( c ) Lysozyme IHC demonstrating increased lysozyme positive cell numbers in tumours from Lgr5 Apc Huwe1 mice . Scale bars = 200 µm ( low magnification ) , 100 µm ( high magnification inset ) . ( d ) OLFM4 IHC demonstrating expanded stem cell population in Lgr5 Apc Huwe1 tumours . Scale bars = 200 µm ( low magnification ) , 100 µm ( high magnification inset ) .

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(c) Lysozyme IHC demonstrating increased lysozyme positive cell numbers in tumours from Lgr5 Apc Huwe1 mice. Scale bars = 200 µm (low magnification), 100 µm (high magnification inset). (d) OLFM4 IHC demonstrating expanded stem cell population in Lgr5 Apc Huwe1 tumours. Scale bars = 200 µm (low magnification), 100 µm (high magnification inset).


( a ) MCL1 Western blot in protein extracts from Vil Apc and Vil Apc Huwe1hom tumours . Levels of MCL1 protein are significantly increased in tumours lacking HUWE1 ( Mann Whitney , p = 0 . 04 , n = 3 ) .

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(a) MCL1 Western blot in protein extracts from Vil Apc and Vil Apc Huwe1hom tumours. Levels of MCL1 protein are significantly increased in tumours lacking HUWE1 (Mann Whitney, p = 0.04, n = 3).


( b ) Kaplan - Meier survival plot of cohorts of induced Lgr5 Apc Huwe1 and Lgr5 Apc Huwe1 Mcl1 mice . Deletion of one copy of Mcl1 led to a significant increase in survival of these animals ( Log Rank , p = 0 . 0052 , n = 9 vs 10 ) .

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(b) Kaplan-Meier survival plot of cohorts of induced Lgr5 Apc Huwe1 and Lgr5 Apc Huwe1 Mcl1 mice. Deletion of one copy of Mcl1 led to a significant increase in survival of these animals (Log Rank, p = 0.0052, n = 9 vs 10).


( c ) Ratio of adenoma / indolent lesions observed in Lgr5 Apc Huwe1 and Lgr5 Apc Huwe1 Mcl1het mice . Note the decreased ration of adenoma / indolent lesions observed upon Mcl1 deletion ( Mann - Whitney , p = 0 . 0281 , n = 8 v 8 ) .

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(c) Ratio of adenoma / indolent lesions observed in Lgr5 Apc Huwe1 and Lgr5 Apc Huwe1 Mcl1het mice. Note the decreased ration of adenoma / indolent lesions observed upon Mcl1 deletion (Mann-Whitney, p=0.0281, n = 8 v 8).


A ) GST pulldown used to analyse TBC1D14 interactors . Recombinant GST or GST - TBC1D14 was incubated with or without HEK 293A lysate . Bound proteins were eluted from the beads using Laemmli sample buffer and subjected to SDS - PAGE , with visible bands being excised from the gel and analysed by mass spectrometry .

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A) GST pulldown used to analyse TBC1D14 interactors. Recombinant GST or GST-TBC1D14 was incubated with or without HEK 293A lysate. Bound proteins were eluted from the beads using Laemmli sample buffer and subjected to SDS-PAGE, with visible bands being excised from the gel and analysed by mass spectrometry.


B ) Lysates of HEK293A cells expressing either GFP or GFP - TBC1D14 were subjected to immunoprecipitation ( IP ) with GFP - Trap , and then immunoblotted for GFP , TRAPPC12 and TRAPPC4 .

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B) Lysates of HEK293A cells expressing either GFP or GFP-TBC1D14 were subjected to immunoprecipitation (IP) with GFP-Trap, and then immunoblotted for GFP, TRAPPC12 and TRAPPC4.


C ) Lysates of cells expressing GFP or GFP - TRAPPC3 were subjected to IP with GFP - Trap , and then immunoblotted for TRAPPC4 , GFP and TBC1D14 .

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C) Lysates of cells expressing GFP or GFP-TRAPPC3 were subjected to IP with GFP-Trap, and then immunoblotted for TRAPPC4, GFP and TBC1D14.


D ) Cells expressing GFP - TBC1D14 , generating a tubulated endosomal compartment , were fed Alexa647 labelled Transferrin ( white , blue in merge ) for 15 minutes in full medium , fixed , stained for endogenous TRAPPC4 ( red ) and analysed by confocal microscopy . White arrowheads indicate transfected cells , yellow arrowheads depict regions of colocalisation in inset . Scale bars = 10 μm .

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D) Cells expressing GFP-TBC1D14, generating a tubulated endosomal compartment, were fed Alexa647 labelled Transferrin (white, blue in merge) for 15 minutes in full medium, fixed, stained for endogenous TRAPPC4 (red) and analysed by confocal microscopy. White arrowheads indicate transfected cells, yellow arrowheads depict regions of colocalisation in inset. Scale bars = 10 μm.


E ) Cells treated as in D were stained for endogenous TRAPPC12 ( red ) and analysed by confocal microscopy . Scale bars = 10

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E) Cells treated as in D were stained for endogenous TRAPPC12 (red) and analysed by confocal microscopy. Scale bars = 10 μm


F ) Cells in complete medium were fixed , stained for endogenous TBC1D14 ( green ) , RAB1B ( red ) and TRAPPC4 ( white , blue in merge ) and analysed by confocal microscopy . Inset shows protein localisation in juxtanuclear area . Yellow arrowheads show regions of colocalisation Scale bars = 10 μm . Data shown are representative from 3 independent experiments .

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F) Cells in complete medium were fixed, stained for endogenous TBC1D14 (green), RAB1B (red) and TRAPPC4 (white, blue in merge) and analysed by confocal microscopy. Inset shows protein localisation in juxtanuclear area. Yellow arrowheads show regions of colocalisation Scale bars = 10 μm. Data shown are representative from 3 independent experiments.


A ) HEK293A cells transfected with GFP ( upper panels ) or GFP - TBC1D14 ( lower panels ) ( green ) were labelled with anti - RAB11A ( red ) and anti - RAB1B ( white , blue in merge ) antibodies . Arrows depict transfected cells . Inset : GFP - TBC1D14 transfected cell showing triple co - localisation between GFP - TBC1D14 , RAB11A and RAB1B ( white arrows ) and double co - localisation between GFP - TBC1D14 and RAB11A ( yellow arrows ) .

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A) HEK293A cells transfected with GFP (upper panels) or GFP-TBC1D14 (lower panels) (green) were labelled with anti-RAB11A (red) and anti-RAB1B (white, blue in merge) antibodies. Arrows depict transfected cells. Inset: GFP-TBC1D14 transfected cell showing triple co-localisation between GFP-TBC1D14, RAB11A and RAB1B (white arrows) and double co-localisation between GFP-TBC1D14 and RAB11A (yellow arrows).


B ) Co - localisation of GFP - TBC1D14 , TRAPPC4 ( red ) and RAB11A ( white , blue in merge ) . White arrowheads indicate regions of triple co - localisation .

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B) Co-localisation of GFP-TBC1D14, TRAPPC4 (red) and RAB11A (white, blue in merge). White arrowheads indicate regions of triple co-localisation.


C ) Co - localisation of GFP - TBC1D14 , TRAPPC4 ( red ) and RAB1B ( white , blue in merge ) . White arrowheads indicate regions of triple co - localisation , yellow arrowheads regions of double co - localisation between GFP - TBC1D14 and TRAPPC4 . All scale bars = 20 μm .

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C) Co-localisation of GFP-TBC1D14, TRAPPC4 (red) and RAB1B (white, blue in merge). White arrowheads indicate regions of triple co-localisation, yellow arrowheads regions of double co-localisation between GFP-TBC1D14 and TRAPPC4. All scale bars = 20 μm.


B ) HEK 293A cell lysates ( from approximately 8 × 106 cells per construct ) expressing the indicated GFP fusions were subjected to IP and immunoblotting for GFP , TRAPPC12 and TRAPPC4 . Data are representative of 3 independent experiments .

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B) HEK 293A cell lysates (from approximately 8 × 106 cells per construct) expressing the indicated GFP fusions were subjected to IP and immunoblotting for GFP, TRAPPC12 and TRAPPC4. Data are representative of 3 independent experiments.


A ) Cells expressing the indicated GFP - fusions ( green ) were loaded with Alexa647 - Transferrin ( red ; arrowheads indicate transfected cells ) for 15 minutes , fixed and the morphology of the transferrin positive recycling endosomes analysed by confocal microscopy . Scale bars = 10 μm . Arrows in inset indicate the juxtanuclear

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A) Cells expressing the indicated GFP-fusions (green) were loaded with Alexa647-Transferrin (red; arrowheads indicate transfected cells) for 15 minutes, fixed and the morphology of the transferrin positive recycling endosomes analysed by confocal microscopy. Scale bars = 10 μm. Arrows in inset indicate the juxtanuclear ERC


B ) Cells transfected as in ( A ) were loaded with Alexa - 647 transferrin for 15 minutes and the fluorescent transferrin chased out for the indicated time periods . At the end of the timecourse the cells were fixed and the transferrin content of GFP positive cells analysed by flow cytometry . Results are expressed as a percentage of Tfn fluorescence at t = 0 and are the mean of 3 independent experiments , ± s . e . m .

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B) Cells transfected as in (A) were loaded with Alexa-647 transferrin for 15 minutes and the fluorescent transferrin chased out for the indicated time periods. At the end of the timecourse the cells were fixed and the transferrin content of GFP positive cells analysed by flow cytometry. Results are expressed as a percentage of Tfn fluorescence at t=0 and are the mean of 3 independent experiments, ± s.e.m.


C ) Cells transfected as in ( A ) were stained for the cis - Golgi marker GM130 . Arrowheads depict transfected cells . Scale bars = 10 μm . Bar chart is quantification of fragmented Golgi complex ( > 100 cells per condition from three independent experiments , ± s . e . m . * * p < 0 . 01 , one way ANOVA with Sidak ' s post - test ) .

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C) Cells transfected as in (A) were stained for the cis-Golgi marker GM130. Arrowheads depict transfected cells. Scale bars = 10 μm. Bar chart is quantification of fragmented Golgi complex (> 100 cells per condition from three independent experiments, ± s.e.m. ** p < 0.01, one way ANOVA with Sidak's post-test).


D ) HeLa C1 cells transfected with CFP or CFP - TBR were treated with D / D solubiliser ( Clontech ) or D / D solubiliser plus 2 . 5 μg / ml Brefeldin A ( BFA ) for the indicated time periods , trypsinised , fixed and their GFP content analysed by flow cytometry . Results are expressed as a percentage of GFP levels at t = 0 , and the mean of 3 independent experiments ± s . e . m .

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D) HeLa C1 cells transfected with CFP or CFP-TBR were treated with D/D solubiliser (Clontech) or D/D solubiliser plus 2.5 μg/ml Brefeldin A (BFA) for the indicated time periods, trypsinised, fixed and their GFP content analysed by flow cytometry. Results are expressed as a percentage of GFP levels at t=0, and the mean of 3 independent experiments ± s.e.m.


A ) HEK293A cells expressing GFP , GFP - TBC1D14 224 - 669 or GFP - TBR were treated in duplicate with EBSS , EBSS plus 100 nM BafA1 or not for two hours , lysed and subjected to immunoblotting for LC3B , tubulin and GFP . The amount of LC3B / tubulin for each condition from three independent experiments is shown on the bar graph , ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , one way ANOVA with Sidak ' s multiple comparison test .

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A) HEK293A cells expressing GFP, GFP-TBC1D14 224-669 or GFP-TBR were treated in duplicate with EBSS, EBSS plus 100 nM BafA1 or not for two hours, lysed and subjected to immunoblotting for LC3B, tubulin and GFP. The amount of LC3B/tubulin for each condition from three independent experiments is shown on the bar graph, ±s.e.m. * p<0.05, ** p<0.01, one way ANOVA with Sidak's multiple comparison test.


B ) HEK293A GFP - LC3B ( 2GL9 ) cells were transfected with mCherry or mCherry - TBR , treated with EBSS for two hours , fixed and analysed by confocal microscopy .

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B) HEK293A GFP-LC3B (2GL9) cells were transfected with mCherry or mCherry-TBR, treated with EBSS for two hours, fixed and analysed by confocal microscopy.


C ) HEK293A cells were treated as in ( B ) , fixed , stained for WIPI2 and analysed by confocal microscopy .

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C) HEK293A cells were treated as in (B), fixed, stained for WIPI2 and analysed by confocal microscopy.


D ) HEK293A cells stably expressing GFP - DFCP1 were treated as in ( B ) , fixed and analysed by confocal microscopy . Scale bars = 20 μm . For quantification of ( B ) , ( C ) and ( D ) , 10 fields of view containing transfected cells were imaged for each of three experiments , and the number of GFP - LC3B , WIPI2 or GFP - DFCP1 puncta per cell enumerated using Imaris software ( Bitplane ) .

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D) HEK293A cells stably expressing GFP-DFCP1 were treated as in (B), fixed and analysed by confocal microscopy. Scale bars = 20 μm. For quantification of (B), (C) and (D), 10 fields of view containing transfected cells were imaged for each of three experiments, and the number of GFP-LC3B, WIPI2 or GFP-DFCP1 puncta per cell enumerated using Imaris software (Bitplane).


B ) Samples prepared according to ( A ) ( 8 × 107 HEK293A cells per transfection ) were lysed in TNTE , split equally and denatured with 1 % SDS ( D ) or not ( ND ) and subjected to 1 hour streptavidin resin pulldown at room temperature . The bound proteins were eluted in 2x Laemmli sample buffer plus 3 mM Biotin and immunoblotted for the indicated proteins .

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B) Samples prepared according to (A) (8 × 107 HEK293A cells per transfection) were lysed in TNTE, split equally and denatured with 1% SDS (D) or not (ND) and subjected to 1 hour streptavidin resin pulldown at room temperature. The bound proteins were eluted in 2x Laemmli sample buffer plus 3 mM Biotin and immunoblotted for the indicated proteins.


C ) siRNA transfected control cells ( RISC Free ) or TRAPPC8 knockdown ( TRAPPC8 ) ( 1x10cm plate per condition ) were lysed in TNTE and used in pulldown assays with immobilised recombinant GST or GST - TBR . Bound proteins were subjected to immunoblot analysis using TRAPPC8 , TRAPPC4 and tubulin antibodies . Ponceau staining was used to verify loading of the GST fusion proteins ( GST - TBR indicated with arrowhead ) . The bar chart expresses the relative amount of TRAPPC4 isolated from siTRAPPC8 compared to siRISC Free control cells from three independent experiments , error bars are ± s . e . m . * * p < 0 . 01 , unpaired T -

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C) siRNA transfected control cells (RISC Free) or TRAPPC8 knockdown (TRAPPC8) (1x10cm plate per condition) were lysed in TNTE and used in pulldown assays with immobilised recombinant GST or GST-TBR. Bound proteins were subjected to immunoblot analysis using TRAPPC8, TRAPPC4 and tubulin antibodies. Ponceau staining was used to verify loading of the GST fusion proteins (GST-TBR indicated with arrowhead). The bar chart expresses the relative amount of TRAPPC4 isolated from siTRAPPC8 compared to siRISC Free control cells from three independent experiments, error bars are ± s.e.m. ** p<0.01, unpaired T-test


D ) HEK293T cells stably depleted of TRAPPC8 ( shC8 ) or not ( shCtrl ) were transfected with constructs encoding GFP or GFP - TRAPPC3 and subjected to GFP Trap IP . Precipitated proteins were immunoblotted for GFP , TRAPPC4 and TRAPPC8 . Bar chart : the amount of TRAPPC4 isolated was normalised to the amount of GFP - TRAPPC3 precipitated from three independent experiments and plotted ± s . e . m . , ns = non - significant , unpaired T - test .

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D) HEK293T cells stably depleted of TRAPPC8 (shC8) or not (shCtrl) were transfected with constructs encoding GFP or GFP-TRAPPC3 and subjected to GFP Trap IP. Precipitated proteins were immunoblotted for GFP, TRAPPC4 and TRAPPC8. Bar chart: the amount of TRAPPC4 isolated was normalised to the amount of GFP-TRAPPC3 precipitated from three independent experiments and plotted ± s.e.m., ns = non-significant, unpaired T-test.


A ) HEK293A cells treated with transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 and RAB11A and B ( knockdown confirmed by immunoblot analysis ) were fed with Alexa - 647 Transferrin for 15 minutes in full medium , and the fluorescent transferrin chased out for the indicated time periods . At the end of the time course , cells were trypsinised , fixed and the Alexa - 647 fluorescence analysed by flow cytometry . Results are plotted as percentage of transferrin fluorescence at t = 0 for the RISC free control cells , expressed as the mean of three independent experiments ± s . e . m .

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A) HEK293A cells treated with transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 and RAB11A and B (knockdown confirmed by immunoblot analysis) were fed with Alexa-647 Transferrin for 15 minutes in full medium, and the fluorescent transferrin chased out for the indicated time periods. At the end of the time course, cells were trypsinised, fixed and the Alexa-647 fluorescence analysed by flow cytometry. Results are plotted as percentage of transferrin fluorescence at t=0 for the RISC free control cells, expressed as the mean of three independent experiments ± s.e.m.


B ) Cells transiently transfected with RISC Free control siRNA , or siRNA duplexes directed against TRAPPC8 or RAB1A and B were stained for the cis - Golgi markers GM130 or RAB1B and analysed by confocal microscopy . Scale bars = 20 μm in main panels , 10 μm in inset panels . Bar graph : > 100 cells per siRNA were scored for fragmented or normal juxtanuclear Golgi stacks from three independent experiments . Error bars ± s . e . m . , * p < 0 . 05 , * * p < 0 . 01 , one way ANOVA with Sidak ' s multiple comparison test .

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B) Cells transiently transfected with RISC Free control siRNA, or siRNA duplexes directed against TRAPPC8 or RAB1A and B were stained for the cis-Golgi markers GM130 or RAB1B and analysed by confocal microscopy. Scale bars = 20 μm in main panels, 10 μm in inset panels. Bar graph: > 100 cells per siRNA were scored for fragmented or normal juxtanuclear Golgi stacks from three independent experiments. Error bars ± s.e.m., * p<0.05, ** p<0.01, one way ANOVA with Sidak's multiple comparison test.


C ) HeLa C1 cells transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 or RAB1A and B ( knockdown confirmed by immunoblot analysis ) , were treated with D / D solubiliser for the indicated time periods , trypsinised , fixed and their GFP content analysed by flow cytometry . The line graph shows the GFP fluorescence as a percentage of t = 0 , ± s . e . m . from three independent experiments .

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C) HeLa C1 cells transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 or RAB1A and B (knockdown confirmed by immunoblot analysis), were treated with D/D solubiliser for the indicated time periods, trypsinised, fixed and their GFP content analysed by flow cytometry. The line graph shows the GFP fluorescence as a percentage of t=0, ± s.e.m. from three independent experiments.


A ) ATG9 positive membranes were immunoisolated from approximately 2 × 107 fed ( F ) or starved ( S ) HEK293A cells using a monoclonal hamster anti - ATG9 ( ATG9 ) or control hamster IgM and immunoblotted for ATG9 using a rabbit polyclonal antibody , TRAPPC8 , TRAPPC4 and actin .

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A) ATG9 positive membranes were immunoisolated from approximately 2 × 107 fed (F) or starved (S) HEK293A cells using a monoclonal hamster anti-ATG9 (ATG9) or control hamster IgM and immunoblotted for ATG9 using a rabbit polyclonal antibody, TRAPPC8, TRAPPC4 and actin.


B ) HEK293A cells transfected with GFP or GFP - TBR ( green ) were stained for ATG9 using hamster anti - ATG9 ( red ) and analysed by confocal microscopy . Scale bar = 20

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B) HEK293A cells transfected with GFP or GFP-TBR (green) were stained for ATG9 using hamster anti-ATG9 (red) and analysed by confocal microscopy. Scale bar = 20 μm


C ) HEK293A cells transiently transfected with RISC Free siRNA or siRNA against TRAPPC8 were stained for RAB1B ( green ) and ATG9 using hamster anti - ATG9 ( red ) and imaged using confocal microscopy .

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C) HEK293A cells transiently transfected with RISC Free siRNA or siRNA against TRAPPC8 were stained for RAB1B (green) and ATG9 using hamster anti-ATG9 (red) and imaged using confocal microscopy.


D ) Stills at t = 30 minutes from time lapse imaging of 9B9 cells ( stably expressing GFP - LC3 and mRFP - ATG9 ) expressing either CFP or CFP - TBR ( blue ) . Scale bars = 20 μm . Inset panels show mRFP - ATG9 and GFP - LC3 contact events at the indicated time points . Bar chart shows number of times a GFP - LC3 spot was within 1 μm of an mRFP - ATG9 spot ( a contact event ) , expressed as total mRFP - ATG9 contacts per GFP - LC3 spot , n = 18 cells per condition pooled from 4 independent experiments .

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D) Stills at t = 30 minutes from time lapse imaging of 9B9 cells (stably expressing GFP-LC3 and mRFP-ATG9) expressing either CFP or CFP-TBR (blue). Scale bars = 20 μm. Inset panels show mRFP-ATG9 and GFP-LC3 contact events at the indicated time points. Bar chart shows number of times a GFP-LC3 spot was within 1 μm of an mRFP-ATG9 spot (a contact event), expressed as total mRFP-ATG9 contacts per GFP-LC3 spot, n = 18 cells per condition pooled from 4 independent experiments.


A ) Cells depleted of ULK1 ( siULK1 ) or not ( RISC Free ) were transfected with GFP or GFP - TBR ( green ) , starved ( EBSS ) or not ( - EBSS ) and stained for ATG9 using hamster anti - ATG9 ( red ) . Arrows indicate transfected cells . Western blot indicates ULK1 knockdown . Scale bars = 20 μm .

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 B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O

A) Cells depleted of ULK1 (siULK1) or not (RISC Free) were transfected with GFP or GFP-TBR (green), starved (EBSS) or not (-EBSS) and stained for ATG9 using hamster anti-ATG9 (red). Arrows indicate transfected cells. Western blot indicates ULK1 knockdown. Scale bars = 20 μm.


B ) Cells depleted of ULK1 ( siULK1 ) or not ( RISC Free ) were transfected with myc - RAB1B or myc - RAB1B ( S22N ) ( green ) , starved ( EBSS ) or not ( - EBSS ) and stained for ATG9 using hamster anti - ATG9 ( red ) Arrows indicate transfected cells . Western blot indicates ULK1 knockdown . Scale bars = 20 μm .

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

B) Cells depleted of ULK1 (siULK1) or not (RISC Free) were transfected with myc-RAB1B or myc-RAB1B (S22N) (green), starved (EBSS) or not (-EBSS) and stained for ATG9 using hamster anti-ATG9 (red) Arrows indicate transfected cells. Western blot indicates ULK1 knockdown. Scale bars = 20 μm.


( B ) ParB clusters on F plasmid in vivo . Typical E . coli cells ( DLT3594 ) displays foci of ParBF - mVenus protein ( top ) expressed from the endogenous genetic locus of the F plasmid ( F1 - 10B - mVenus ) . The nucleoid is labelled with Hu - mCherry ( central ) . The overlay ( bottom ) combines the two fluorescent channels . Over 99 % of cells harbor ParBF foci . White bars : 1

O O O B-MEASURED_VAR 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 O O O O O O O B-MEASURED_VAR O O O O O O

(B) ParB clusters on F plasmid in vivo. Typical E. coli cells (DLT3594) displays foci of ParBF-mVenus protein (top) expressed from the endogenous genetic locus of the F plasmid (F1-10B-mVenus). The nucleoid is labelled with Hu-mCherry (central). The overlay (bottom) combines the two fluorescent channels. Over 99 % of cells harbor ParBF foci. White bars: 1 µm


( C ) ParBF binding outside parSF on the F plasmid is compatible with a power - law decay . High resolution ChIP - seq performed on DLT3586 carrying the F plasmid ( F1 - 10B ) . The ParB density , normalized to 1 at the first bp downstream the last parSF binding repeat after background subtraction , is displayed over 14 - Kbp on the right side of parSF . Monte Carlo simulations and analytic formula are represented in red and dotted black lines , respectively . MC simulations were performed with a Freely - Jointed Chain of linear length L = 15 - Kbp and a cluster radius σ = 75nm . The two other parameters , the Kuhn length a = 10 - bp and the total number of proteins on the F plasmid Nt = 360 ( related to the normalization constant of the protein concentration κ = 0 . 41 ) were fitted from the ChIP - seq data ( see text and Fig . EV2 ) . As a benchmark for simulations , the analytics are obtained from Eq . ( 1 ) with the same parameters . Inset ; The ParBF binding profile ( black line ) is represented as the number of nucleotide reads over 80 - Kbp centered at parS . The number of reads in the Input sample ( grey line ) is normalized to the total number of reads in the IP

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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

(C) ParBF binding outside parSF on the F plasmid is compatible with a power-law decay. High resolution ChIP-seq performed on DLT3586 carrying the F plasmid (F1-10B). The ParB density, normalized to 1 at the first bp downstream the last parSF binding repeat after background subtraction, is displayed over 14-Kbp on the right side of parSF. Monte Carlo simulations and analytic formula are represented in red and dotted black lines, respectively. MC simulations were performed with a Freely-Jointed Chain of linear length L=15-Kbp and a cluster radius σ=75nm. The two other parameters, the Kuhn length a=10-bp and the total number of proteins on the F plasmid Nt=360 (related to the normalization constant of the protein concentration κ=0.41) were fitted from the ChIP-seq data (see text and Fig. EV2). As a benchmark for simulations, the analytics are obtained from Eq.(1) with the same parameters. Inset; The ParBF binding profile (black line) is represented as the number of nucleotide reads over 80-Kbp centered at parS. The number of reads in the Input sample (grey line) is normalized to the total number of reads in the IP sample


Typical E . coli cell displays foc expressed from the endogenous genetic locus of the F plasmid ( F1 - 10B - mVenus ) . The nucleoid is labelled with Hu - mCherry ( central ) . The overlay ( bottom ) combines the two fluorescent channels parSF inserted at the xylE locus on E . coli chromosome from

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

Typical E. coli cell displays foc expressed from the endogenous genetic locus of the F plasmid (F1-10B-mVenus). The nucleoid is labelled with Hu-mCherry (central). The overlay (bottom) combines the two fluorescent channels parSF inserted at the xylE locus on E. coli chromosome from DLT358


ParBF binding outside parSF on the F plasmid is compatible with a power - law decay . High resolution ChIP - seq performed on DLT3586 carrying the F plasmid ( F1 - 10B ) . The ParB density , normalized to 1 at the first bp downstream the last parSF binding repeat after background subtraction , is displayed over 14 - Kbp on the right side of parSF . Monte Carlo simulations and analytic formula are represented in red and dotted black lines , respectively . MC simulations were performed with a Freely - Jointed Chain of linear length L = 15 - Kbp and a cluster radius σ = 75nm . The two other parameters , the Kuhn length a = 10 - bp and the total number of proteins on the F plasmid Nt = 360 ( related to the normalization constant of the protein concentration κ = 0 . 41 ) were fitted from the ChIP - seq dat parSF inserted at the xylE locus on E . coli chromosome fro

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

ParBF binding outside parSF on the F plasmid is compatible with a power-law decay. High resolution ChIP-seq performed on DLT3586 carrying the F plasmid (F1-10B). The ParB density, normalized to 1 at the first bp downstream the last parSF binding repeat after background subtraction, is displayed over 14-Kbp on the right side of parSF. Monte Carlo simulations and analytic formula are represented in red and dotted black lines, respectively. MC simulations were performed with a Freely-Jointed Chain of linear length L=15-Kbp and a cluster radius σ=75nm. The two other parameters, the Kuhn length a=10-bp and the total number of proteins on the F plasmid Nt=360 (related to the normalization constant of the protein concentration κ=0.41) were fitted from the ChIP-seq dat parSF inserted at the xylE locus on E. coli chromosome fro DLT207


( A ) Normalized and rescaled ParBF binding profiles at different ParBF / parSF ratio . ChIP - seq density on the right side of parSF inserted at xylE were measured in DLT2075 induced ( 16 , 28 ) or not ( 0 . 4 ) with IPTG ( 100 and 500 µM ) , or carrying HCN plasmids pZC302 ( 0 . 04 ) or pJYB57 ( 0 . 016 ) , normalized as in Fig . 1C , E with the amplitudes of the curves rescaled by the indicated factors ( 1 . 2 , 10 or 50 ) to overlap with the curves of highest amplitude . The ParB / parS ratio is calculated relative to the one of F plasmid as determined from Western blot analyses ( Appendix Fig . S2B ) . Monte Carlo simulations and analytical formula are plotted with the same parameters as in Fig . 1E . Note ( i ) that the dips at ~ 9 - kbp are not visible for the low levels of available ParB since the signal is close to the basal level and ( ii ) that the ChIP - seq data at 100 µM IPTG induction ( 16 ) are the same as in Fig . 1E . Inset ; Same as in the main to display the density without rescaling . ( B ) ParBF are dispersed in the cell upon titration by HCN plasmids . ParBF - mVenus expressed from pJYB294 were imaged as in Fig . 1D in DLT3577 ( left ) and DLT3576 ( right ) carrying pZC302 and pJYB57 , respectively . The number of extra parSF per cell , indicated on top of each raw , are estimated from the copy number per cell of HCN plasmids carrying 10 specific binding sites . White bars : 1 µm . ( C ) The size of ParBF clusters is independent of the intracellular ParBF concentration . We considered two possible evolutions of the cluster size upon variations of ParB amount in the framework of " Nucleation & caging " with corresponding schematics drawn on the right . For direct comparison with ( A ) , all curves are displayed with a rescaling of the amplitude corresponding to the WT expression level . Top : constant ParB concentration ; supposing that clusters are compact , the cluster radius σ would depend on the number m of ParB like $ \ \ sigma = m ^ { \ frac { 1 } { 3 } } $ . Predictions profiles , plotted at different ratio of ParB / parS , vary within the range of the experimental levels tested . Bottom : constant cluster size ; ParB concentrations vary but the range of exploration remains the same resulting in

O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR 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 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 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 O O O O O O O B-MEASURED_VAR 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 O O O 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-MEASURED_VAR O O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR 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 B-MEASURED_VAR 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

(A) Normalized and rescaled ParBF binding profiles at different ParBF/parSF ratio. ChIP-seq density on the right side of parSF inserted at xylE were measured in DLT2075 induced (16, 28) or not (0.4) with IPTG (100 and 500 µM), or carrying HCN plasmids pZC302 (0.04) or pJYB57 (0.016), normalized as in Fig. 1C, E with the amplitudes of the curves rescaled by the indicated factors (1.2, 10 or 50) to overlap with the curves of highest amplitude. The ParB/parS ratio is calculated relative to the one of F plasmid as determined from Western blot analyses (Appendix Fig. S2B). Monte Carlo simulations and analytical formula are plotted with the same parameters as in Fig. 1E. Note (i) that the dips at ~9-kbp are not visible for the low levels of available ParB since the signal is close to the basal level and (ii) that the ChIP-seq data at 100 µM IPTG induction (16) are the same as in Fig. 1E. Inset; Same as in the main to display the density without rescaling. (B) ParBF are dispersed in the cell upon titration by HCN plasmids. ParBF-mVenus expressed from pJYB294 were imaged as in Fig. 1D in DLT3577 (left) and DLT3576 (right) carrying pZC302 and pJYB57, respectively. The number of extra parSF per cell, indicated on top of each raw, are estimated from the copy number per cell of HCN plasmids carrying 10 specific binding sites. White bars: 1 µm. (C) The size of ParBF clusters is independent of the intracellular ParBF concentration. We considered two possible evolutions of the cluster size upon variations of ParB amount in the framework of "Nucleation & caging" with corresponding schematics drawn on the right. For direct comparison with (A), all curves are displayed with a rescaling of the amplitude corresponding to the WT expression level. Top: constant ParB concentration; supposing that clusters are compact, the cluster radius σ would depend on the number m of ParB like$\ \sigma = m^{\frac{1}{3}}$. Predictions profiles, plotted at different ratio of ParB/parS, vary within the range of the experimental levels tested. Bottom: constant cluster size; ParB concentrations vary but the range of exploration remains the same resulting in overlappin


( A ) The formation of secondary ParBF - DNA complexes requires the box II motif . EMSA were performed with a 144 - bp 32P - labelled DNA fragments ( C144 ) carrying a single 16 - bp parS binding motif . Reaction mixtures containing 100 µg . ml - 1 sonicated salmon sperm DNA were incubated in the absence ( - ) or the presence of increasing concentrations ( grey triangle ; 10 , 30 , 100 , 300 , and 1000 nM ) of ParBF or ParBF - 3R * . Positions of free and bound probes are indicated on the left . B1 represents complexes involving the specific interaction on the 16 - bp binding site , while B " 2 and B " 3 complexes represent secondary complexes involving the parSF site with one or two additional nsDNA - binding interactions , respectively ( Sanchez et al ,

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

(A) The formation of secondary ParBF-DNA complexes requires the box II motif. EMSA were performed with a 144-bp 32P-labelled DNA fragments (C144) carrying a single 16-bp parS binding motif. Reaction mixtures containing 100 µg.ml-1 sonicated salmon sperm DNA were incubated in the absence (-) or the presence of increasing concentrations (grey triangle; 10, 30, 100, 300, and 1000 nM) of ParBF or ParBF-3R*. Positions of free and bound probes are indicated on the left. B1 represents complexes involving the specific interaction on the 16-bp binding site, while B"2 and B"3 complexes represent secondary complexes involving the parSF site with one or two additional nsDNA-binding interactions, respectively (Sanchez et al,


( B ) ParBF cluster formation requires the box II motif . Epifluorescence microscopy of ParBF - 3R * - mVenus from DLT3566 is displayed as in Fig . 1D . White bars : 1

O O O B-MEASURED_VAR 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

(B) ParBF cluster formation requires the box II motif. Epifluorescence microscopy of ParBF-3R*-mVenus from DLT3566 is displayed as in Fig. 1D. White bars: 1 µm


( C ) ParBF in vivo DNA binding in the vicinity of parSF sites requires the box II motif . ChIP - seq was performed on DLT3726 carrying parSF in the xylE chromosomal locus and expressing ParBF - 3R * variant . ParBF - 3R * DNA binding profile displayed the number of nucleotide reads as a function of the E . coli genomic coordinates . The peak at parSF covered approximately 950 - bp , which corresponds to the 402 - bp between the 1st and 10th specific binding sites and ~ 280 - bp on each sides ( representing the average size of the DNA library ; see Appendix Fig . S3E ) . No ParBF - 3R * enrichment was found on parSF - flanking DNA and elsewhere on the chromosome . Inset , zoom in on the right side of parSF over 5 - Kbp with the ParB density , normalized to 1 at the first bp after the last parS binding repeat , plotted as a function of the distance from parSF . Note that a highly similar DNA binding pattern is obtained with ParBF - 3R * - mVenus ( strain DLT3566 ; Appendix Fig . S3C )

O O O B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O O O B-MEASURED_VAR 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 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 O B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR 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

(C) ParBF in vivo DNA binding in the vicinity of parSF sites requires the box II motif. ChIP-seq was performed on DLT3726 carrying parSF in the xylE chromosomal locus and expressing ParBF-3R* variant. ParBF-3R* DNA binding profile displayed the number of nucleotide reads as a function of the E. coli genomic coordinates. The peak at parSF covered approximately 950-bp, which corresponds to the 402-bp between the 1st and 10th specific binding sites and ~280-bp on each sides (representing the average size of the DNA library; see Appendix Fig. S3E). No ParBF-3R* enrichment was found on parSF-flanking DNA and elsewhere on the chromosome. Inset, zoom in on the right side of parSF over 5-Kbp with the ParB density, normalized to 1 at the first bp after the last parS binding repeat, plotted as a function of the distance from parSF. Note that a highly similar DNA binding pattern is obtained with ParBF -3R*-mVenus (strain DLT3566; Appendix Fig. S3C)


( A ) Schematic representation of the genomic locus of the chromosome 1 of V . cholerae with the three parS sites , named parS1 - 3 . The rRNA operon ( blue rectangle ) spans the genomic coordinates 53823 to 59123 ( B ) ChIP - seq performed on strain N16961 is displayed as the number of nucleotide reads in function of the genomic coordinates . Correspondence to the parS1 - 3 location represented in ( A ) is indicated by grey dotted lines . The number of reads in the Input sample ( grey line ) is normalized to the total number of reads in the IP sample . ( C ) We modeled the ChIP - Seq data as in Fig . 1C - E by means of MC simulations with a Freely jointed chain of size N = 2000 monomers of size a = 16 - bp . Data are normalized after background subtraction to the read value at parS1 ( genomic coordinate 62438 ) . The best fit was achieved with σ = 25nm and an amplitude κ = 0 . 15 leading to Nt ~ 50 ParB on the chromosome . In the MC simulation , we accounted for the finite width of the distribution around parS sites by including the average fragment size of the DNA

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 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 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 O O O O O O O O O O O O O O O O O O O O O O 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-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 B-MEASURED_VAR 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

(A) Schematic representation of the genomic locus of the chromosome 1 of V. cholerae with the three parS sites, named parS1-3. The rRNA operon (blue rectangle) spans the genomic coordinates 53823 to 59123 (B) ChIP-seq performed on strain N16961 is displayed as the number of nucleotide reads in function of the genomic coordinates. Correspondence to the parS1-3 location represented in (A) is indicated by grey dotted lines. The number of reads in the Input sample (grey line) is normalized to the total number of reads in the IP sample. (C) We modeled the ChIP-Seq data as in Fig.1C-E by means of MC simulations with a Freely jointed chain of size N=2000 monomers of size a=16-bp. Data are normalized after background subtraction to the read value at parS1 (genomic coordinate 62438). The best fit was achieved with σ=25nm and an amplitude κ=0.15 leading to Nt~50 ParB on the chromosome. In the MC simulation, we accounted for the finite width of the distribution around parS sites by including the average fragment size of the DNA librar


ChIP - sequencing assays were performed on DLT2075 ( xylE : : parSF ) expressing ParBF grown in exponential ( expo ) or stationary ( stat ) phases with addition of rifampicin when indicated ( + Rif ) . The assays have been performed in duplicate for the + Rif and once for the stationary phase experiments . ( A ) ParBF DNA binding around parSF is independent of active transcription . The color - coded ParBF profiles are represented over 50 - Kbp as the relative ParB density normalized to 1 at the first bp after the last parSF binding site . Loci A , C , E and F are defined in Appendix Fig . S1A . ( B ) The dips and peaks are highly similar in the three indicated conditions . Same as in ( A ) with zoom in on the right side of parSF up to 9 - Kpb and normalization to 1 at genomic coordinate 230 . The dotted line corresponds to the analytics description of " Nucleation and caging " ( see details in Fig . 1C - E ) . ( C ) ParBF binding profile upstream of the locus A . Same as in ( A ) with zoom in from - 6 . 5 to - 16 . 5 - Kbp by normalization to 1 at genomic coordinate - 6 . 5 - Kbp ( upstream of the dip at the locus A ) . The ParBF DNA binding profile remains compatible to a power - law , represented by the analytics description ( dotted line ) , upstream of the locus A in stationary phase ( black ) and in exponential phase ( blue ) . Also , the dips and peaks are highly similar in both conditions . These data are not in favor of the " 1D - spreading " or the " Spreading and bridging " models that predicts a basal uniform distribution or a linear decrease after a barrier , respectively ( Broedersz et al , 2014 ) . ( D ) The promoter region at locus A prevents ParBF DNA binding . Chip - seq assays were performed in isogenic xylE : : parSF strains ( DLT2075 ; black curve ) in which the locus A is replaced by a kanamycin gene ( DLT3651 ; red curve ) . The assay in the ∆ ( locus A ) genomic context has been performed once . The relative ParB density as a function of the distance from parSF

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 B-MEASURED_VAR 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O

ChIP-sequencing assays were performed on DLT2075 (xylE::parSF) expressing ParBF grown in exponential (expo) or stationary (stat) phases with addition of rifampicin when indicated (+Rif). The assays have been performed in duplicate for the +Rif and once for the stationary phase experiments. (A) ParBF DNA binding around parSF is independent of active transcription. The color-coded ParBF profiles are represented over 50-Kbp as the relative ParB density normalized to 1 at the first bp after the last parSF binding site. Loci A, C, E and F are defined in Appendix Fig. S1A. (B) The dips and peaks are highly similar in the three indicated conditions. Same as in (A) with zoom in on the right side of parSF up to 9-Kpb and normalization to 1 at genomic coordinate 230. The dotted line corresponds to the analytics description of "Nucleation and caging" (see details in Fig. 1C-E). (C) ParBF binding profile upstream of the locus A. Same as in (A) with zoom in from -6.5 to -16.5-Kbp by normalization to 1 at genomic coordinate -6.5-Kbp (upstream of the dip at the locus A). The ParBF DNA binding profile remains compatible to a power-law, represented by the analytics description (dotted line), upstream of the locus A in stationary phase (black) and in exponential phase (blue). Also, the dips and peaks are highly similar in both conditions. These data are not in favor of the "1D-spreading" or the "Spreading and bridging" models that predicts a basal uniform distribution or a linear decrease after a barrier, respectively (Broedersz et al, 2014). (D) The promoter region at locus A prevents ParBF DNA binding. Chip-seq assays were performed in isogenic xylE::parSF strains (DLT2075; black curve) in which the locus A is replaced by a kanamycin gene (DLT3651; red curve). The assay in the ∆(locus A) genomic context has been performed once. The relative ParB density as a function of the distance from parSF is


ParBF exchange between foci was measured by FRAP an from two - foci cell of DLT1215 carrying pJYB234 A - Representative images of a photobleached cell during a FRAP experiment . The 488 nm laser was pulsed ( Bleach ) on one of the two foci at ~ 2 . 4 sec ( black arrow ) . Red and blue arrows correspond to the bleached and unbleached focus , respectively . Time is indicated in second ( upper right ) . The cell outline is drawn in red . Scale bar : 1

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

ParBF exchange between foci was measured by FRAP an from two-foci cell of DLT1215 carrying pJYB234 A- Representative images of a photobleached cell during a FRAP experiment. The 488 nm laser was pulsed (Bleach) on one of the two foci at ~2.4 sec (black arrow). Red and blue arrows correspond to the bleached and unbleached focus, respectively. Time is indicated in second (upper right). The cell outline is drawn in red. Scale bar: 1 μm


ParBF exchange between foci was measured by FRAP and FLIM ( fluorescence lifetime imaging microscopy ) from two - foci cell of DLT1215 carrying pJYB234 B - Quantification of ParBF - mVenus fluorescence intensity over time . The dynamics of fluorescence intensity is shown from averaging 18 independent measurements of the bleached ( FRAP , red line ) and unbleached ( FLIM , blue line ) foci . Foci fluorescence intensity in each experiment was normalized to the average intensity of each focus before

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 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 O O O O O O O O

ParBF exchange between foci was measured by FRAP and FLIM (fluorescence lifetime imaging microscopy) from two-foci cell of DLT1215 carrying pJYB234 B- Quantification of ParBF-mVenus fluorescence intensity over time. The dynamics of fluorescence intensity is shown from averaging 18 independent measurements of the bleached (FRAP, red line) and unbleached (FLIM, blue line) foci. Foci fluorescence intensity in each experiment was normalized to the average intensity of each focus before photobleaching


Human DCs were incubated with a non - cytolytic dose ( 0 . 2 μg / ml ) of purified PLY for 45 min . Immunofluorescence staining shows that PLY ( green ) co - localize with MRC - 1 in DCs and EEA - 1 ( purple , early endosomes ) . All scale bars , 5 μm . Images are representative of three independent experiments .

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

Human DCs were incubated with a non-cytolytic dose (0.2 μg/ml) of purified PLY for 45 min. Immunofluorescence staining shows that PLY (green) co-localize with MRC-1 in DCs and EEA-1 (purple, early endosomes). All scale bars, 5 μm. Images are representative of three independent experiments.


Human DCs were incubated with a non - cytolytic dose ( 0 . 2 μg / ml ) of purified LLO for 45 min . Immunofluorescence staining shows that LLO ( green ) co - localize with MRC - 1 ( red ) in DCs and EEA - 1 ( purple , early endosomes ) . All scale bars , 5 μm . Images are representative of three independent experiments .

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

Human DCs were incubated with a non-cytolytic dose (0.2 μg/ml) of purified LLO for 45 min. Immunofluorescence staining shows that LLO (green) co-localize with MRC-1 (red) in DCs and EEA-1 (purple, early endosomes). All scale bars, 5 μm. Images are representative of three independent experiments.


Human DCs were incubated with a non - cytolytic dose ( 0 . 2 μg / ml ) of purified SLO for 45 min . Immunofluorescence staining shows that SLO ( green ) co - localize with MRC - 1 ( red ) in DCs and EEA - 1 ( purple , early endosomes ) . All scale bars , 5 μm . Images are representative of three independent experiments .

Human DCs were incubated with a non-cytolytic dose (0.2 μg/ml) of purified SLO for 45 min. Immunofluorescence staining shows that SLO (green) co-localize with MRC-1 (red) in DCs and EEA-1 (purple, early endosomes). All scale bars, 5 μm. Images are representative of three independent experiments.


( A ) ELISA showing the dose - dependent binding of plate - bound MRC - 1 peptides P2 , P3 , and the control peptides CP1 and CP2 to PLY ( 0 - 0 . 5 μM ) . BSA was used as negative control to show the binding specificity . Data are mean ± s . e . m . of two independent experiments , each containing three replicates per condition .

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

(A) ELISA showing the dose-dependent binding of plate-bound MRC-1 peptides P2, P3, and the control peptides CP1 and CP2 to PLY (0-0.5 μM). BSA was used as negative control to show the binding specificity. Data are mean ± s.e.m. of two independent experiments, each containing three replicates per condition.


( B ) Hemolysis assay ( n = 4 ) of 1 μg / ml purified PLY in the presence of increasing concentrations of MRC - 1 peptides , P2 , scrambled P2 , P3 and control peptide CP2 ( 1 - 1000 μM ) . Data represent mean ± s . e . m . * denotes P < 0 . 05 by one - way ANOVA with Dunnett ' s post hoc test for multiple comparisons . Exact P values are shown in Appendix Table S4 .

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 O O O O 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) Hemolysis assay (n=4) of 1 μg/ml purified PLY in the presence of increasing concentrations of MRC-1 peptides, P2, scrambled P2, P3 and control peptide CP2(1-1000 μM). Data represent mean ± s.e.m. * denotes P < 0.05 by one-way ANOVA with Dunnett's post hoc test for multiple comparisons. Exact P values are shown in Appendix Table S4.


( C ) LDH cytotoxicity assay in human THP - 1 macrophages stimulated with purified PLY , LLO or SLO ( 0 . 5 μg / ml ) in the presence or absence of 100 μM peptides P2 , scrambled P2 , P3 or control peptide CP2 for 18 h . Cholesterol ( 100 μM ) was used as positive control to inhibit hemolysis . Data are mean ± s . e . m from 4 independent experiments . * * * * denotes P < 0 . 0001 by two - way ANOVA with Bonferroni post hoc test for multiple comparisons . n . s . denotes not significant . Exact P values are shown in Appendix Table S4 .

O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) LDH cytotoxicity assay in human THP-1 macrophages stimulated with purified PLY, LLO or SLO (0.5 μg/ml) in the presence or absence of 100 μM peptides P2, scrambled P2, P3 or control peptide CP2 for 18 h. Cholesterol (100 μM) was used as positive control to inhibit hemolysis. Data are mean ± s.e.m from 4 independent experiments. **** denotes P< 0.0001 by two-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.


( D ) Binding of FITC - labelled peptides P2 and CP2 to wild - type pneumococci , TIGR4 ( T4 ) and isogenic PLY mutant ( T4Δply ) was visualized by fluorescence microscopy . Scale bars , 10 μm . In magnified images , scale bars , 1 μm . Images are representative of three independent experiments .

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

(D) Binding of FITC-labelled peptides P2 and CP2 to wild-type pneumococci, TIGR4 (T4) and isogenic PLY mutant (T4Δply) was visualized by fluorescence microscopy. Scale bars, 10 μm. In magnified images, scale bars, 1 μm. Images are representative of three independent experiments.


( E ) The hemolytic activity of wild - type pneumococci , TIGR4 ( T4 ) and PLY mutant , T4Δply in the presence of 100 μM peptide P2 and CP2 . Data are the mean ± s . e . m . of three independent experiments . * * * denotes P < 0 . 001 by one - way ANOVA with Bonferroni post hoc test for multiple comparisons . n . s . denotes not significant . Exact P values are shown in Appendix Table S4 .

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

(E) The hemolytic activity of wild-type pneumococci, TIGR4 (T4) and PLY mutant, T4Δply in the presence of 100 μM peptide P2 and CP2. Data are the mean ± s.e.m. of three independent experiments. *** denotes P < 0.001 by one-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.


( A ) IL - 8 released by human THP - 1 macrophages stimulated with purified PLY , LLO or SLO ( 0 . 5 μg / ml ) in the presence or absence of 100 μM peptides P2 , P3 or control peptide CP2 for 18 h . Cholesterol ( 100 μM ) was used as positive control to inhibit hemolysis . Data are mean ± s . e . m from three independent experiments . * * * * denotes P < 0 . 0001 by two - way ANOVA with Bonferroni post hoc test for multiple comparisons . n . s . denotes not significant . Exact P values are shown in Appendix Table S4 .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IL-8 released by human THP-1 macrophages stimulated with purified PLY, LLO or SLO (0.5 μg/ml) in the presence or absence of 100 μM peptides P2, P3 or control peptide CP2 for 18 h. Cholesterol (100 μM) was used as positive control to inhibit hemolysis. Data are mean ± s.e.m from three independent experiments. **** denotes P< 0.0001 by two-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.


( C ) 3D volume images of the GFP - lung epithelial models at 1 h and 3 h post stimulation with 1 μg / ml PLY in the presence or absence of 100 μM peptide P2 or the control peptide CP2 . Images are representative of two independent experiments with n = 3 models / condition .

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

(C) 3D volume images of the GFP-lung epithelial models at 1 h and 3 h post stimulation with 1 μg/ml PLY in the presence or absence of 100 μM peptide P2 or the control peptide CP2. Images are representative of two independent experiments with n=3 models/condition.


( D ) Invasion of wild - type pneumococci T4 ( TIGR4 ) or its isogenic PLY mutant T4Δply into the lung epithelial models ( n = 3 / condition ) in the presence or absence of 100 μM peptide P2 or the control peptide CP2 at 2h post infection was measured using CFU viability assay following gentamicin killing of extracellular bacteria . Anti - PLY was used as control to test the effect of blocking PLY . Data in d and e are mean ± s . e . m . of n = 3 models / condition from two independent experiments . % bacterial entry = ( bacteria uptaken / input ) x100 . * * denotes P < 0 . 01 by one - way ANOVA with Dunnett ' s post hoc test for multiple comparisons . n . s . denotes not significant . Exact P values are shown in Appendix Table S4 .

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

(D) Invasion of wild-type pneumococci T4 (TIGR4) or its isogenic PLY mutant T4Δply into the lung epithelial models (n=3/condition) in the presence or absence of 100 μM peptide P2 or the control peptide CP2 at 2h post infection was measured using CFU viability assay following gentamicin killing of extracellular bacteria. Anti-PLY was used as control to test the effect of blocking PLY. Data in d and e are mean ± s.e.m. of n=3 models/condition from two independent experiments. % bacterial entry = (bacteria uptaken/input) x100. ** denotes P < 0.01 by one-way ANOVA with Dunnett's post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.


( E ) Human DCs were infected with type 4 and type 2 pneumococci , T4 and D39 , respectively , at MOI of 10 in the presence or absence of 100 μM peptides , P2 or CP2 , and intracellular bacteria were counted at 3 h post infection following gentamicin killing of extracellular bacteria . Cytochalasin D ( 0 . 5 mM ) was used as negative control to inhibit phagocytosis . Anti - PLY was used as control to test the effect of blocking PLY . Data are mean ± s . e . m . of three independent experiments . * * * * denotes P < 0 . 0001 by two - way ANOVA with Bonferroni post hoc test for multiple comparisons . n . s . denotes not significant . Exact P values are shown in Appendix Table S4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Human DCs were infected with type 4 and type 2 pneumococci, T4 and D39, respectively, at MOI of 10 in the presence or absence of 100 μM peptides, P2 or CP2, and intracellular bacteria were counted at 3 h post infection following gentamicin killing of extracellular bacteria. Cytochalasin D (0.5 mM) was used as negative control to inhibit phagocytosis. Anti-PLY was used as control to test the effect of blocking PLY. Data are mean ± s.e.m. of three independent experiments. **** denotes P < 0.0001 by two-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.


( F ) DCs were infected with the unencapsulated pneumococcal strain T4R in the presence of 100 μM peptides , P2 or CP2 at MOI of 10 for 2 h . Immunofluorescence microscopy images show that in DCs treated with peptide P2 ( but not the control peptide CP2 ) , intracellular T4R ( green ) do not co - localize with MRC - 1 ( red ) , but with the autophagy protein LC3B ( cyan ) . Images are representative of three independent experiments . Scale bars , 10 μm . In magnified images , scale bars , 5 μm . Arrows indicate regions of co - localization of intracellular T4R with MRC - 1 and LC3B . ( G ) Quantification of percentage of intracellular S . pneumoniae ( n = 50 ) in infected DCs that co - localize with MRC - 1 and LC3B . Data are mean ± s . e . m from two independent experiments . * * * * denotes P < 0 . 0001 by two - way ANOVA with Bonferroni post hoc test for multiple comparisons . n . s . denotes not significant . P values are shown in Appendix Table S4 .

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

(F) DCs were infected with the unencapsulated pneumococcal strain T4R in the presence of 100 μM peptides, P2 or CP2 at MOI of 10 for 2 h. Immunofluorescence microscopy images show that in DCs treated with peptide P2 (but not the control peptide CP2), intracellular T4R (green) do not co-localize with MRC-1 (red), but with the autophagy protein LC3B (cyan). Images are representative of three independent experiments. Scale bars, 10 μm. In magnified images, scale bars, 5 μm. Arrows indicate regions of co-localization of intracellular T4R with MRC-1 and LC3B. (G) Quantification of percentage of intracellular S. pneumoniae (n=50) in infected DCs that co-localize with MRC-1 and LC3B. Data are mean±s.e.m from two independent experiments. **** denotes P <0.0001 by two-way ANOVA with Bonferroni post hoc test for multiple comparisons. n.s. denotes not significant. P values are shown in Appendix Table S4.


( A ) Survival percentage of 3 - 4 dpf zebrafish embryos ( n ≥ 156 ) upon infection with S . pneumoniae T4 alone or its isogenic PLY mutant , T4Δply . Injection with E3 growth medium served as mock control . * * * denotes P < 0 . 0005 and * * * * denotes P < 0 . 0001 by Mantel Cox test . Exact P values are shown in Appendix Table S4 .

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

(A) Survival percentage of 3-4 dpf zebrafish embryos (n ≥156) upon infection with S. pneumoniae T4 alone or its isogenic PLY mutant, T4Δply. Injection with E3 growth medium served as mock control. *** denotes P < 0.0005 and **** denotes P < 0.0001 by Mantel Cox test. Exact P values are shown in Appendix Table S4.


( B ) Zebrafish survival percentage upon infection with T4 alone or together with peptide P2 or CP2 or P2 - conjugated CaP NPs ( P2 - NPs ) . * * * denotes P < 0 . 0005 and * * * * denotes P < 0 . 0001 by Mantel Cox test . Exact P values are shown in Appendix Table S4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Zebrafish survival percentage upon infection with T4 alone or together with peptide P2 or CP2 or P2-conjugated CaP NPs (P2-NPs). *** denotes P < 0.0005 and **** denotes P < 0.0001 by Mantel Cox test. Exact P values are shown in Appendix Table S4.


( C ) Survival of mice ( n = 10 ) upon intranasal infection with 2x106 CFU of S . pneumoniae T4 together with peptide P2 or CP2 or P2 - NPs over 3 days post infection . Infected mice were checked twice daily in the morning at 9 am ( early check ) and in evening at 7 pm ( late check ) for clinical symptoms . Unloaded NPs and the isogenic PLY mutant strain ( T4Δply ) served as negative controls . * denotes P < 0 . 05 and * * denotes P < 0 . 005 by Mantel Cox test . Exact P values are shown in Appendix Table S4 .

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

(C) Survival of mice (n=10) upon intranasal infection with 2x106 CFU of S. pneumoniae T4 together with peptide P2 or CP2 or P2-NPs over 3 days post infection. Infected mice were checked twice daily in the morning at 9 am (early check) and in evening at 7 pm (late check) for clinical symptoms. Unloaded NPs and the isogenic PLY mutant strain (T4Δply) served as negative controls. * denotes P < 0.05 and ** denotes P < 0.005 by Mantel Cox test. Exact P values are shown in Appendix Table S4.


Bacterial load in the lungs of infected mice ( n = 10 ) were measured post sacrifice . Data represent mean ± s . e . m . * denotes P < 0 . 05 , * * denotes P < 0 . 01 and * * * * denotes P < 0 . 0001 by one - way ANOVA with Bonferroni ' s post hoc test for multiple comparisons . n . s . denotes not significant . Exact P values are shown in Appendix Table S4 .

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

Bacterial load in the lungs of infected mice (n=10) were measured post sacrifice. Data represent mean ± s.e.m. * denotes P < 0.05, ** denotes P < 0.01 and **** denotes P <0.0001 by one-way ANOVA with Bonferroni's post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.


levels of pro - inflammatory cytokines TNF - α in the lungs of infected mice ( n = 10 ) were measured post sacrifice . Data represent mean ± s . e . m . * denotes P < 0 . 05 , * * denotes P < 0 . 01 and * * * * denotes P < 0 . 0001 by one - way ANOVA with Bonferroni ' s post hoc test for multiple comparisons . n . s . denotes not significant . Exact P values are shown in Appendix Table S4 .

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

levels of pro-inflammatory cytokines TNF-α in the lungs of infected mice (n=10) were measured post sacrifice. Data represent mean ± s.e.m. * denotes P < 0.05, ** denotes P < 0.01 and **** denotes P <0.0001 by one-way ANOVA with Bonferroni's post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.


levels of pro - inflammatory cytokines IL - 12 in the lungs of infected mice ( n = 10 ) were measured post sacrifice . Data represent mean ± s . e . m . * denotes P < 0 . 05 , * * denotes P < 0 . 01 and * * * * denotes P < 0 . 0001 by one - way ANOVA with Bonferroni ' s post hoc test for multiple comparisons . n . s . denotes not significant . Exact P values are shown in Appendix Table S4 .

levels of pro-inflammatory cytokines IL-12 in the lungs of infected mice (n=10) were measured post sacrifice. Data represent mean ± s.e.m. * denotes P < 0.05, ** denotes P < 0.01 and **** denotes P <0.0001 by one-way ANOVA with Bonferroni's post hoc test for multiple comparisons. n.s. denotes not significant. Exact P values are shown in Appendix Table S4.


C - E . Luciferase reporter assay in HeLa cells using the synthetic miR - Control or miR - 182 - 3p in combination with the wild type ( C ) or the mutant 3 ' UTR of TRF2 construct ( D ) or the wild type 3 ' UTR of TRF1 ( E ) . Data information : data are shown as mean ± SD . Three independent experiments were performed ( n = 3 ) . P values are determined by Mann - Whitney test .

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 I-CONTROLLED_VAR I-CONTROLLED_VAR 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 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

C-E. Luciferase reporter assay in HeLa cells using the synthetic miR-Control or miR-182-3p in combination with the wild type (C) or the mutant 3'UTR of TRF2 construct (D) or the wild type 3'UTR of TRF1 (E). Data information: data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by Mann-Whitney test.


F , G . Western blotting for TRF2 expression in Telomerase Positive ( HeLa , HCT116 , MDA - MB - 231 , MDA - MB - 436 ) and ALT positive ( U2 - OS , Saos - 2 ) cells transiently transfected with miR - Control or miR - 182 - 3p . Upper panel shows the quantification of TRF2 expression . Bottom panel , representative images are shown , actin was used as loading control . Data information : data are shown as mean ± SD . Three independent experiments were performed ( n = 3 ) . P values are determined by Mann - Whitney test .

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

F,G. Western blotting for TRF2 expression in Telomerase Positive (HeLa, HCT116, MDA-MB-231, MDA-MB-436) and ALT positive (U2-OS, Saos-2) cells transiently transfected with miR-Control or miR-182-3p. Upper panel shows the quantification of TRF2 expression. Bottom panel, representative images are shown, actin was used as loading control. Data information: data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by Mann-Whitney test.


H . U2 - OS cells transiently transfected with the miR - Control , miR - 182 - 3p or miR - 182 - 3p inhibitor were assayed by quantitative immunofluorescence for TRF2 three days post - transfection . Left panel , representative images . Scale bar : 10µm . Right panel , quantification of TRF2 fluorescence intensity . a . f . u . arbitrary fluorescence units . N = number of analysed nuclei . Red bar indicates mean value . Data information : Three independent experiments were performed ( n = 3 ) . P values are determined by Student ' s t - test ; for ( H )

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

H. U2-OS cells transiently transfected with the miR-Control, miR-182-3p or miR-182-3p inhibitor were assayed by quantitative immunofluorescence for TRF2 three days post-transfection. Left panel, representative images. Scale bar: 10µm. Right panel, quantification of TRF2 fluorescence intensity. a.f.u. arbitrary fluorescence units. N=number of analysed nuclei. Red bar indicates mean value. Data information: Three independent experiments were performed (n=3). P values are determined by Student's t-test; for (H)


I . U2 - OS cells transfected as described in ( H ) were assayed by immuno - fluorescence combined with telomeric FISH . Left panel , representative images of co - localizations between TRF2 and telomeres ( white arrowheads ) . Scale bar : 10µm . Right panel , co - localizations were analysed using imageJ software . N = number of analysed nuclei . Data information : For I , data are shown as mean ± SD . Three independent experiments were performed ( n = 3 ) . P values are determined by Mann - Whitney 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 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 O O O O O O O O O O O O O O O O O O O O

I. U2-OS cells transfected as described in (H) were assayed by immuno-fluorescence combined with telomeric FISH. Left panel, representative images of co-localizations between TRF2 and telomeres (white arrowheads). Scale bar: 10µm. Right panel, co-localizations were analysed using imageJ software. N=number of analysed nuclei. Data information: For I, data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by Mann-Whitney test.


A . Immunofluorescence analysis of γH2AX combined with telomeric FISH ( TIFs ) was performed in MDA - MB - 231 cells transfected with the indicated miRNAs or siRNAs . The mean number of TIFs per nucleus was analysed . B . Representative images and enlargements of co - localizations ( white arrowheads ) relative to the experiment described in A . Scale bar : 10µm . Data information : For A data are shown as mean ± SD . Three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test . At least 60 nuclei were analysed for each experimental condition . All the experiments were performed three days post - transfection with the indicated miRNAs or siRNAs .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunofluorescence analysis of γH2AX combined with telomeric FISH (TIFs) was performed in MDA-MB-231 cells transfected with the indicated miRNAs or siRNAs. The mean number of TIFs per nucleus was analysed. B. Representative images and enlargements of co-localizations (white arrowheads) relative to the experiment described in A. Scale bar: 10µm. Data information: For A data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test. At least 60 nuclei were analysed for each experimental condition. All the experiments were performed three days post-transfection with the indicated miRNAs or siRNAs.


C . Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe ( PIFs ) was performed in MDA - MB - 231 cells transfected with the indicated miRNAs or siRNAs . The γH2AX positive cells with ≥ 1 PIFs per nucleus were analysed . D . Representative images of co - localizations ( white arrowheads ) relative to the experiment described in C . Scale bar : 10µm . Data information : For C , data are shown as mean ± SD . Three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test . At least 60 nuclei were analysed for each experimental condition . All the experiments were performed three days post - transfection with the indicated miRNAs or siRNAs .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe (PIFs) was performed in MDA-MB-231 cells transfected with the indicated miRNAs or siRNAs. The γH2AX positive cells with ≥1 PIFs per nucleus were analysed. D. Representative images of co-localizations (white arrowheads) relative to the experiment described in C. Scale bar: 10µm. Data information: For C, data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test. At least 60 nuclei were analysed for each experimental condition. All the experiments were performed three days post-transfection with the indicated miRNAs or siRNAs.


E . Quantification of TIFs in MDA - MB - 231 cells over - expressing TRF2 or an empty vector ( pBabe ) , transfected with indicated miRNAs . The mean number of TIFs per nucleus was quantified . F . Representative images and enlargements relative to the experiment described in E . White arrowheads indicate co - localizations events . Scale bar : 10µm . Data information : data are shown as mean ± SD . Three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test . At least 60 nuclei were analysed for each experimental condition . All the experiments were performed three days post - transfection with the indicated

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

E. Quantification of TIFs in MDA-MB-231 cells over-expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The mean number of TIFs per nucleus was quantified. F. Representative images and enlargements relative to the experiment described in E. White arrowheads indicate co-localizations events. Scale bar: 10µm. Data information: data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test. At least 60 nuclei were analysed for each experimental condition. All the experiments were performed three days post-transfection with the indicated miRNAs


G . Quantification of PIFs in MDA - MB - 231 cells over - expressing TRF2 or an empty vector ( pBabe ) , transfected with indicated miRNAs . The γH2AX positive cells with ≥ 1 PIFs per nucleus were analysed . H . Representative images relative to the experiment described in G . White arrowheads indicate co - localizations events . Scale bar : 10µm . Data information : For G data are shown as mean ± SD . Three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test . At least 60 nuclei were analysed for each experimental condition . All the experiments were performed three days post - transfection with the indicated

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

G. Quantification of PIFs in MDA-MB-231 cells over-expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The γH2AX positive cells with ≥1 PIFs per nucleus were analysed. H. Representative images relative to the experiment described in G. White arrowheads indicate co-localizations events. Scale bar: 10µm. Data information: For G data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test. At least 60 nuclei were analysed for each experimental condition. All the experiments were performed three days post-transfection with the indicated miRNAs


A , B . MDA - MB - 436 and MDA - MB - 231 cells underwent two rounds of transfection with miR - Control , miR - 182 - 3p or miR - 182 - 3p inhibitor . Starting from the day of the second transfection , cell confluence was monitored by Incucyte every 24h up to a maximum of 3 days . The percentage of cell confluence was analysed . Data information : For A , B data are shown as mean ± SEM . three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,B. MDA-MB-436 and MDA-MB-231 cells underwent two rounds of transfection with miR-Control, miR-182-3p or miR-182-3p inhibitor. Starting from the day of the second transfection, cell confluence was monitored by Incucyte every 24h up to a maximum of 3 days. The percentage of cell confluence was analysed. Data information: For A,B data are shown as mean ± SEM. three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test.


C , D . Cell number of MDA - MB - 436 ( C ) and MDA - MB - 231 ( D ) cells and TRF2 expression were analysed by automatic cell count and by western blotting at the end of the experiment described in A and B . Actin was used as loading control . Data information : For C , D data are shown as mean ± SD . three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test .

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

C,D. Cell number of MDA-MB-436 (C) and MDA-MB-231 (D) cells and TRF2 expression were analysed by automatic cell count and by western blotting at the end of the experiment described in A and B. Actin was used as loading control. Data information: For C,D data are shown as mean ± SD. three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test.


E . Two - dimensional scatter plots of Annexin V analysis performed in MDA - MB - 436 at the end of the second cycle of transfection with miR - Control , miR - 182 - 3p or miR - 182 - 3p inhibitor . Red boxes indicate early and late apoptotic cells . F . Quantification of Annexin V positive cells ( % ) of experiment described in E . G . Two - dimensional scatter plots of Annexin V analysis performed in MDA - MB - 231 as described in E . H . Quantification of Annexin V positive cells ( % ) of experiment described in G . Data information : For F , H data are shown as mean ± SD . P values are determined by unpaired two - tailed t test . For F , H two different biological replicates were performed .

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

E. Two-dimensional scatter plots of Annexin V analysis performed in MDA-MB-436 at the end of the second cycle of transfection with miR-Control, miR-182-3p or miR-182-3p inhibitor. Red boxes indicate early and late apoptotic cells. F. Quantification of Annexin V positive cells (%) of experiment described in E. G. Two-dimensional scatter plots of Annexin V analysis performed in MDA-MB-231 as described in E. H. Quantification of Annexin V positive cells (%) of experiment described in G. Data information: For F,H data are shown as mean ± SD. P values are determined by unpaired two-tailed t test. For F, H two different biological replicates were performed.


I , J . MDA - MB - 436 cells over - expressing TRF2 or an empty vector ( pBabe ) were transiently transfected with indicated miRNAs and cell count ( I ) or apoptosis ( J ) analysis was performed 72h post - transfection . Data information : For , I , J , data are shown as mean ± SD . For I , three independent experiments were performed ( n = 3 ) . P values are determined by unpaired two - tailed t test . For J , two different biological replicates were performed .

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

I,J. MDA-MB-436 cells over-expressing TRF2 or an empty vector (pBabe) were transiently transfected with indicated miRNAs and cell count (I) or apoptosis (J) analysis was performed 72h post-transfection. Data information: For ,I,J, data are shown as mean ± SD. For I, three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test. For J, two different biological replicates were performed.


A . Western blotting for TRF2 expression in BJ cells transiently transfected with miR - Control or miR - 182 - 3p . The graph represents the quantification of three independent experiments . Representative images are shown , actin was used as loading control . Unspecific bands are indicated with ( * ) .

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

A. Western blotting for TRF2 expression in BJ cells transiently transfected with miR-Control or miR-182-3p. The graph represents the quantification of three independent experiments. Representative images are shown, actin was used as loading control. Unspecific bands are indicated with (*).


B , C . Mean of γH2AX foci per nucleus was analysed in BJ cells 72h post - transfection with the indicated miRNAs . Representative images of γH2AX foci are shown in C . Data information : student t test was used to calculate statistical significance . Scale bars ( 10µm ) . P values are indicated .

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 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

B, C. Mean of γH2AX foci per nucleus was analysed in BJ cells 72h post-transfection with the indicated miRNAs. Representative images of γH2AX foci are shown in C. Data information: student t test was used to calculate statistical significance. Scale bars (10µm). P values are indicated.


D . Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe ( TIFs ) was performed in BJ cells 72h post - transfection with the indicated miRNAs . Left panel : the mean number of TIFs per nucleus was analysed . Right panel : representative images and enlargements of co - localizations . Data information : a student t test was used to calculate statistical significance . Scale bars ( 10µm ) . P values are indicated .

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 O O O 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. Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe (TIFs) was performed in BJ cells 72h post-transfection with the indicated miRNAs. Left panel: the mean number of TIFs per nucleus was analysed. Right panel: representative images and enlargements of co-localizations. Data information: a student t test was used to calculate statistical significance. Scale bars (10µm). P values are indicated.


E . Cells number of BJ cells was analysed by automatic cell count at the end of the second round of transfection with miR - Control or miR - 182 - 3p . Data information : a student t test was used to calculate statistical significance . P values are indicated .

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

E. Cells number of BJ cells was analysed by automatic cell count at the end of the second round of transfection with miR-Control or miR-182-3p. Data information: a student t test was used to calculate statistical significance. P values are indicated.


F . FACS analysis to evaluate cell cycle progression by Propidium Iodide ( PI ) staining in BJ cells treated as indicated in 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

F. FACS analysis to evaluate cell cycle progression by Propidium Iodide (PI) staining in BJ cells treated as indicated in E.


G . β - Galactosidase assay in BJ cells after two rounds of transfection with mimic miR - Control or miR - 182 - 3p . Left panel : analysis of β - Galactosidase positive cells . Right panel : representative images . Data information : a student t test was used to calculate statistical significance . P values are indicated .

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

G. β-Galactosidase assay in BJ cells after two rounds of transfection with mimic miR-Control or miR-182-3p. Left panel: analysis of β-Galactosidase positive cells. Right panel: representative images. Data information: a student t test was used to calculate statistical significance. P values are indicated.


H - J . IL - 6 ( H ) , CXCL1 ( I ) , IL - 8 ( J ) factors were analysed by ELISA to evaluated the Senescence - associated secretory phenotype ( SASP ) in BJ cells treated as indicated in G . Data information : a student t test was used to calculate statistical significance . P values are indicated .

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

H-J. IL-6 (H), CXCL1 (I), IL-8 (J) factors were analysed by ELISA to evaluated the Senescence-associated secretory phenotype (SASP) in BJ cells treated as indicated in G. Data information: a student t test was used to calculate statistical significance. P values are indicated.


A , B . MDA - MB - 231 ( A ) and MDA - MB - 436 ( B ) tumor xenografts were treated with LNPs - empty , LNPs - miR - Control or by LNPs - miR - 182 - 3p when the tumors became palpable . Mice were treated 6 times by intravenous tail vein injections with 20µg of LNPs - miR - Control , LNPs - miR - 182 - 3p or equivalent volume of LNPs - empty as indicated in the scheduling . The mean of tumor volumes ( n = 5 per group ) is shown . Data information : For A , B data are shown as mean ± SD . P values are determined by unpaired two - tailed t

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O 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,B. MDA-MB-231 (A) and MDA-MB-436 (B) tumor xenografts were treated with LNPs-empty, LNPs-miR-Control or by LNPs-miR-182-3p when the tumors became palpable. Mice were treated 6 times by intravenous tail vein injections with 20µg of LNPs-miR-Control, LNPs-miR-182-3p or equivalent volume of LNPs-empty as indicated in the scheduling. The mean of tumor volumes (n=5 per group) is shown. Data information: For A, B data are shown as mean ± SD. P values are determined by unpaired two-tailed t test


C , D . Tumors from mice treated in A and B were processed to measure miR - 182 - 3p expression by TaqMan qPCR . Data information : For C , D the line in the middle of the box plot denote a median value , the limits of box represent the interquartile range ( 25th to 75th percentiles ) , while , the whiskers denote the minimum to maximum values . , P values are determined by unpaired two - tailed t test ;

O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O 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,D. Tumors from mice treated in A and B were processed to measure miR-182-3p expression by TaqMan qPCR. Data information: For C, D the line in the middle of the box plot denote a median value, the limits of box represent the interquartile range (25th to 75th percentiles), while, the whiskers denote the minimum to maximum values. , P values are determined by unpaired two-tailed t test;