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

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

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(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 box II motif . Epifluorescence microscopy of ParBF - 3R * - mVenus from DLT3566 is displayed as in Fig . 1D . bars : 1

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

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

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

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

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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 FLIM ( lifetime imaging microscopy ) from two - foci of DLT1215 carrying pJYB234 B - of ParBF - mVenus over time . dynamics of is shown from averaging 18 independent measurements of bleached ( FRAP , red ) unbleached ( FLIM , blue ) foci . Foci in each experiment was normalized to average of each focus before

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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 shows that PLY ( green ) co - localize with MRC - 1 in DCs EEA - 1 ( purple , early endosomes ) . scale bars , 5 μm . are representative of three independent experiments .

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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 shows that LLO ( green ) co - localize with MRC - 1 ( red ) in DCs EEA - 1 ( purple , early endosomes ) . scale bars , 5 μm . are representative of three independent experiments .

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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 shows that SLO ( green ) co - localize with MRC - 1 ( red ) in DCs EEA - 1 ( purple , early endosomes ) . scale bars , 5 μm . 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 dose - dependent binding of plate - bound MRC - 1 peptides P2 , P3 , control peptides CP1 CP2 to PLY ( 0 - 0 . 5 μM ) . BSA was used as negative control to show binding specificity . Data are mean ± s . e . m . of two independent experiments , each containing three replicates per condition .

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(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 ( n = 4 ) of 1 μg / ml purified PLY in presence of increasing concentrations of MRC - 1 peptides , P2 , scrambled P2 , P3 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 for multiple comparisons . Exact P values are shown in Appendix Table S4 .

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(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 in human THP - 1 macrophages stimulated with purified PLY , LLO or SLO ( 0 . 5 μg / ml ) in 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 for multiple comparisons . n . s . denotes not significant . Exact P values are shown in Appendix Table S4 .

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(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 CP2 to wild - type pneumococci , TIGR4 ( T4 ) isogenic PLY mutant ( T4Δply ) was visualized by microscopy . Scale bars , 10 μm . In magnified , scale bars , 1 μm . are representative of three independent experiments .

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

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

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O B-GENEPROD O O B-ORGANISM O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 for multiple comparisons . n . s . denotes not significant . Exact P values are shown in Appendix Table S4 .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of GFP - lung epithelial models at 1 h 3 h post stimulation with 1 μg / ml PLY in presence or absence of 100 μM peptide P2 or control peptide CP2 . are representative of two independent experiments with n = 3 models / condition .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O 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 lung epithelial models ( n = 3 / condition ) in presence or absence of 100 μM peptide P2 or control peptide CP2 at 2h post infection was measured using CFU viability following gentamicin killing of extracellular . Anti - PLY was used as control to effect of blocking PLY . Data in d e are mean ± s . e . m . of n = 3 models / condition from two independent experiments . % entry = ( uptaken / input ) x100 . * * denotes P < 0 . 01 by one - way ANOVA with Dunnett ' s post hoc for multiple comparisons . n . s . denotes not significant . Exact P values are shown in Appendix Table S4 .

O O O B-EXP_ASSAY O O O O B-ORGANISM I-ORGANISM O B-ORGANISM O O O O B-GENEPROD O B-ORGANISM O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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 type 2 pneumococci , T4 D39 , respectively , at MOI of 10 in presence or absence of 100 μM peptides , P2 or CP2 , intracellular were counted at 3 h post infection following gentamicin killing of extracellular . Cytochalasin D ( 0 . 5 mM ) was used as negative control to inhibit phagocytosis . Anti - PLY was used as control to 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 for multiple comparisons . n . s . denotes not significant . Exact P values are shown in Appendix Table S4 .

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

O O O B-CELL_TYPE O O O O B-ORGANISM I-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of 3 - 4 dpf zebrafish ( 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 * * * * denotes P < 0 . 0001 by Mantel Cox . Exact P values are shown in Appendix Table S4 .

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

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

O O O B-ORGANISM B-EXP_ASSAY O O O B-ORGANISM O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O 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 morning at 9 am ( early check ) in evening at 7 pm ( late check ) for clinical symptoms . Unloaded NPs isogenic PLY mutant ( T4Δply ) served as negative controls . * denotes P < 0 . 05 * * denotes P < 0 . 005 by Mantel Cox . Exact P values are shown in Appendix Table S4 .

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

(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.


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

B-EXP_ASSAY O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


of pro - inflammatory cytokines IL - 12 in lungs of infected mice ( n = 10 ) were measured post sacrifice . Data represent mean ± s . e . m . * denotes P < 0 . 05 , * * denotes P < 0 . 01 * * * * denotes P < 0 . 0001 by one - way ANOVA with Bonferroni ' s post hoc 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 in HeLa using synthetic miR - Control or miR - 182 - 3p in combination with wild type ( C ) or mutant 3 ' UTR of TRF2 construct ( D ) or 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 .

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

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 ) ALT positive ( U2 - OS , Saos - 2 ) transiently transfected with miR - Control or miR - 182 - 3p . Upper panel shows of TRF2 expression . Bottom panel , representative 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 .

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

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 transiently transfected with miR - Control , miR - 182 - 3p or miR - 182 - 3p inhibitor were assayed by quantitative immunofluorescence for TRF2 three days post - transfection . Left panel , representative . Scale bar : 10µm . Right panel , of TRF2 . a . f . u . arbitrary 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 - ; for ( H )

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

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 transfected as described in ( H ) were assayed by immuno - combined with telomeric FISH . Left panel , representative of co - localizations between TRF2 telomeres ( 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 .

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

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 of γH2AX combined with telomeric FISH ( TIFs ) was performed in MDA - MB - 231 transfected with indicated miRNAs or siRNAs . mean number of TIFs per nucleus was analysed . B . Representative enlargements of co - localizations ( arrowheads ) relative to 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 - t . At least 60 nuclei were analysed for each experimental condition . experiments were performed three days post - transfection with indicated miRNAs or siRNAs .

O O B-EXP_ASSAY O B-GENEPROD O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR 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 of γH2AX combined with a SatIII FISH probe ( PIFs ) was performed in MDA - MB - 231 transfected with indicated miRNAs or siRNAs . γH2AX positive with ≥ 1 PIFs per nucleus were analysed . D . Representative of co - localizations ( arrowheads ) relative to 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 - t . At least 60 nuclei were analysed for each experimental condition . experiments were performed three days post - transfection with indicated miRNAs or siRNAs .

O O B-EXP_ASSAY O B-GENEPROD O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR 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 . of TIFs in MDA - MB - 231 over - expressing TRF2 or an empty vector ( pBabe ) , transfected with indicated miRNAs . mean number of TIFs per nucleus was quantified . F . Representative enlargements relative to experiment described in E . 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 - t . At least 60 nuclei were analysed for each experimental condition . experiments were performed three days post - transfection with indicated

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR 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 . of PIFs in MDA - MB - 231 over - expressing TRF2 or an empty vector ( pBabe ) , transfected with indicated miRNAs . γH2AX positive with ≥ 1 PIFs per nucleus were analysed . H . Representative relative to experiment described in G . 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 - t . At least 60 nuclei were analysed for each experimental condition . experiments were performed three days post - transfection with indicated

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

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

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

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 . number of MDA - MB - 436 ( C ) MDA - MB - 231 ( D ) TRF2 expression were analysed by automatic by western blotting at end of experiment described in A 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 - t .

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

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

O O O O O O O O B-GENEPROD I-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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 over - expressing TRF2 or an empty vector ( pBabe ) were transiently transfected with indicated miRNAs ( I ) or apoptosis ( J ) 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 - t . For J , two different biological replicates were performed .

O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 transiently transfected with miR - Control or miR - 182 - 3p . graph represents of three independent experiments . Representative are shown , actin was used as loading control . Unspecific bands are indicated with ( * ) .

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

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 72h post - transfection with indicated miRNAs . Representative of γH2AX foci are shown in C . Data information : student t was used to calculate statistical significance . Scale bars ( 10µm ) . P values are indicated .

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

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

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

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

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

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 to evaluate cycle progression by Propidium Iodide ( PI ) in BJ treated as indicated in E .

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


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

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

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 Senescence - associated secretory phenotype ( SASP ) in BJ treated as indicated in G . Data information : a student t was used to calculate statistical significance . P values are indicated .

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

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 ) MDA - MB - 436 ( B ) xenografts were treated with LNPs - empty , LNPs - miR - Control or by LNPs - miR - 182 - 3p when 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 scheduling . mean of 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 - t

O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B were processed to measure miR - 182 - 3p expression by TaqMan qPCR . Data information : For C , D in middle of box plot denote a median value , limits of box represent interquartile range ( 25th to 75th percentiles ) , while , whiskers denote minimum to maximum values . , P values are determined by unpaired two - t ;

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

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;


E . Representative of of indicated markers on samples from mice bearing MDA - MB - 231 human breast cancer xenografts . Scale bar : 50μm . F . histograms show : expression of TRF2 , calculated as immunoreactivity score ( IRS ) by , of positive to γH2AX , Tunel or CD31 . analyses were performed on 3 mice per group , points represent number of field analysed for each condition . Data information : For F , data are shown as mean ± SD . ( F ) , P values are determined by Mann - Whitney .

O O O O O O O O O O B-ORGANISM O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE B-ORGANISM B-DISEASE I-DISEASE B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Representative images of IHC analysis of the indicated markers on tumor samples from mice bearing MDA-MB-231 human breast cancer xenografts. Scale bar: 50μm. F. The histograms show: the expression of TRF2, calculated as immunoreactivity score (IRS) by IHC, and the count of positive cells to γH2AX, Tunel or CD31 staining. The analyses were performed on 3 mice per group, the points represent the number of field analysed for each condition. Data information: For F, data are shown as mean ± SD. (F), P values are determined by Mann-Whitney test.


G , H . Luminescent MDA - MB - 436 were injected into brain monitored by IVIS imaging system . After one week from implant , treatment with LNPs - miR - Control LNPs - miR - 182 - 3p was performed as indicated in A B . Representative from in vivo ( upper panel ) or ex - vivo ( bottom panel ) brain tumors are shown in G . Boxplots ( H ) show measurement of photons for each brain ( n = 5 per group ) acquired at indicated times . Data information : For H , in middle of box plot denote a median value , limits of box represent interquartile range ( 25th to 75th percentiles ) , while , whiskers denote minimum to maximum values . For H , P values are determined by unpaired two - t ;

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, H. Luminescent MDA-MB-436 cells were injected into the brain and monitored by IVIS imaging system. After one week from implant, treatment with LNPs-miR-Control and LNPs-miR-182-3p was performed as indicated in A and B. Representative images from in vivo (upper panel) or ex-vivo (bottom panel) brain tumors are shown in G. Boxplots (H) show the measurement of photons for each brain tumor (n=5 per group) acquired at the indicated times. Data information: For H, 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. For H, P values are determined by unpaired two-tailed t test;


A , B . PDTCs # 1 # 2 underwent two rounds of transfection with miR - Control or miR - 182 - 3p . Three days after second transfection , miR - 182 - 3p TRF2 expression were analysed by TaqMan qPCR western blotting , respectively . Actin was used as loading control . Data information : data are shown as mean ± SD . P values are determined by unpaired two - t For experiments showed in A , B two or three biological replicates were performed , respectively .

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

A,B. PDTCs #1 and #2 underwent two rounds of transfection with miR-Control or miR-182-3p. Three days after the second transfection, miR-182-3p and TRF2 expression were analysed by TaqMan qPCR and western blotting, respectively. Actin was used as loading control. Data information: data are shown as mean ± SD. P values are determined by unpaired two-tailed t test For the experiments showed in A, B two or three biological replicates were performed, respectively.


C , D . Left panel , of each PDTCs was measured by ImageJ . Right panel , representative are shown . Scale bar : 50μm . At least 85 3D were analysed for each experimental condition . Data information : data are shown as mean ± SD . P values are determined by unpaired two - t For experiments showed in C , D two or three biological replicates were performed , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Left panel, area of each PDTCs was measured by ImageJ. Right panel, representative images are shown. Scale bar: 50μm. At least 85 3D cells were analysed for each experimental condition. Data information: data are shown as mean ± SD. P values are determined by unpaired two-tailed t test For the experiments showed in C, D two or three biological replicates were performed, respectively.


E . NSG mice implanted with breast PDTX ( # 2 ) were treated with LNPs - empty , LNPs - miR - Control or LNPs - miR - 182 - 3p as indicated in scheduling . Caliper measurement of tumors were taken at indicated days . mean of volumes ( n = 5 per group ) is shown . Data information : data are shown as mean ± SD . P values are determined by unpaired two - t ;

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

E. NSG mice implanted with breast PDTX (#2) were treated with LNPs-empty, LNPs-miR-Control or LNPs-miR-182-3p as indicated in the scheduling. Caliper measurement of tumors were taken at the indicated days. The mean of tumor volumes (n=5 per group) is shown. Data information: data are shown as mean ± SD. P values are determined by unpaired two-tailed t test;


F . miR - 182 - 3p expression of tumors from mice treated in ( E ) was assayed by TaqMan qPCR . Data information : data are shown as mean ± SD . P values are determined by unpaired two - t

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

F. miR-182-3p expression of tumors from mice treated in (E) was assayed by TaqMan qPCR. Data information: data are shown as mean ± SD. P values are determined by unpaired two-tailed t test


G . Representative of of indicated markers from tumors of experiment showed in E . Scale bar : 50μm . H . histograms show : expression of TRF2 measured as immunoreactivity score ( IRS ) , of positive to γH2AX Tunel . was performed on 3 mice per group , points represent number of field analysed for each condition . Data information : For H , data are shown as mean ± SD . For ( H ) , P values are determined by Mann - Whitney .

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

G. Representative images of IHC analysis of the indicated markers from tumors of the experiment showed in E. Scale bar: 50μm. H. The histograms show: the expression levels of TRF2 measured as immunoreactivity score (IRS), the percentage of positive cells to γH2AX and Tunel. The analysis was performed on 3 mice per group, the points represent the number of field analysed for each condition. Data information: For H, data are shown as mean ± SD. For (H), P values are determined by Mann-Whitney test.


A . Representative of intestine sections from mice previously treated with LNPs - Empty or LNPs - miR - 182 - 3p . H & E ( scale bar : 200μm ) with TRF2 or γH2AX antibodies are shown ( scale bar : 50μm ) . B , C . of TRF2 expression as immunoreactivity score ( IRS ) ( B ) of γH2AX positive ( % ) ( C ) on intestine samples . D . Representative H & E ( scale bar : 200μm ) , TRF2 γH2AX of skin samples corresponding to LNPs - Empty or LNPs - miR - 182 - 3p treated ( scale bar : 50μm ) . E , F . of TRF2 expression as immunoreactivity score ( IRS ) ( E ) of γH2AX positive ( % ) ( F ) on skin samples . G . Representative H & E ( scale bar : 200μm ) , TRF2 γH2AX of bone marrow samples corresponding to LNPs - Empty or LNPs - miR - 182 - 3p treated ( scale bar : 50μm ) . H , I . of TRF2 expression as immunoreactivity score ( IRS ) ( H ) of γH2AX positive ( % ) ( I ) on bone marrow samples . Data information : For B , C , E , F , H , I , data are shown as mean ± SD . A Mann - Whitney t was used to calculate statistical significance . Four mice per group were analysed points represent number of field analysed for each condition .

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

A. Representative images of intestine sections from mice previously treated with LNPs-Empty or LNPs-miR-182-3p. H&E staining (scale bar: 200μm) and IHC analysis with TRF2 or γH2AX antibodies are shown (scale bar: 50μm). B,C. Quantification of TRF2 expression as immunoreactivity score (IRS) (B) and of γH2AX positive cells (%) (C) on intestine samples. D. Representative H&E (scale bar: 200μm), TRF2 and γH2AX images of skin samples corresponding to LNPs-Empty or LNPs-miR-182-3p treated animals (scale bar: 50μm). E,F. Quantification of TRF2 expression as immunoreactivity score (IRS) (E) and of γH2AX positive cells (%) (F) on skin samples. G. Representative H&E (scale bar: 200μm), TRF2 and γH2AX images of bone marrow samples corresponding to LNPs-Empty or LNPs-miR-182-3p treated animals (scale bar: 50μm). H,I. Quantification of TRF2 expression as immunoreactivity score (IRS) (H) and of γH2AX positive cells (%) (I) on bone marrow samples. Data information: For B,C,E,F,H,I, data are shown as mean ± SD. A Mann-Whitney test t test was used to calculate statistical significance. Four mice per group were analysed the points represent the number of field analysed for each condition.


( B ) A fragment encompassing 432 - 520 of Atg13 ( wt ) or corresponding FV mutant ( FV ) was expressed in E . coli incubated in excess with an immobilized GST ‐ tagged fragment of Atg1 ( 501 - 897 ) purified from E . coli ( GST ‐ Atg1 ) . Input ( 5 . 5 % ) bound were analysed by coomassie blue , which stains proportional to their . of marker ( in kDa ) is indicated on left .

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(B) A fragment encompassing amino acids 432-520 of Atg13 (wt) or the corresponding FV mutant (FV) was expressed in E. coli and incubated in excess with an immobilized GST‐tagged fragment of Atg1 (amino acids 501-897) purified from E. coli (GST‐Atg1). Input (5.5%) and bound proteins were analysed by coomassie blue staining, which stains proteins proportional to their size. The size of marker proteins (in kDa) is indicated on the left.


( C ) ATG1 ‐ TAP atg13Δ containing endogenously GFP ‐ tagged Atg17 , Atg29 or Vac8 wild ‐ type ( wt ) or Atg13 FV ‐ mutant ( F468A ; V469A ) were grown to mid log phase . Atg1 was immunoprecipitated its association with Atg13 GFP ‐ tagged was analysed by immunoblotting . Extract inputs are shown in Supplementary Figure S1B .

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(C) ATG1‐TAP atg13Δ cells containing endogenously GFP‐tagged Atg17, Atg29 or Vac8 and wild‐type (wt) or the Atg13 FV‐mutant (F468A; V469A) were grown to mid log phase. Atg1 was immunoprecipitated and its association with Atg13 and the GFP‐tagged proteins was analysed by immunoblotting. Extract inputs are shown in Supplementary Figure S1B.


( D ) atg1Δ atg1Δatg13Δ expressing HA ‐ tagged Atg1 wild ‐ type ( wt ) containing an empty plasmid were grown to mid log phase ( rich ) , then treated with rapamycin ( rapa ) or starved for 4 h in SD ‐ N medium ( starv ) . Atg1 was immunoprecipitated its association with Atg13 analysed by immunoblotting . Note that phosphorylated Atg13 isolated from grown in rich medium migrates slightly slower on SDS - PAGE , extent corresponds to shift typically observed under our gel conditions .

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(D) atg1Δ and atg1Δatg13Δ cells expressing HA‐tagged Atg1 and wild‐type (wt) cells containing an empty plasmid were grown to mid log phase (rich), and then treated with rapamycin (rapa) or starved for 4 h in SD‐N medium (starv). Atg1 was immunoprecipitated and its association with Atg13 analysed by immunoblotting. Note that phosphorylated Atg13 isolated from cells grown in rich medium migrates slightly slower on SDS-PAGE, and the extent corresponds to the shift typically observed under our gel conditions.


( E ) Yeast containing endogenously tagged Atg1 ‐ TAP with without endogenously tagged Atg13 ‐ GFP , or conversely containing endogenously tagged Atg13 ‐ TAP Atg1 ‐ GFP expressed from a centromeric plasmid were grown to mid log phase ( rich ) treated with rapamycin ( rapa ) or starved for 4 h in SD ‐ N medium ( starv ) . Atg1 ‐ TAP Atg13 ‐ TAP were affinity purified their association with GFP ‐ tagged was analysed by western blotting .

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(E) Yeast cells containing endogenously tagged Atg1‐TAP with and without endogenously tagged Atg13‐GFP, or conversely cells containing endogenously tagged Atg13‐TAP and Atg1‐GFP expressed from a centromeric plasmid were grown to mid log phase (rich) and treated with rapamycin (rapa) or starved for 4 h in SD‐N medium (starv). Atg1‐TAP and Atg13‐TAP were affinity purified and their association with GFP‐tagged proteins was analysed by western blotting.


( F ) Histone3 ‐ HA tagged Atg1 was expressed together with wild ‐ type ( wt ) or Atg13 ‐ FV mutant fused to Suv methylase ( Suv ) in atg1Δatg13Δ , or for control together with Pbs2 ‐ Suv in atg1Δ . Logarithmically growing cultures were treated with rapamycin ( rapa ) for 0 or 120 min , methylation was monitored by preparing extracts western blotting with an anti ‐ trimethylation ‐ specific antibody . Note that Suv ‐ , GFP ‐ or TAP ‐ tagged Atg13 are fully functional , however , do not show characteristic phosphorylation ‐ induced reduced mobility observed with untagged under our gel conditions .

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(F) Histone3‐HA tagged Atg1 was expressed together with wild‐type (wt) or the Atg13‐FV mutant fused to Suv methylase (Suv) in atg1Δatg13Δ cells, or for control together with Pbs2‐Suv in atg1Δ cells. Logarithmically growing cultures were treated with rapamycin (rapa) for 0 or 120 min, and methylation was monitored by preparing cell extracts and western blotting with an anti‐trimethylation‐specific antibody. Note that Suv‐, GFP‐ or TAP‐tagged Atg13 are fully functional, however, do not show the characteristic phosphorylation‐induced reduced mobility observed with the untagged protein under our gel conditions.


( G ) atg1Δatg13Δ containing TAP ‐ tagged wild ‐ type Atg1 ( wt ) or Atg1 kinase ‐ dead ( kd ) K54A mutant wild ‐ type Atg13 , Atg13 ‐ FV mutant or an empty plasmid were grown to mid log phase . Atg1 was immunoprecipitated its autophosphorylation activity was measured by autoradiography in vitro .

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(G) atg1Δatg13Δ cells containing TAP‐tagged wild‐type Atg1 (wt) or the Atg1 kinase‐dead (kd) K54A mutant and wild‐type Atg13, the Atg13‐FV mutant or an empty plasmid were grown to mid log phase. Atg1 was immunoprecipitated and its autophosphorylation activity was measured by autoradiography in vitro.


( H ) pho8Δ60pho13Δatg13Δ expressing wild ‐ type Atg13 ( wt ) , Atg13 ‐ FV mutant ( FV ) or an empty plasmid were grown to mid log phase starved for 4 h in SD ‐ N medium . Pho8Δ60 ‐ specific alkaline phosphatase ( ALP ) activity ( nmol / min / mg ) was measured in three independent experiments as described in ' Materials methods ' , plotted as relative ALP activity with standard deviation ( s . d . ) compared to wild ‐ type controls .

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(H) pho8Δ60pho13Δatg13Δ cells expressing wild‐type Atg13 (wt), the Atg13‐FV mutant (FV) or an empty plasmid were grown to mid log phase and starved for 4 h in SD‐N medium. Pho8Δ60‐specific alkaline phosphatase (ALP) activity (nmol/min/mg) was measured in three independent experiments as described in 'Materials and methods', and plotted as relative ALP activity with standard deviation (s.d.) compared to the wild‐type controls.


( I ) atg13 expressing GFP ‐ Atg8 containing wild ‐ type Atg13 , Atg13 ‐ FV mutant or an empty plasmid were grown to mid log phase with without starvation for 4 h in SD ‐ N . Processing of endogenous Ape1 cleavage of GFP ‐ Atg8 was analysed by western blotting , quantified by calculating of versus uncleaved Ape1 or GFP ‐ Atg8 , respectively . asterisk marks a non ‐ specific band detected by Atg13 antibody . Figure source data can be found with Supplementary data .

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

(I) atg13 cells expressing GFP‐Atg8 and containing wild‐type Atg13, the Atg13‐FV mutant or an empty plasmid were grown to mid log phase with and without starvation for 4 h in SD‐N. Processing of endogenous Ape1 and cleavage of GFP‐Atg8 was analysed by western blotting, and quantified by calculating the ratio of cleaved versus uncleaved Ape1 or GFP‐Atg8, respectively. The asterisk marks a non‐specific band detected by the Atg13 antibody. Figure source data can be found with the Supplementary data.


( B ) GST ‐ Atg8 or GST ‐ Ubiquitin ( Ub ) was purified from E . coli bound to GSH beads . Wild ‐ type Atg1 ‐ TAP or VE EYE mutants were purified from yeast , incubated with immobilized GST bound analysed by western blotting .

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(B) GST‐Atg8 or GST‐Ubiquitin (Ub) was purified from E. coli and bound to GSH beads. Wild‐type Atg1‐TAP or the VE and EYE mutants were purified from yeast, incubated with the immobilized GST proteins and bound proteins analysed by western blotting.


( C ) ( C ) GST ‐ Atg8 GST ‐ Ubiquitin were immobilized as described in ( B ) , probed for their ability to bind Atg1 ‐ TAP isolated from wild ‐ type or atg13Δ .

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(C) (C) GST‐Atg8 and GST‐Ubiquitin were immobilized as described in (B), and probed for their ability to bind Atg1‐TAP isolated from wild‐type or atg13Δ cells.


( D ) atg1Δatg13Δ expressing as indicated wild ‐ type Atg1 ‐ TAP or VE or EYE mutants , wild ‐ type Atg13 or Atg13 ‐ FV mutant were grown to mid log phase . Atg1 was immunoprecipitated its association with Atg13 was determined by western blotting .

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(D) atg1Δatg13Δ cells expressing as indicated wild‐type Atg1‐TAP or the VE or EYE mutants, and wild‐type Atg13 or the Atg13‐FV mutant were grown to mid log phase. Atg1 was immunoprecipitated and its association with Atg13 was determined by western blotting.


( E ) GST , GST ‐ LC3B , GST ‐ GATE16 GST ‐ GABARAP were purified from E . coli bound to beads . HEK293 lysates prepared from expressing wild ‐ type ( wt ) HA ‐ ULK1 or DFP mutant were incubated with beads , bound analysed by western blotting .

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(E) GST, GST‐LC3B, GST‐GATE16 and GST‐GABARAP were purified from E. coli and bound to beads. HEK293 cell lysates prepared from cells expressing wild‐type (wt) HA‐ULK1 or the DFP mutant were incubated with the beads, and bound proteins analysed by western blotting.


( F ) HEK293 were co ‐ transfected with GFP or GFP ‐ GABARAP along with either wild ‐ type HA ‐ ULK1 or DFP mutant . GFP GFP ‐ GABARAP were immobilized on GFP ‐ Trap resin HA ‐ ULK1 binding was analysed by western blotting . Figure source data can be found with Supplementary data .

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(F) HEK293 cells were co‐transfected with GFP or GFP‐GABARAP along with either wild‐type HA‐ULK1 or the DFP mutant. GFP and GFP‐GABARAP were immobilized on GFP‐Trap resin and HA‐ULK1 binding was analysed by western blotting. Figure source data can be found with the Supplementary data.


( A ) atg1Δatg13Δ containing an empty control plasmid , wild ‐ type or indicated Atg1 mutants , Atg13 wild ‐ type , Atg13 ‐ FVmutant or an empty plasmid were grown to mid log phase . Processing of endogenous Ape1 was analysed by western blotting , quantified as described in legend of Figure 1I . Expression of different was controlled by immunoblotting with antibodies against Atg1 or Atg13 . asterisk marks a non ‐ specific band detected by Atg13 antibody .

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(A) atg1Δatg13Δ cells containing an empty control plasmid, wild‐type or the indicated Atg1 mutants, and Atg13 wild‐type, the Atg13‐FVmutant or an empty plasmid were grown to mid log phase. Processing of endogenous Ape1 was analysed by western blotting, and quantified as described in the legend of Figure 1I. Expression of the different proteins was controlled by immunoblotting with antibodies against Atg1 or Atg13. The asterisk marks a non‐specific band detected by the Atg13 antibody.


( B ) pho8Δ60pho13Δatg1Δ expressing wild ‐ type ( wt ) , kinase ‐ dead ( kd ) , Atg1 ‐ VE ( VE ) or ‐ EYE ( EYE ) mutants or an empty plasmid were grown to mid log phase starved for 4 h in SD ‐ N medium . Pho8Δ60 ‐ specific alkaline phosphatase ( ALP ) activity ( nmol / min / mg ) was measured in three independent experiments as described in ' Materials methods ' , plotted as relative ALP activity with standard deviation ( s . d . ) compared to wild ‐ type controls .

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(B) pho8Δ60pho13Δatg1Δ cells expressing wild‐type (wt), kinase‐dead (kd), Atg1‐VE (VE) or ‐EYE (EYE) mutants or an empty plasmid were grown to mid log phase and starved for 4 h in SD‐N medium. Pho8Δ60‐specific alkaline phosphatase (ALP) activity (nmol/min/mg) was measured in three independent experiments as described in 'Materials and methods', and plotted as relative ALP activity with standard deviation (s.d.) compared to the wild‐type controls.


( C ) as in ( A ) expressing GFP ‐ Atg8 were grown to mid log phase with starvation for 4 h in SD ‐ N medium . processing of GFP ‐ Atg8 was analysed quantified as in Figure 1I .

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(C) Cells as in (A) expressing GFP‐Atg8 were grown to mid log phase with starvation for 4 h in SD‐N medium. The processing of GFP‐Atg8 was analysed and quantified as in Figure 1I.


( D ) Exponentially growing atg1Δ atg1Δatg8Δ strains expressing PAS marker Ape1 ‐ RFP either GFP ‐ tagged wild ‐ type Atg1 or Atg1 ‐ VE mutant were examined by microscopy . Bar = 5 μm .

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(D) Exponentially growing atg1Δ and atg1Δatg8Δ strains expressing the PAS marker Ape1‐RFP and either GFP‐tagged wild‐type Atg1 or the Atg1‐VE mutant were examined by fluorescence microscopy. Bar=5 μm.


( E ) atg1Δ containing TAP ‐ tagged wild ‐ type or indicated Atg1 ‐ mutants were grown to mid log phase . Atg1 was immunoprecipitated its in vitro autophosphorylation activity was measured by autoradiography . kinase ‐ dead ( kd ) Atg1 ‐ mutant K54A serves as a negative control . Figure source data can be found with Supplementary data .

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(E) atg1Δ cells containing TAP‐tagged wild‐type or the indicated Atg1‐mutants were grown to mid log phase. Atg1 was immunoprecipitated and its in vitro autophosphorylation activity was measured by autoradiography. The kinase‐dead (kd) Atg1‐mutant K54A serves as a negative control. Figure source data can be found with the Supplementary data.


( A ) Exponentially growing ypt7Δ were starved in SD ‐ N medium for 4 h , lysed extract separated into a ( S ) a 5000 g pellet fraction . pellet was treated with ( + ) or without ( − ) TX ‐ 100 , centrifuged supernatant ( S ) pellet ( P ) fractions analysed by western blotting with anti ‐ Atg1 , anti ‐ Pep12 anti ‐ Pgk1 antibodies .

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(A) Exponentially growing ypt7Δ cells were starved in SD‐N medium for 4 h, lysed and the extract separated into a cytoplasmic (S) and a 5000 g membrane pellet fraction. The pellet was treated with (+) or without (−) TX‐100, centrifuged and the supernatant (S) and pellet (P) fractions analysed by western blotting with anti‐Atg1, anti‐Pep12 and anti‐Pgk1 antibodies.


( B ) Exponentially growing ypt7Δatg1Δ expressing wild ‐ type Atg1 or Atg1 ‐ VE mutant were starved in SD ‐ N for 4 h , lysed extract separated into ( S ) ( P ) fractions . fractions were analysed by western blotting with anti ‐ Atg1 , anti ‐ Pgk1 anti ‐ Ape1 antibodies , of Atg1 to Ape1 in pellet fractions was quantified indicated as a compared to wild ‐ type .

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(B) Exponentially growing ypt7Δatg1Δ cells expressing wild‐type Atg1 or the Atg1‐VE mutant protein were starved in SD‐N for 4 h, lysed and the extract separated into cytoplasmic (S) and membrane (P) fractions. The fractions were analysed by western blotting with anti‐Atg1, anti‐Pgk1 and anti‐Ape1 antibodies, and the ratio of Atg1 to Ape1 in the pellet fractions was quantified and indicated as a ratio compared to wild‐type cells.


( C ) HEK293 were co ‐ transfected with GFP ‐ GABARAP HA ‐ ULK1 , starved for 2 h , subsequently immunostained for HA ( red ) WIPI2 ( ) . arrows mark putative autophagosomes that stain positive for three markers . Bar = 10 μm .

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(C) HEK293 cells were co‐transfected with GFP‐GABARAP and HA‐ULK1, starved for 2 h, and subsequently immunostained for HA (red) and WIPI2 (white). The arrows mark putative autophagosomes that stain positive for all three markers. Bar=10 μm.


( D ) HEK293 stably expressing GFP ‐ LC3 were transfected with wild ‐ type HA ‐ ULK1 or DFP mutant , immunostained for HA WIPI2 after 2 h starvation . HA ‐ ULK1 WIPI2 spot numbers were counted using Imaris software in over 70 ( from one out of two experiments ) plotted as of ULK1 to WIPI2 . * * * Indicates a statistically significant P ‐ value of 0 . 0001 . One of two independent experiments is shown .

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(D) HEK293 cells stably expressing GFP‐LC3 were transfected with wild‐type HA‐ULK1 or the DFP mutant, and immunostained for HA and WIPI2 after 2 h starvation. HA‐ULK1 and WIPI2 spot numbers were counted using Imaris software in over 70 cells (from one out of two experiments) and plotted as the ratio of ULK1 to WIPI2 puncta. ***Indicates a statistically significant P‐value of 0.0001. One of the two independent experiments is shown.


( E ) of HA ‐ ULK1 WIPI2 spot numbers in HEK293 expressing wild ‐ type HA ‐ ULK1 or DFP mutant . were treated as above , spot number counted using Imaris software . A representative immunofluorescence image is shown in Supplementary Figure S3F . * * * * indicate P ‐ values of 0 . 0001 or 0 . 0158 , respectively . Figure source data can be found with Supplementary data .

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(E) Quantification of HA‐ULK1 and WIPI2 spot numbers in HEK293 cells expressing wild‐type HA‐ULK1 or the DFP mutant. Cells were treated as above, and the spot number counted using Imaris software. A representative immunofluorescence image is shown in Supplementary Figure S3F. *** and * indicate P‐values of 0.0001 or 0.0158, respectively. Figure source data can be found with the Supplementary data.


( A ) atg1Δ containing either wild ‐ type Atg1 or kinase ‐ dead Atg1 ‐ T226A mutant tagged with GFP were grown to mid log phase followed by starvation for 24 h in SD ‐ N . Samples were taken at indicated time points GFP cleavage was analysed by western blotting . Processing of endogenous Ape1 under starvation conditions monitors autophagic flux .

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(A) atg1Δ cells containing either wild‐type Atg1 or the kinase‐dead Atg1‐T226A mutant tagged with GFP were grown to mid log phase followed by starvation for 24 h in SD‐N. Samples were taken at indicated time points and GFP cleavage was analysed by western blotting. Processing of endogenous Ape1 under starvation conditions monitors the autophagic flux.


( B ) atg1Δ containing either GFP ‐ tagged wild ‐ type Atg1 or Atg1 ‐ VE mutant were grown analysed as in ( A ) .

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(B) atg1Δ cells containing either GFP‐tagged wild‐type Atg1 or the Atg1‐VE mutant were grown and analysed as in (A).