1 is shown as Pearson correlation coefficients (P.C.C.) of the signals. Graph represents data from three independent experiments with ≥ 70 cells per condition (mean ± SD). *P<0.05 (One-way-ANOVA with Bonferroni's multiple comparison test).


E Automated quantification Number of LAMP1 - positive vesicles per cell is shown . Graph represents data from three independent experiments with ≥ 70 cells per condition ( mean ± SD ) .

O O O O O O O O O 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 Automated quantification Number of LAMP1-positive vesicles per cell is shown. Graph represents data from three independent experiments with ≥ 70 cells per condition (mean ± SD).


F Western blot analysis of lysates of cells treated As a positive control , cells were treated with the mTOR - inhibitor Torin1 ( 1 µM ) for 2 h . GAPDH was probed as loading control .

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F Western blot analysis of lysates of cells treated As a positive control, cells were treated with the mTOR-inhibitor Torin1 (1 µM) for 2 h. GAPDH was probed as loading control.


G C . elegans wild - type ( N2 ) and mutant strains for ubc - 25 ( ok1732 ) , for scav - 3 ( qx193 ) or the ubc - 25 and scav - 3 double mutant ( ok1732 ; qx193 ) carrying a EGFP - Gal3 transgene were imaged in adult worms ( L4 + 24 h ) . 3D reconstructions of the hypodermis . Small and big units represent 1 and 5 µm , respectively .

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G C.elegans wild-type (N2) and mutant strains for ubc-25(ok1732), for scav-3(qx193) or the ubc-25 and scav-3 double mutant (ok1732;qx193) carrying a EGFP-Gal3 transgene were imaged in adult worms (L4 + 24 h). 3D reconstructions of the hypodermis. Small and big units represent 1 and 5 µm, respectively.


H Average volume of permeabilized lysosomes within the region of hypodermis corresponding to 100 µm of body length was quantified . At least 20 animals were scored in each strain . * P < 0 . 05 ; * * P < 0 . 01 ; * * * * P < 0 . 0001 ( One - way - ANOVA with Dunnett ' s multiple comparison test or Student ' s two - tailed unpaired t test ) .

H Average volume of permeabilized lysosomes within the region of hypodermis corresponding to 100 µm of body length was quantified. At least 20 animals were scored in each strain. *P<0.05; **P<0.01; ****P<0.0001 (One-way-ANOVA with Dunnett's multiple comparison test or Student's two-tailed unpaired t test).


I Populations of worms with different extent of lysosome permeabilization were analyzed . Graph represents percentage of worms with less than 5 , 5 to 20 or more than 20 permeabilized lysosomes in the region of hypodermis corresponding to 100 µm of body length .

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I Populations of worms with different extent of lysosome permeabilization were analyzed. Graph represents percentage of worms with less than 5, 5 to 20 or more than 20 permeabilized lysosomes in the region of hypodermis corresponding to 100 µm of body length.


B Measured free PF - 670 exposure in the brain tissue of mice and NHPs . Drug exposure in NHPs after administration of 10 mpk PF - 670 ( AUC = 3 . 6μMh ) is ~ 7 - fold higher than that in mice given 32 mpk PF - 670 ( AUC = 0 . 5 μMh ) ( mean ± SEM ) .

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

B Measured free PF-670 exposure in the brain tissue of mice and NHPs. Drug exposure in NHPs after administration of 10 mpk PF-670 (AUC=3.6μMh) is ~7-fold higher than that in mice given 32 mpk PF-670 (AUC=0.5 μMh) (mean±SEM).


C , D Double - plotted actograms of NHPs ' activity for 16 days . NHPs were treated with 10 mpk PF - 670 for 3 consecutive days at the same solar time of day : 4 pm for the DD experiment ( C ) and 4 : 30 pm ( ZT11 ) for the LD experiment ( D ) , respectively , which are highlighted as red lines . White and black rectangles indicate the times of light going on and off ( LD 12 : 12 ) .

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C, D Double-plotted actograms of NHPs' activity for 16 days. NHPs were treated with 10 mpk PF-670 for 3 consecutive days at the same solar time of day: 4 pm for the DD experiment (C) and 4:30 pm (ZT11) for the LD experiment (D), respectively, which are highlighted as red lines. White and black rectangles indicate the times of light going on and off (LD 12:12).


E , F Phase delay induced by the 3 - day dosing under DD ( E ) or LD ( F ) . The phase of activity onset in NHPs ( 10 mpk ) is more delayed than that in mice ( 32 mpk ) under DD ( E ) , but not under LD ( F ) ( P = 0 . 03 , one - way analysis of variance ( ANOVA ) ; n . s . , no significant difference ) . Veh denotes vehicle . The error bars represent mean ± SEM . The mouse data are adopted from ( Kim et al , 2013 ) . G The quantification of ( E ) and ( F ) indicates that light has a stronger attenuating effect on the PF - 670 - induced phase delay in NHPs than in mice ( P = 0 . 036 , two - way ANOVA ) .

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E, F Phase delay induced by the 3-day dosing under DD (E) or LD (F). The phase of activity onset in NHPs (10 mpk) is more delayed than that in mice (32 mpk) under DD (E), but not under LD (F) (P=0.03, one-way analysis of variance (ANOVA); n.s., no significant difference). Veh denotes vehicle. The error bars represent mean±SEM. The mouse data are adopted from (Kim et al, 2013). G The quantification of (E) and (F) indicates that light has a stronger attenuating effect on the PF-670-induced phase delay in NHPs than in mice (P=0.036, two-way ANOVA).


C Double - plotted actogram of NHPs ' activity for 16 days . The light is on at 6 am and off at 6 pm except for Days 8 to 10 when the light is on at 8 am and off at 8 pm ( i . e . LD is delayed by 2 h ) .

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C Double-plotted actogram of NHPs' activity for 16 days. The light is on at 6 am and off at 6 pm except for Days 8 to 10 when the light is on at 8 am and off at 8 pm (i.e. LD is delayed by 2 h).


D The model successfully predicts the phase shift induced by 2 h jet lag ( Days 8 - 10 ) and its recovery after jet lag ( Days 11 - 13 ) ( n = 7 ; mean ±

O O O O O O O O O O O O O O O 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 The model successfully predicts the phase shift induced by 2 h jet lag (Days 8-10) and its recovery after jet lag (Days 11-13) (n=7; mean±SEM


E NHPs were treated with 10 mpk PF - 670 for 3 consecutive days at ZT4 in LD 12 : 12 , which are highlighted as the red line .

E NHPs were treated with 10 mpk PF-670 for 3 consecutive days at ZT4 in LD 12:12, which are highlighted as the red line.


B The simulated PRCs of diurnal NHPs have a higher ratio of maximal magnitudes between advance and delay zones ( A / D = 1 . 25 ) than that of the experimentally measured nocturnal mouse PRC ( A / D = 0 . 41 Here , the red line and red range represent the mean ± SD of the simulated 12 h light PRCs of the NHP model with different gating and adaptation Similarly , the light PRC of diurnal humans has a higher A / D ratio than that of nocturnal

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B The simulated PRCs of diurnal NHPs have a higher ratio of maximal magnitudes between advance and delay zones (A/D=1.25) than that of the experimentally measured nocturnal mouse PRC (A/D=0.41 Here, the red line and red range represent the mean±SD of the simulated 12 h light PRCs of the NHP model with different gating and adaptation Similarly, the light PRC of diurnal humans has a higher A/D ratio than that of nocturnal mice


E Consistently , higher experimentally measured PER2 levels in the SCN at the dosing time leads to a larger PF - 670 - induced phase delay which was also captured by the NHP model when 20 mpk dosing was used .

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E Consistently, higher experimentally measured PER2 levels in the SCN at the dosing time leads to a larger PF-670-induced phase delay which was also captured by the NHP model when 20 mpk dosing was used.


( a ) Confocal time - lapse images of an MCF10A cell - in - cell structure expressing GFP - LC3 and H2B - mCherry . The arrow marks recruitment of LC3 to the entotic vacuole ( see Supplementary Movie S1 ) . Scale bar , 15 μm .

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(a) Confocal time-lapse images of an MCF10A cell-in-cell structure expressing GFP-LC3 and H2B-mCherry. The arrow marks recruitment of LC3 to the entotic vacuole (see Supplementary Movie S1). Scale bar, 15 μm.


( b ) Images of MCF10A cells expressing GFP - LC3G120A and mCherry - LC3 ; note no recruitment of GFP - LC3G120A to the entotic vacuole . Scale bar , 15 μm . ( c ) LC3 is recruited from the host - cell cytosol onto the entotic vacuole membrane . Data points are the mean fluorescence intensity of GFP - LC3 on vacuoles versus cytoplasm for two independent cell - in - cell structures ( see Supplementary Movie S2 ) .

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(b) Images of MCF10A cells expressing GFP-LC3G120A and mCherry-LC3; note no recruitment of GFP-LC3G120A to the entotic vacuole. Scale bar, 15 μm. (c) LC3 is recruited from the host-cell cytosol onto the entotic vacuole membrane. Data points are the mean fluorescence intensity of GFP-LC3 on vacuoles versus cytoplasm for two independent cell-in-cell structures (see Supplementary Movie S2).


( d ) Correlative video - light - electron microscopy of an MCF10A GFP - LC3 cell - in - cell structure . Cells were imaged by time - lapse microscopy , followed by fixation after LC3 recruitment ( see Supplementary Movie S3 ) . Top left , post - fixation images of phase - contrast and GFP - fluorescence signals were taken demonstrating LC3 recruitment . Bottom left and right , electron microscopy ( EM ) of the same cell shows the entotic vacuole is a single membrane ( arrow in right image , which is a higher magnification of the area outlined in the bottom left image )

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(d) Correlative video-light-electron microscopy of an MCF10A GFP-LC3 cell-in-cell structure. Cells were imaged by time-lapse microscopy, followed by fixation after LC3 recruitment (see Supplementary Movie S3). Top left, post-fixation images of phase-contrast and GFP-fluorescence signals were taken demonstrating LC3 recruitment. Bottom left and right, electron microscopy (EM) of the same cell shows the entotic vacuole is a single membrane (arrow in right image, which is a higher magnification of the area outlined in the bottom left image)


( e ) Percentage of MCF10A cell - in - cell structures , with and without autophagy inhibition , that show LC3 recruitment associated with death of the internalized cell . Data represent mean ± s . e . m . from at least three independent experiments ; n , total number of death events ; * * * P0 . 0001 .

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(e) Percentage of MCF10A cell-in-cell structures, with and without autophagy inhibition, that show LC3 recruitment associated with death of the internalized cell. Data represent mean±s.e.m. from at least three independent experiments; n, total number of death events; ***P0.0001.


( f , g ) Time - lapse images of MCF10A cell - in - cell structures expressing either 2 × FYVE - mCherry ( red ) and GFP - LC3 ( green ; f ) or Lamp1 - GFP ( green ) and mCherry - LC3 ( red ; g ) . The arrows indicate recruitment to the entotic vacuole ( see Supplementary Movies S4 and S5 ) . Times are indicated as h : min : s ( a , f ) or min ( g ) . Scale bars , 5 μm .

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(f,g) Time-lapse images of MCF10A cell-in-cell structures expressing either 2×FYVE-mCherry (red) and GFP-LC3 (green; f) or Lamp1-GFP (green) and mCherry-LC3 (red; g). The arrows indicate recruitment to the entotic vacuole (see Supplementary Movies S4 and S5). Times are indicated as h:min:s (a,f) or min (g). Scale bars, 5 μm.


( h ) Representative images of cell - in - cell structures in which only host cells express Cathepsin - B - mCherry . Left , a live inner cell with no Cathepsin B in the vacuole ( arrow ) ; middle , a live internalized cell with Cathepsin B from the host inside of the vacuole ( arrowhead ) ; right , a dead internalized cell with Cathepsin B from the host throughout the corpse ( asterisk ) . Scale bar , 15 μm .

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(h) Representative images of cell-in-cell structures in which only host cells express Cathepsin-B-mCherry. Left, a live inner cell with no Cathepsin B in the vacuole (arrow); middle, a live internalized cell with Cathepsin B from the host inside of the vacuole (arrowhead); right, a dead internalized cell with Cathepsin B from the host throughout the corpse (asterisk). Scale bar, 15 μm.


( i ) Five representative timings of GFP - LC3 recruitment to entotic vacuoles ( green bar ) and death of internalized cells ( cross ) from time - lapse microscopy . ( j ) Initiation and duration of 2 × FYVE domain ( red bar , n = 11 ) , LC3 ( green bar , n = 20 ) and Lamp1 ( orange bar , n = 7 ) recruitment to entotic vacuoles , determined using double - expressing cells shown in f , g . Error bars represent s . d .

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(i) Five representative timings of GFP-LC3 recruitment to entotic vacuoles (green bar) and death of internalized cells (cross) from time-lapse microscopy. (j) Initiation and duration of 2×FYVE domain (red bar, n=11), LC3 (green bar, n=20) and Lamp1 (orange bar, n=7) recruitment to entotic vacuoles, determined using double-expressing cells shown in f,g. Error bars represent s.d.


( a ) Left , model depicting the multiple cell fates of internalized cells ; the arrow thickness represents the relative frequency of events . Right , images of MCF10A cell - in - cell structures ( 1 , host cell ; 2 , internalized cell ) that show a cell death ( top images ) or release ( bottom images ) event . Scale bar , 15 μm .

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(a) Left, model depicting the multiple cell fates of internalized cells; the arrow thickness represents the relative frequency of events. Right, images of MCF10A cell-in-cell structures (1, host cell; 2, internalized cell) that show a cell death (top images) or release (bottom images) event. Scale bar, 15 μm.


( b ) Quantification of MCF10A internalized cell fate over 20 h following autophagy inhibition . Data represent the mean from at least three independent experiments ; n , total number of structures analysed ; * * * P0 . 007 , * * P0 . 004 , * P0 . 01 .

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(b) Quantification of MCF10A internalized cell fate over 20 h following autophagy inhibition. Data represent the mean from at least three independent experiments; n, total number of structures analysed; ***P0.007, **P0.004, *P0.01.


( c , d ) Quantification of LC3 recruitment ( c ) , and cell fate ( d ) , of mixed cell - in - cell structures for which host or internalized cells were treated with ATG5 siRNA . Data represent mean ± s . e . m . from three separate experiments ; n , total number of structures analysed ; * P0 . 01 , * * P0 . 004 .

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(c,d) Quantification of LC3 recruitment (c), and cell fate (d), of mixed cell-in-cell structures for which host or internalized cells were treated with ATG5 siRNA. Data represent mean±s.e.m.from three separate experiments; n, total number of structures analysed; *P0.01, **P0.004.


( e ) Left , quantification of type of internalized cell death after autophagy inhibition . Data represent the mean of three independent experiments ; n , total death events analysed ; * * P0 . 03 . Right , representative images of non - apoptotic and apoptotic death of an internalized cell ; the arrow points to fragmented apoptotic nuclei . Scale bars , 15 μm .

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(e) Left, quantification of type of internalized cell death after autophagy inhibition. Data represent the mean of three independent experiments; n, total death events analysed; **P0.03. Right, representative images of non-apoptotic and apoptotic death of an internalized cell; the arrow points to fragmented apoptotic nuclei. Scale bars, 15 μm.


( f ) Quantification of mCherry / GFP ratio of live MCF10A single cells or internalized cells expressing tandem GFP - mCherry - LC3 , with or without siRNA treatment as indicated . Data represent mean ± s . d . ; numbers of cells analysed for each data point ( from left to right ) 30 , 30 , 30 , 25 , 30 , 25 , 25 , 25 , 30 , 27 ; * P0 . 03 , * * P0 . 003 , * * * P0 . 0004 .

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(f) Quantification of mCherry/GFP ratio of live MCF10A single cells or internalized cells expressing tandem GFP-mCherry-LC3, with or without siRNA treatment as indicated. Data represent mean±s.d.; numbers of cells analysed for each data point (from left to right) 30, 30, 30, 25, 30, 25, 25, 25, 30, 27; *P0.03, **P0.003, ***P0.0004.


( g ) Quantification of autophagosome and autolysosome area in control MCF10A ( n = 10 ) and internalized cell ( n = 10 ) cytoplasm imaged by electron microscopy ; * * P0 . 005 . ( h ) Representative electron micrographs of cell - in - cell structures ( left ) and internalized cell cytoplasm ( middle and right ) showing autophagosomes ( arrow ) and autolysosomes ( arrowhead ) .

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(g) Quantification of autophagosome and autolysosome area in control MCF10A (n=10) and internalized cell (n=10) cytoplasm imaged by electron microscopy; **P0.005. (h) Representative electron micrographs of cell-in-cell structures (left) and internalized cell cytoplasm (middle and right) showing autophagosomes (arrow) and autolysosomes (arrowhead).


( a ) The fate of internalized cells ( control MCF10A and MCF10A expressing E7 and Bcl2 ) was measured with and without autophagy inhibition . Data show the percentage of internalized cell release with autophagy inhibition , compared with each control without autophagy inhibition ; mean ± s . e . m . from at least three independent experiments ( total cell numbers analysed : ATG5 siRNA , 261 cells ; Bcl2 ATG5 siRNA , 97 cells ; 3 - MA , 170 cells ; Bcl2 3 - MA , 112 cells ) ; * P0 . 05 , * * P0 . 02 .

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(a) The fate of internalized cells (control MCF10A and MCF10A expressing E7 and Bcl2) was measured with and without autophagy inhibition. Data show the percentage of internalized cell release with autophagy inhibition, compared with each control without autophagy inhibition; mean±s.e.m. from at least three independent experiments (total cell numbers analysed: ATG5 siRNA, 261 cells; Bcl2 ATG5 siRNA, 97 cells; 3-MA, 170 cells; Bcl2 3-MA, 112 cells); *P0.05, **P0.02.


( b ) Effects of Y27632 , 3 - MA and VPS34 and ATG5 siRNA on MCF10A cell - in - cell formation in suspension for 7 h ; > 300 cells per condition were scored for cell - in - cell formation ; ± s . e . m . from three independent experiments ; * * P0 . 004 .

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(b) Effects of Y27632, 3-MA and VPS34 and ATG5 siRNA on MCF10A cell-in-cell formation in suspension for 7 h; >300 cells per condition were scored for cell-in-cell formation; ±s.e.m.from three independent experiments; **P0.004.


( c ) Representative images of an MCF10A E7 + Bcl2 cell - in - cell structure formed in soft agar . The arrow marks LC3 recruitment around internalized cells . Scale bar , 5 μm .

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(c) Representative images of an MCF10A E7+Bcl2 cell-in-cell structure formed in soft agar. The arrow marks LC3 recruitment around internalized cells. Scale bar, 5 μm.


( d ) Quantification of MCF10A E7 + Bcl2 cell - in - cell structures 48 h after seeding into soft agar ( white bars ) , and colony formation after 2 weeks ( grey bars ) . Data represent mean ± s . e . m . from three independent experiments ; * P0 . 03 , * * P0 . 004 , * * * P0 . 001 . ( e ) Representative wells for data in d . The insets show higher magnifications .

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(d) Quantification of MCF10A E7+Bcl2 cell-in-cell structures 48 h after seeding into soft agar (white bars), and colony formation after 2 weeks (grey bars). Data represent mean±s.e.m.from three independent experiments; *P0.03, **P0.004, ***P0.001. (e) Representative wells for data in d. The insets show higher magnifications.


( f ) Quantification of MCF10A E7 + Bcl2 colony formation with Atg5 knockdown with two separate siRNAs . Data represent mean ± s . e . m . from three independent experiments ; * P0 . 001 . ( g ) Representative wells for data in f . The insets show higher magnifications . See also Supplementary Movie S6 .

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(f) Quantification of MCF10A E7+Bcl2 colony formation with Atg5 knockdown with two separate siRNAs. Data represent mean±s.e.m.from three independent experiments; *P0.001. (g) Representative wells for data in f. The insets show higher magnifications. See also Supplementary Movie S6.


( a ) Time - lapse images of MCF10A cell - in - cell structures with an internalized cell that undergoes apoptosis . The arrows mark the recruitment of LC3 around apoptotic fragments ( see Supplementary Movie S7 ) . Scale bar , 10 μm . ( b ) Quantification of LC3 recruitment to apoptotic internalized cells in the presence of ATG5 or FIP200 siRNA ; n , number of cells analysed ; * * * P0 . 0001 , chi - squared test .

(a) Time-lapse images of MCF10A cell-in-cell structures with an internalized cell that undergoes apoptosis. The arrows mark the recruitment of LC3 around apoptotic fragments (see Supplementary Movie S7). Scale bar, 10 μm. (b) Quantification of LC3 recruitment to apoptotic internalized cells in the presence of ATG5 or FIP200 siRNA; n, number of cells analysed; ***P0.0001, chi-squared test.


( c ) Apoptotic U937 cells expressing H2B - mCherry phagocytized by J774 macrophages expressing GFP - LC3 . Confocal images show LC3 recruitment to an engulfed corpse ( arrow ; see Supplementary Movie S8 ) . Scale bar , 15 μm . ( d ) Quantification of LC3 recruitment to apoptotic phagosomes in control and Atg5 - shRNA - expressing cells ; * * * P0 . 0001 , chi - squared test .

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(c) Apoptotic U937 cells expressing H2B-mCherry phagocytized by J774 macrophages expressing GFP-LC3. Confocal images show LC3 recruitment to an engulfed corpse (arrow; see Supplementary Movie S8). Scale bar, 15 μm. (d) Quantification of LC3 recruitment to apoptotic phagosomes in control and Atg5-shRNA-expressing cells; ***P0.0001, chi-squared test.


( e ) Correlative light - electron microscopy of an apoptotic phagosome with GFP - LC3 recruitment ( top left image , white arrow ) . The LC3 - loaded phagosome consists of a single membrane ( black arrow in right image , which is a higher magnification of the area outlined in the bottom left image ; EM , electron microscopy ; AP , apoptotic cell nucleus ) .

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(e) Correlative light-electron microscopy of an apoptotic phagosome with GFP-LC3 recruitment (top left image, white arrow). The LC3-loaded phagosome consists of a single membrane (black arrow in right image, which is a higher magnification of the area outlined in the bottom left image; EM, electron microscopy; AP, apoptotic cell nucleus).


( f ) Representative images of apoptotic cell phagocytosis and degradation in control ( n = 21 ) and Atg5 shRNA ( n = 21 ) J774 GFP - LC3 cells ; * * * P0 . 0001 , chi - squared test . Scale bar , 10 μm . See also Supplementary Fig . S3 .

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(f) Representative images of apoptotic cell phagocytosis and degradation in control (n=21) and Atg5 shRNA (n=21) J774 GFP-LC3 cells; ***P0.0001, chi-squared test. Scale bar, 10 μm. See also Supplementary Fig. S3.


( a ) Representative images of a J774 macrophage expressing GFP - LC3 incubated in media containing red dextran . The arrow marks LC3 recruitment to a dextran - containing macropinosome . Scale bar , 2 μm . ( b ) Quantification of LC3 recruitment to macropinosomes in control and Atg5 shRNA J774 macrophages ; n , number of macropinosomes analysed ; * * * P0 . 0001 , chi - squared test . See Supplementary Movie S9 .

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(a) Representative images of a J774 macrophage expressing GFP-LC3 incubated in media containing red dextran. The arrow marks LC3 recruitment to a dextran-containing macropinosome. Scale bar, 2 μm. (b) Quantification of LC3 recruitment to macropinosomes in control and Atg5 shRNA J774 macrophages; n, number of macropinosomes analysed; ***P0.0001, chi-squared test. See Supplementary Movie S9.


( c ) Representative image of LC3 recruitment to macropinosomes ( arrows ) in MCF10A cells expressing GFP - LC3 incubated with red dextran . Scale bar , 2 μm . ( d ) Quantification of LC3 recruitment to macropinosomes in MCF10A cells treated with siRNA against FIP200 or ATG5 ; n , number of macropinosomes analysed ; * * * P0 . 0001 , chi - squared test .

(c) Representative image of LC3 recruitment to macropinosomes (arrows) in MCF10A cells expressing GFP-LC3 incubated with red dextran. Scale bar, 2 μm. (d) Quantification of LC3 recruitment to macropinosomes in MCF10A cells treated with siRNA against FIP200 or ATG5; n, number of macropinosomes analysed; ***P0.0001, chi-squared test.


( e ) J774 GFP - LC3 macrophages were incubated with 3 μm uncoated latex beads and imaged by confocal microscopy for 1 h , followed by the addition of LysoTracker red to mark acidified compartments . A macropinosome recruited LC3 ( arrow ) whereas engulfed latex beads did not ( arrowheads ) . Scale bar , 5 μm . See Supplementary Movie S10 .

(e) J774 GFP-LC3 macrophages were incubated with 3 μm uncoated latex beads and imaged by confocal microscopy for 1 h, followed by the addition of LysoTracker red to mark acidified compartments. A macropinosome recruited LC3 (arrow) whereas engulfed latex beads did not (arrowheads). Scale bar, 5 μm. See Supplementary Movie S10.


( a ) A C . elegans embryo expressing mCherry : : RAB - 5 and GFP : : LGG - 1 . The arrows point to two apoptotic phagosomes . See Supplementary Movie S11 . Scale bar , 10 μm . ( b ) Cropped time - lapse images showing RAB - 5 and LGG - 1 recruitment to an apoptotic phagosome ( from a , left arrow ) .

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(a) A C. elegans embryo expressing mCherry::RAB-5 and GFP::LGG-1. The arrows point to two apoptotic phagosomes. See Supplementary Movie S11. Scale bar, 10 μm. (b) Cropped time-lapse images showing RAB-5 and LGG-1 recruitment to an apoptotic phagosome (from a, left arrow).


( c ) Representative images of apoptotic phagosomes in embryos from control - RNAi - or bec - 1 - RNAi - fed worms . The insets show higher magnifications of the areas outlined in the main panels . ( d ) Quantification of GFP : : LGG - 1 - and mCherry : : RAB - 5 - positive phagosomes in control - or bec - 1 - RNAi embryos determined by time - lapse imaging . Control , 21 embryos , 52 phagosomes ; bec - 1 , 9 embryos , 25 phagosomes ; * * * P0 . 0001 , chi - squared test .

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(c) Representative images of apoptotic phagosomes in embryos from control-RNAi- or bec-1-RNAi-fed worms. The insets show higher magnifications of the areas outlined in the main panels. (d) Quantification of GFP::LGG-1- and mCherry::RAB-5-positive phagosomes in control- or bec-1-RNAi embryos determined by time-lapse imaging. Control, 21 embryos, 52 phagosomes; bec-1, 9 embryos, 25 phagosomes; ***P0.0001, chi-squared test.


( e ) Representative DIC images from control - and bec - 1 - RNAi embryos at different stages of development ( minutes after first division ) . The yellow arrowheads mark apoptotic corpses . Scale bar , 10 μm . ( f ) Quantification of apoptotic corpses in control - and bec - 1 - RNAi embryos ; data show ± s . d . ; * * * P0 . 0001 .

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(e) Representative DIC images from control- and bec-1-RNAi embryos at different stages of development (minutes after first division). The yellow arrowheads mark apoptotic corpses. Scale bar, 10 μm. (f) Quantification of apoptotic corpses in control- and bec-1-RNAi embryos; data show ± s.d.; ***P0.0001.


( g ) Representative images of CED - 1 : : GFP embryos with control or bec - 1 RNAi . The yellow arrowheads mark engulfed corpses surrounded by CED - 1 : : GFP . Scale bar , 5 μm .

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(g) Representative images of CED-1::GFP embryos with control or bec-1 RNAi. The yellow arrowheads mark engulfed corpses surrounded by CED-1::GFP. Scale bar, 5 μm.


Immunoblot analysis of CEP55 expression in a panel of human breast cancer lines ( n = 25 ) Tubulin served as loading controls .

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Immunoblot analysis of CEP55 expression in a panel of human breast cancer lines (n=25) Tubulin served as loading controls.


Immunoblot analysis of CEP55 expression in progression series of MCF10A lines ( MCF10AT and MCF10CA ) , respectively . COX - IV served as loading controls .

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Immunoblot analysis of CEP55 expression in progression series of MCF10A lines (MCF10AT and MCF10CA), respectively. COX-IV served as loading controls.


A panel of selected breast cancer and near normal cell lines was reverse - transfected with 5 nM pooled CEP55 siRNAs and cell viability determined after 6 days . Cell viability relative to its own respective control transfected with scramble siRNA was calculated . Graph represents the mean ± SEM of three independent experiments .

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A panel of selected breast cancer and near normal cell lines was reverse-transfected with 5 nM pooled CEP55 siRNAs and cell viability determined after 6 days. Cell viability relative to its own respective control transfected with scramble siRNA was calculated. Graph represents the mean± SEM of three independent experiments.


Immunoblot analysis of CEP55 expression in CEP55 knockdown MDA - MB - 231 cells . Two isogenic lines ( sh # 2 and sh # 8 ) were obtained using two different shRNA sequences as described in method section . COX - IV served as loading control .

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Immunoblot analysis of CEP55 expression in CEP55 knockdown MDA-MB-231 cells. Two isogenic lines (sh#2 and sh#8) were obtained using two different shRNA sequences as described in method section. COX-IV served as loading control.


Effect of CEP55 knockdown on cell proliferation in MDA - MB - 231 cells assessed using the IncuCyte ZOOM ® live cell imager ( phase - only processing module ) . The percentage of cell confluence was determined using an IncuCyte mask analyzer . Graph represents the mean ± SEM of three independent experiments .

Effect of CEP55 knockdown on cell proliferation in MDA-MB-231 cells assessed using the IncuCyte ZOOM® live cell imager (phase-only processing module). The percentage of cell confluence was determined using an IncuCyte mask analyzer. Graph represents the mean±SEM of three independent experiments.


Representative images of colony forming capacity at 14 days determined using crystal violet staining in control and CEP55 knockdown MDA - MB - 231 cells .

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Representative images of colony forming capacity at 14 days determined using crystal violet staining in control and CEP55 knockdown MDA-MB-231 cells.


Six - week - old female NOD / SCID cohorts of mice were injected in the 4th inguinal mammary fat - pad with the control and CEP55 knockdown cells . Growth rate ( area , mm2 ) of the tumors was measured using digital calliper . Differences in growth were determined using Student ' s t test . Graph represents the mean tumor area ± SEM , n = 6 mice / group .

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Six-week-old female NOD/SCID cohorts of mice were injected in the 4th inguinal mammary fat-pad with the control and CEP55 knockdown cells. Growth rate (area, mm2) of the tumors was measured using digital calliper. Differences in growth were determined using Student's t test. Graph represents the mean tumor area±SEM, n=6 mice/group.


Immunoblots analysis of CEP55 protein expression in MCF10A cells stably transfected with a Flag - CEP55 expression construct and two isogenic lines were obtained ( CEP55 - # 16 and CEP55 - # 17 ) with its respective empty vector ( EV ) transfected parental cells . COX - IV served as a loading control .

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Immunoblots analysis of CEP55 protein expression in MCF10A cells stably transfected with a Flag-CEP55 expression construct and two isogenic lines were obtained (CEP55-#16 and CEP55-#17) with its respective empty vector (EV) transfected parental cells. COX-IV served as a loading control.


Effect of CEP55 overexpression on cell proliferation in MCF10A assessed using the IncuCyte ZOOM ® live cell imager as described in panel E . Graph represents the mean ± SEM of two independent experiments .

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Effect of CEP55 overexpression on cell proliferation in MCF10A assessed using the IncuCyte ZOOM® live cell imager as described in panel E. Graph represents the mean±SEM of two independent experiments.


Representative images of colony forming capacity at 14 days determined using crystal violet staining in empty vector ( EV ) and CEP55 - overexpressing ( CEP55 # 17 and CEP55 # 18 ) lines .

Representative images of colony forming capacity at 14 days determined using crystal violet staining in empty vector (EV) and CEP55-overexpressing (CEP55#17 and CEP55#18) lines.


Representative images of single plane phase - contrast and Z - stacked immunofluorescence of empty vector ( EV ) and CEP55 - overexpressing MCF10A cells grown on Matrigel for 14 days . Red : Cytokeratin - 19 ; Green : F - actin stained by Phallodin and Blue : DAPI . Z - stack images were acquired through Zeiss LSM 780 confocal microscope - ZMBH .

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Representative images of single plane phase-contrast and Z-stacked immunofluorescence of empty vector (EV) and CEP55-overexpressing MCF10A cells grown on Matrigel for 14 days. Red: Cytokeratin-19; Green: F-actin stained by Phallodin and Blue: DAPI. Z-stack images were acquired through Zeiss LSM 780 confocal microscope-ZMBH.


( A ) Polyploidy analysis ( > 4N DNA contents ) determined using FACS in control and CEP55 knockdown MDA - MB - 231 cells ( left panel ) . Representative corresponding cytogram showing different phases of cell cycle and the polyploidy subpopulation analyzed using ModFit LT 4 . 0 software ( right panel ) . Yellow peaks represent subpopulation of polyploidy / aneuploidy . Graph represents the mean ± SEM of three independent experiments .

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(A) Polyploidy analysis (>4N DNA contents) determined using FACS in control and CEP55 knockdown MDA-MB-231 cells (left panel). Representative corresponding cytogram showing different phases of cell cycle and the polyploidy subpopulation analyzed using ModFit LT 4.0 software (right panel). Yellow peaks represent subpopulation of polyploidy/aneuploidy. Graph represents the mean±SEM of three independent experiments.


( B ) Analysis of chromosome number in control and CEP55 knockdown MDA - MB - 231 cells ( sh # 8 ) by Giemsa staining of metaphase spreads ( left panel ) . Representative images showing chromosome spreads assessed by Zeiss AxioScope2 microscopy ( right panel ) . n = number of metaphase spreads counted ( mean ± 95 % confidence interval of two independent experiments ) .

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(B) Analysis of chromosome number in control and CEP55 knockdown MDA-MB-231 cells (sh#8) by Giemsa staining of metaphase spreads (left panel). Representative images showing chromosome spreads assessed by Zeiss AxioScope2 microscopy (right panel). n= number of metaphase spreads counted (mean ±95% confidence interval of two independent experiments).


( C ) Representative SNP array plots showing chromosomal alterations following CEP55 - knockdown in MDA - MB - 231 cells . BAF is B allele frequency , value between 0 and 1 and represents the proportion contributed by one SNP allele ( B ) to the total copy number : BAF is an estimate of NB / ( NA + NB ) , where NA and NB are the number of A and B alleles , respectively .

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(C) Representative SNP array plots showing chromosomal alterations following CEP55-knockdown in MDA-MB-231 cells. BAF is B allele frequency, value between 0 and 1 and represents the proportion contributed by one SNP allele (B) to the total copy number: BAF is an estimate of NB/(NA+ NB), where NA and NB are the number of A and B alleles, respectively.


( D ) Percentage of genome altered evaluated using amplified copies ( 6 - 8 copies across genome ) in both parental and CEP55 knockdown cells determined using data from SNP arrays . Graph represents the mean ± SEM of two independent experiments .

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(D) Percentage of genome altered evaluated using amplified copies (6-8 copies across genome) in both parental and CEP55 knockdown cells determined using data from SNP arrays. Graph represents the mean±SEM of two independent experiments.


( E ) SNP array plots showing chromosomal alterations following CEP55 overexpression in MCF10A cells . Red box indicates an extra chromosome 20 in CEP55 - overexpressing cells .

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(E) SNP array plots showing chromosomal alterations following CEP55 overexpression in MCF10A cells. Red box indicates an extra chromosome 20 in CEP55-overexpressing cells.


( G ) Kaplan - Meier survival analysis of METABRIC data showing the relationship between CEP55 expression and survival . Cases were subdivided into tertiles . Log - rank ( Mantel - Cox ) test , P < 0 . 0001 .

(G) Kaplan-Meier survival analysis of METABRIC data showing the relationship between CEP55 expression and survival. Cases were subdivided into tertiles. Log-rank (Mantel-Cox) test, P< 0.0001.


( A ) Representative cytogram of control and CEP55 knockdown MDA - MB - 231 cells showing cell cycle profiles following treatment with and without the B12536 ( 5 nM ) .

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(A) Representative cytogram of control and CEP55 knockdown MDA-MB-231 cells showing cell cycle profiles following treatment with and without the B12536 (5 nM).


Percentage of polyploidy ( > 4N ) following B12536 ( 5 nM ) in control and CEP55 knockdown MDA - MB - 231 cells . Graph represents the mean ± SEM of three independent experiments .

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Percentage of polyploidy (>4N) following B12536 (5 nM) in control and CEP55 knockdown MDA-MB-231 cells. Graph represents the mean±SEM of three independent experiments.


Percentage of polyploidy ( > 4N ) following nocodazole ( 0 . 5 µM ) in control and CEP55 knockdown MDA - MB - 231 cells . Graph represents the mean ± SEM of three independent experiments .

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Percentage of polyploidy (>4N) following nocodazole (0.5 µM) in control and CEP55 knockdown MDA-MB-231 cells. Graph represents the mean±SEM of three independent experiments.


Apoptotic fraction ( Sub - G1 population ) determined by propidium iodide staining of cells treated with PLK1i Graph represents the mean ± SEM of three independent experiments .

Apoptotic fraction (Sub-G1 population) determined by propidium iodide staining of cells treated with PLK1i Graph represents the mean±SEM of three independent experiments.


Apoptotic fraction ( Sub - G1 population ) determined by propidium iodide staining of cells treated with Nocodazole Graph represents the mean ± SEM of three independent experiments .

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Apoptotic fraction (Sub-G1 population) determined by propidium iodide staining of cells treated with Nocodazole Graph represents the mean±SEM of three independent experiments.


( F ) Percentage of sub - G1 fraction following B12536 ( 10 nM ) or nocodazole ( 0 . 5 µM ) in control and CEP55 knockdown Hs578T cells . Graph represents the mean ± SEM of two independent experiments .

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(F) Percentage of sub-G1 fraction following B12536 (10 nM) or nocodazole (0.5 µM) in control and CEP55 knockdown Hs578T cells. Graph represents the mean±SEM of two independent experiments.


( G ) Representative cytogram of empty vector ( EV ) and CEP55 - overexpressing MCF10A cells showing cell cycle profile following treatment with and without the B12536 ( 10 nM ) .

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(G) Representative cytogram of empty vector (EV) and CEP55-overexpressing MCF10A cells showing cell cycle profile following treatment with and without the B12536 (10 nM).


( H ) Percentage of polyploidy following treatment with and without B12536 ( 10 nM ) in empty vector ( EV ) and CEP55 - overepressing MCF10A cells . Graph represents the mean ± SEM of three independent experiments .

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(H) Percentage of polyploidy following treatment with and without B12536 (10 nM) in empty vector (EV) and CEP55-overepressing MCF10A cells. Graph represents the mean±SEM of three independent experiments.


( I ) Corresponding sub - G1 population determined using propidium iodide staining of cells treated with B12536 as described in panel H . Graph represents the mean ± SEM of three independent experiments .

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(I) Corresponding sub-G1 population determined using propidium iodide staining of cells treated with B12536 as described in panel H. Graph represents the mean±SEM of three independent experiments.


( A ) Box and whiskers plot showing average time spent in mitosis and ( B ) mitotic outcomes in control and CEP55 knockdown MDA - MB - 231 cells following treatment with the BI2536 ( 5 nM ) . Time taken to complete mitosis was defined as the time from nuclear envelope breakdown until two daughter cells were observed whereas mitotic slippage or death was defined as cells that prematurely exited mitosis with a flattened and a multinucleated phenotype or died during mitosis , characterized by membrane blebbing . Graph represents the mean ± SEM of two independent experiments . For each experiments n = 50 mitotic cells were counted per condition using Olympus Xcellence IX81 time - lapse microscopy .

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(A) Box and whiskers plot showing average time spent in mitosis and (B) mitotic outcomes in control and CEP55 knockdown MDA-MB-231 cells following treatment with the BI2536 (5 nM). Time taken to complete mitosis was defined as the time from nuclear envelope breakdown until two daughter cells were observed whereas mitotic slippage or death was defined as cells that prematurely exited mitosis with a flattened and a multinucleated phenotype or died during mitosis, characterized by membrane blebbing. Graph represents the mean±SEM of two independent experiments. For each experiments n=50 mitotic cells were counted per condition using Olympus Xcellence IX81 time-lapse microscopy.


( C , D ) Cells were pre - treated for two hours with 50 µM of the pan Caspase inhibitor Z - VAD - FMK , followed by treatment with B12536 ( 5 nM ) , before assessment of cell fate ( C ) and an average time spent in mitosis ( D ) Graph represents the mean ± SEM of two independent experiments .

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(C, D) Cells were pre-treated for two hours with 50 µM of the pan Caspase inhibitor Z-VAD-FMK, followed by treatment with B12536 (5 nM), before assessment of cell fate (C) and an average time spent in mitosis (D) Graph represents the mean±SEM of two independent experiments.


( E ) Cells were synchronized using double thymidine then released into nocodazole ( 0 . 5 µM ) , and protein lysates were collected at the indicated time points . Immunoblot analysis was then performed to determine the expression and activity of mitotic regulators as indicated . Levels of phospho - MEKT286 and dephosphorylation of phospho - CDK1Y15 served as markers of Cdk1 activation / mitotic entry . COX - IV served as a loading control .

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(E) Cells were synchronized using double thymidine then released into nocodazole (0.5 µM), and protein lysates were collected at the indicated time points. Immunoblot analysis was then performed to determine the expression and activity of mitotic regulators as indicated. Levels of phospho-MEKT286 and dephosphorylation of phospho-CDK1Y15 served as markers of Cdk1 activation/mitotic entry. COX-IV served as a loading control.


( G ) Representative mCerulean lifetimes maps of Hs578T control and CEP55 knockdown cells upon nocodazole ( 0 . 5 µM ) treatment at indicated time points . Cells were synchronized using double thymidine for 16 hours prior to nocodazole treatment .

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(G) Representative mCerulean lifetimes maps of Hs578T control and CEP55 knockdown cells upon nocodazole (0.5 µM) treatment at indicated time points. Cells were synchronized using double thymidine for 16 hours prior to nocodazole treatment.


( H ) Quantification of mCerulean lifetimes of control and Hs578T CEP55 knockdow ncells and in response to treatment with nocodazole at indicated time points as shown in G . Blue : 1 ns ; red : 3 ns . Graph represents the mean ± SEM of three independent experiments .

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(H) Quantification of mCerulean lifetimes of control and Hs578T CEP55 knockdow ncells and in response to treatment with nocodazole at indicated time points as shown in G. Blue: 1 ns; red: 3 ns. Graph represents the mean±SEM of three independent experiments.


( I ) Control and CEP55 knockdown MDA - MB - 231 cells were synchronized as above , released into culture medium for six hours and then blocked with nocodazole ( 0 . 5 µM ) for an additional four hours prior to treatment with the Cdk1 inhibitor RO - 3306 for an additional 16 hours . Sub - G1 cells were then identified by propidium iodide staining and quantified by FACS . Graph represents the mean ± SEM of two independent experiments .

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(I) Control and CEP55 knockdown MDA-MB-231 cells were synchronized as above, released into culture medium for six hours and then blocked with nocodazole (0.5 µM) for an additional four hours prior to treatment with the Cdk1 inhibitor RO-3306 for an additional 16 hours. Sub-G1 cells were then identified by propidium iodide staining and quantified by FACS. Graph represents the mean±SEM of two independent experiments.


( J ) Control and CEP55 knockdown MDA - MB - 231 cells were transfected with MAD2 siRNA ( 5 nM ) for 48 hours followed by nocodazole ( 0 . 5 µM ) treatment for an additional 12 hours . Cells were collected , fixed and sub - G1 analysis was performed as described in methods . Graph represents the mean ± SEM of two independent experiments .

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(J) Control and CEP55 knockdown MDA-MB-231 cells were transfected with MAD2 siRNA (5 nM) for 48 hours followed by nocodazole (0.5 µM) treatment for an additional 12 hours. Cells were collected, fixed and sub-G1 analysis was performed as described in methods. Graph represents the mean±SEM of two independent experiments.