( a - f ) GFPAspergillus conidia , incubated overnight at room temperature , were untreated ( a - c ) or exposed ( d - f ) to 10 ng ml − 1 IL - 17A for 4 h followed by anti - IL - 17A - PE . Cells were green in ( a , b ) , red upon staining with anti - IL17A - PE ( b , e ) and double stained in the merged images ( c , f ) . Scale bars , 10 μm .

(a-f) GFPAspergillus conidia, incubated overnight at room temperature, were untreated (a-c) or exposed (d-f) to 10 ng ml−1 IL-17A for 4 h followed by anti-IL-17A-PE. Cells were green in (a,b), red upon staining with anti-IL17A-PE (b,e) and double stained in the merged images (c,f). Scale bars, 10 μm.


( g , h ) IL - 17A promotes biofilm formation in A . fumigatus AF293 strain . SEM images refer to cells untreated ( g ) or pretreated ( h ) with 10 ng ml − 1 IL - 17A for 4 h and subsequently cultured onto plastic discs for 24 h . Scale bars , 20 μm .

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(g,h) IL-17A promotes biofilm formation in A. fumigatus AF293 strain. SEM images refer to cells untreated (g) or pretreated (h) with 10 ng ml−1 IL-17A for 4 h and subsequently cultured onto plastic discs for 24 h. Scale bars, 20 μm.


( i - k ) IL - 17A induces autophagy in Aspergillus . Representative electron micrographs of vacuoles ( v ) in A . fumigatus AF293 unexposed ( k ) or exposed to 10 ng ml − 1 IL - 17A ( j ) or 15 nM rapamycin ( i ) for 4 h before TEM . Scale bars , 500 nm .

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(i-k) IL-17A induces autophagy in Aspergillus. Representative electron micrographs of vacuoles (v) in A. fumigatus AF293 unexposed (k) or exposed to 10 ng ml−1 IL-17A (j) or 15 nM rapamycin (i) for 4 h before TEM. Scale bars, 500 nm.


( l ) IL - 17A binds the Aspergillus Crf1p protein . Dose - dependent binding of IL - 17A to Crf1p ( red circle ) , Gel1p ( light blue circle ) or the Aspergillus - derived polysaccharides β - 1 , 3 - D - glucan ( blue circle ) , α - 1 , 3 - D - glucan ( open circle ) and galactomannan ( grey circle ) . The optical density at 450 nm was corrected by subtracting the optical density at 570 nm ( normalized OD ) in the enzyme - linked immunosorbent assay ( ELISA ) on microtitre - coated plates . Data are mean ± s . d . ( n = 5 ) . P - values ranged from 0 . 01 to 0 . 001 for IL - 17A - treated versus untreated ( 0 ) Crf1p ( one - way analysis of variance , Bonferroni post - test ) .

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

(l) IL-17A binds the Aspergillus Crf1p protein. Dose-dependent binding of IL-17A to Crf1p (red circle), Gel1p (light blue circle) or the Aspergillus-derived polysaccharides β-1,3-D-glucan (blue circle), α-1,3-D-glucan (open circle) and galactomannan (grey circle). The optical density at 450 nm was corrected by subtracting the optical density at 570 nm (normalized OD) in the enzyme-linked immunosorbent assay (ELISA) on microtitre-coated plates. Data are mean±s.d. (n=5). P-values ranged from 0.01 to 0.001 for IL-17A-treated versus untreated (0) Crf1p (one-way analysis of variance, Bonferroni post-test).


( m ) ELISA assay showing failure of IL - 17A to bind the Aspergillus mutant crf1Δ ( blue square ) as opposed to the wild - type strain AF293 ( red square ) . Data are mean ± s . d . ( n = 5 ) . P - values ranged from 0 . 05 to 0 . 01 , wild - type versus mutant strain exposed to 1 or 10 ng ml − 1 IL - 17A ( one - way analysis of variance , Bonferroni post - test ) .

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

(m) ELISA assay showing failure of IL-17A to bind the Aspergillus mutant crf1Δ (blue square) as opposed to the wild-type strain AF293 (red square). Data are mean±s.d. (n =5). P-values ranged from 0.05 to 0.01, wild-type versus mutant strain exposed to 1 or 10 ng ml−1 IL-17A (one-way analysis of variance, Bonferroni post-test).


( n - q ) IL - 17A promotes filamentation in the mutant crf1Δ ( o ) but not the wild - type AF293 ( q ) strain after 24 - h exposure in liquid culture . Scale bars , 500 μm .

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(n-q) IL-17A promotes filamentation in the mutant crf1Δ (o) but not the wild-type AF293 (q) strain after 24-h exposure in liquid culture. Scale bars, 500 μm.


( r , s ) C57BL / 6 mice were infected intranasally with the Aspergillus crf1Δ mutant ( r ) or the AF293 ( s ) strain and monitored for fungal growth ( log10 colony - forming units ( CFU ) ± s . d . ) in the lungs 2 days later . Fungal cells were exposed or not ( none ) to 10 ng ml − 1 IL - 17A for 4 h , extensively washed and injected . Data are mean ± s . d . ( n = 4 ) . The actual P - values are indicated ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(r,s) C57BL/6 mice were infected intranasally with the Aspergillus crf1Δ mutant (r) or the AF293 (s) strain and monitored for fungal growth (log10 colony-forming units (CFU)±s.d.) in the lungs 2 days later. Fungal cells were exposed or not (none) to 10 ng ml−1 IL-17A for 4 h, extensively washed and injected. Data are mean±s.d. (n=4). The actual P-values are indicated (two-tailed t-test).


Live cells DIC images and DAPI nucleoids staining of WT DHFR and I91L + W133V DHFR strains after being grown at 30 ° C , 37 ° C , or 42 ° C ( I91L + W133V was grown at 40 ° C ) in M9 medium supplemented with amino acids for 4 hours ( see Methods ) .

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

Live cells DIC images and DAPI nucleoids staining of WT DHFR and I91L+W133V DHFR strains after being grown at 30°C, 37°C, or 42°C (I91L+W133V was grown at 40°C) in M9 medium supplemented with amino acids for 4 hours (see Methods).


Functional complementation of WT DHFR from an arabinose inducible pBAD plasmid rescues filamentation of mutant strains grown at 42 ° C ( for WT , W133V and V75H + I155A strains ) or at 40 ° C ( for I91L + W133V and V75H + I91L + I155A ) in M9 medium supplemented with amino acids . For the boxplots in panels , F the central band represents the median of the distribution , the box ends represent the 25th and 75th percentile , the whiskers represent the 10th and 90th percentile , while the dots represent the 5th and 95th percentile . Data was usually obtained from 2 - 3 biological replicates . The number of cells used to derive the boxplot distributions in the different panels range usually between 200 to 450 ( please refer to Figure 1 source data for exact number of cells for each dataset ) . See related Figure EV1B .

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

Functional complementation of WT DHFR from an arabinose inducible pBAD plasmid rescues filamentation of mutant strains grown at 42°C (for WT, W133V and V75H+I155A strains) or at 40°C (for I91L+W133V and V75H+I91L+I155A) in M9 medium supplemented with amino acids. For the boxplots in panels , F the central band represents the median of the distribution, the box ends represent the 25th and 75th percentile, the whiskers represent the 10th and 90th percentile, while the dots represent the 5th and 95th percentile. Data was usually obtained from 2-3 biological replicates. The number of cells used to derive the boxplot distributions in the different panels range usually between 200 to 450 (please refer to Figure 1 source data for exact number of cells for each dataset). See related Figure EV1B.


( A ) and ( B ) shows abundance of selected nucleotides and amino acids for mutants I91L + W133V and W133V as well as WT strain treated with 0 . 5μg / ml of Tmp ( Trimethoprim ) after 4 hours of growth at 40 / 42 ° C in M9 minimal medium ( condition of no filamentation ) , Concentration of all metabolites were normalized to WT levels at 4 hours when grown under similar conditions . In minimal medium ( B ) Methionine levels are extremely low Levels of purines ( IMP , AMP ) are also largely rescued with amino acid supplementation , however dTMP , dTDP and dTTP levels remain extremely low . Error bars represent SEM of at least three biological replicates ( see Methods ) . The dashed line in each plot represents value of 1 for WT .

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

(A) and (B) shows abundance of selected nucleotides and amino acids for mutants I91L+W133V and W133V as well as WT strain treated with 0.5μg/ml of Tmp (Trimethoprim) after 4 hours of growth at 40/42°C in M9 minimal medium (condition of no filamentation), Concentration of all metabolites were normalized to WT levels at 4 hours when grown under similar conditions. In minimal medium (B) Methionine levels are extremely low Levels of purines (IMP, AMP) are also largely rescued with amino acid supplementation, however dTMP, dTDP and dTTP levels remain extremely low. Error bars represent SEM of at least three biological replicates (see Methods). The dashed line in each plot represents value of 1 for WT.


( A ) recA ( B ) recN genes measured by quantitative PCR when WT and mutant strains are grown in M9 medium with or without supplementation of amino acids or dTMP . WT and mutant strains were grown for 4 hours of growth in the indicated medium at 37 ° C , while WT treated with different concentrations of Tmp were grown for 4 hours at 42 ° C . Brown bars ( M9 + AA ) in the gray shaded area correspond to filamentation conditions and these are associated with pronounced upregulation of all three SOS genes . On the other hand , conditions with loss of filamentation ( with dTMP or no supplementation ) show much less expression . Error bars represent SD of 2 - 3 biological replicates ( see Methods ) .

O O O O O O O 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 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 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

(A) recA (B) recN genes measured by quantitative PCR when WT and mutant strains are grown in M9 medium with or without supplementation of amino acids or dTMP. WT and mutant strains were grown for 4 hours of growth in the indicated medium at 37°C, while WT treated with different concentrations of Tmp were grown for 4 hours at 42°C. Brown bars (M9+AA) in the gray shaded area correspond to filamentation conditions and these are associated with pronounced upregulation of all three SOS genes. On the other hand, conditions with loss of filamentation (with dTMP or no supplementation) show much less expression. Error bars represent SD of 2-3 biological replicates (see Methods).


WT cells were treated with different concentrations of Tmp at 42 ° C for varying amounts of time in amino acid supplemented M9 medium ( panel B ) or in M9 media supplemented with both amino acids and GMP ( panel C ) , following which they were spotted on M9 + AA plates and allowed to grow at 30 ° C . Colonies were counted next day . In the presence of only amino acids , there was no loss in viability for any concentration of Tmp ( panel B ) , despite extensive filamentation ( Figure EV1E ) . In contrast , in the presence of amino acids and GMP , the cells showed sharp loss in viability when grown at high Tmp concentrations . In both panels , error bars represent SD of two biological replicates . The dashed line represents cfu at one - hour timepoint .

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 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 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 B-CONTROLLED_VAR 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

WT cells were treated with different concentrations of Tmp at 42°C for varying amounts of time in amino acid supplemented M9 medium (panel B) or in M9 media supplemented with both amino acids and GMP (panel C), following which they were spotted on M9+AA plates and allowed to grow at 30°C. Colonies were counted next day. In the presence of only amino acids, there was no loss in viability for any concentration of Tmp (panel B), despite extensive filamentation (Figure EV1E). In contrast, in the presence of amino acids and GMP, the cells showed sharp loss in viability when grown at high Tmp concentrations. In both panels, error bars represent SD of two biological replicates. The dashed line represents cfu at one-hour timepoint.


Expression of SOS response genes recA , recN and sulA when mutant strains and Tmp treated WT cells are grown in M9 medium supplemented with amino acids ( black bars ) or in the presence of both amino acids and GMP ( gray bars ) at 42 ° C ( I91L + W133V was grown at 40 ° C ) . Error bars represent SD of 2 - 3 biological replicates .

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Expression of SOS response genes recA, recN and sulA when mutant strains and Tmp treated WT cells are grown in M9 medium supplemented with amino acids (black bars) or in the presence of both amino acids and GMP (gray bars) at 42°C (I91L+W133V was grown at 40°C). Error bars represent SD of 2-3 biological replicates.


Comparison of growth rates of WT and mutant DHFR strains at 42 ° C ( 40 ° C for I91L + W133V and V75H + I91L + I155A ) in minimal medium that is supplemented with amino acids and / or 1mM dTMP . Except for W133V and to a lesser extent for V75H + I155A , the effect of dTMP on growth rates is only modest . Error bars represent SEM of 3 biological replicates .

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

Comparison of growth rates of WT and mutant DHFR strains at 42°C (40°C for I91L+W133V and V75H+I91L+I155A) in minimal medium that is supplemented with amino acids and/or 1mM dTMP. Except for W133V and to a lesser extent for V75H+I155A, the effect of dTMP on growth rates is only modest. Error bars represent SEM of 3 biological replicates.


in vitro activity of purified Thymidylate Kinase ( Tmk ) as a function of dTMP concentration shows Michaelis - Menten ( MM ) like kinetics . ATP concentration is saturating at 1mM . The KM of dTMP is 13μM . The error bars represent SD of three technical replicates .

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

in vitro activity of purified Thymidylate Kinase (Tmk) as a function of dTMP concentration shows Michaelis-Menten (MM) like kinetics. ATP concentration is saturating at 1mM. The KM of dTMP is 13μM. The error bars represent SD of three technical replicates.


The green points were acquired during growth of WT , W133V and I91L + W133V mutants in the presence of different concentrations of thymidine , which follow the red curve . The inset plot shows the same graph with selected datapoints for I91L + W133V mutant . The cyan circle shows I91L + W133V mutant in the absence of any metabolite supplementation , while red and black points indicate metabolite levels following thymidine and dTMP supplementation respectively .

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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O

The green points were acquired during growth of WT, W133V and I91L+W133V mutants in the presence of different concentrations of thymidine, which follow the red curve. The inset plot shows the same graph with selected datapoints for I91L+W133V mutant. The cyan circle shows I91L+W133V mutant in the absence of any metabolite supplementation, while red and black points indicate metabolite levels following thymidine and dTMP supplementation respectively.


Steady state levels of different metabolites obtained from metabolomics were used to derive activity curves for ( A ) Adenylate Kinase ( AK ) ( B ) Uridylate Kinase ( PyrH ) ( C ) Adenylosuccinate Lyase ( PurB ) and ( D ) GAR Transformylase ( PurN ) . AK , PyrH and PurB appear to follow hyperbolic MM kinetics in vivo , while PurN shows Hill - like dependence with cooperativity . In panel ( A ) , the red data points were acquired during growth in amino acid supplemented M9 medium in the absence of external AMP and are derived from WT and various stabilizing and destabilizing point mutations of AK ( Adkar et al . , 2019 ) as well as DHFR mutants used in the present study , while black points were derived exclusively from cultures of AK mutants supplemented with 1mM external AMP ( Adkar et al . , 2019 ) . Data points for all other panels ( B - D ) are derived from mutant DHFR strains in the present study . Since SAICAR , AICAR , GAR and FGAR levels were undetectable in WT , all data points in ( C - D ) are relative to those observed in I91L + W133V mutant following 4 hours of growth at 40 ° C . Data points in all panels represent metabolite levels for individual biological replicates without averaging .

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

Steady state levels of different metabolites obtained from metabolomics were used to derive activity curves for (A) Adenylate Kinase (AK) (B) Uridylate Kinase (PyrH) (C) Adenylosuccinate Lyase (PurB) and (D) GAR Transformylase (PurN). AK, PyrH and PurB appear to follow hyperbolic MM kinetics in vivo, while PurN shows Hill-like dependence with cooperativity. In panel (A), the red data points were acquired during growth in amino acid supplemented M9 medium in the absence of external AMP and are derived from WT and various stabilizing and destabilizing point mutations of AK (Adkar et al., 2019) as well as DHFR mutants used in the present study, while black points were derived exclusively from cultures of AK mutants supplemented with 1mM external AMP (Adkar et al., 2019). Data points for all other panels (B-D) are derived from mutant DHFR strains in the present study. Since SAICAR, AICAR, GAR and FGAR levels were undetectable in WT, all data points in (C-D) are relative to those observed in I91L+W133V mutant following 4 hours of growth at 40°C. Data points in all panels represent metabolite levels for individual biological replicates without averaging.


( A ) Motility assay of C . c . ΔpseI cells expressing predicted Leg or Pse synthases ( LegI or PseI , in yellow or red , respectively ) from Plac on pSRK - Gm . Overnight cultures were spotted on PYE soft ( 0 . 3 % ) agar plates with gentamycin and IPTG ( 0 . 5 mM ) and incubated for 3 days at 30 ° C . Only predicted PseI - like synthase coding sequences ( CDSs ) restore motility of the C . c . ΔpseI cells . Note that the predicted synthases labeled in white do not share similarities with LegI or PseI of C . jejuni . Motility assays are representative of three independent experiments .

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(A) Motility assay of C.c. ΔpseI cells expressing predicted Leg or Pse synthases (LegI or PseI, in yellow or red, respectively) from Plac on pSRK-Gm. Overnight cultures were spotted on PYE soft (0.3%) agar plates with gentamycin and IPTG (0.5 mM) and incubated for 3 days at 30°C. Only predicted PseI-like synthase coding sequences (CDSs) restore motility of the C.c. ΔpseI cells. Note that the predicted synthases labeled in white do not share similarities with LegI or PseI of C. jejuni. Motility assays are representative of three independent experiments.


( B ) Immunoblot of cell extracts from cells in ( A ) probed with antibodies to C . crescentus FljK ( FljKCc ) . The migration of flagellins in ΔpseI cells is shifted towards lower molecular mass suggesting that post - translational modification of flagellin is defective in ΔpseI mutant . Molecular sizes are indicated by the blues lines ( in kDa ) . Bs : Brevundimonas subvibrioides , Bl : Brevundimonas lutea , Bv : Brevundimonas viscosa , Dv : Dermabacter vaginalis , Li : Leptospira interrogans , Tp : Treponema pallidum , Td : Treponema denticola , AbACICU : Acinetobacter baumannii ACICU , AbLUH : Acinetobacter baumannii LUH , AbLAC : Acinetobacter baumannii LAC - 4 , Lp : Legionella pneumophila , PaY82 : Pseudomonas aeruginosa Y82 , PaPAPS475 : Pseudomonas aeruginosa PAPS475 , Sj : Shewanella japonicum , Basu : Bacillus subtilis , Ks : Kurthia sibirica , Cb : Clostridium botulinum , Gk : Geobacillus kaustophilus , Mh : Moorella humiferrea , Myco : Mycobacterium sp . KS0706 , HrrPV6 : Halorubrum sp . PV6 , Ms : Methanobrevibacter smithii . All the genes come from synthetic fragments codon optimized for E . coli ( except the synthase CDS from Gk that is codon optimized for C . c . ) . Empty carets indicate the position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Yellow boxes indicate PseI functional orthologs . Immunoblots are representative of at least two 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot of cell extracts from cells in (A) probed with antibodies to C. crescentus FljK (FljKCc). The migration of flagellins in ΔpseI cells is shifted towards lower molecular mass suggesting that post-translational modification of flagellin is defective in ΔpseI mutant. Molecular sizes are indicated by the blues lines (in kDa). Bs: Brevundimonas subvibrioides, Bl: Brevundimonas lutea, Bv: Brevundimonas viscosa, Dv: Dermabacter vaginalis, Li: Leptospira interrogans, Tp: Treponema pallidum, Td: Treponema denticola, AbACICU: Acinetobacter baumannii ACICU, AbLUH: Acinetobacter baumannii LUH, AbLAC: Acinetobacter baumannii LAC-4, Lp: Legionella pneumophila, PaY82: Pseudomonas aeruginosa Y82, PaPAPS475: Pseudomonas aeruginosa PAPS475, Sj: Shewanella japonicum, Basu: Bacillus subtilis, Ks: Kurthia sibirica, Cb: Clostridium botulinum, Gk: Geobacillus kaustophilus, Mh: Moorella humiferrea, Myco: Mycobacterium sp. KS0706, HrrPV6: Halorubrum sp. PV6, Ms: Methanobrevibacter smithii. All the genes come from synthetic fragments codon optimized for E. coli (except the synthase CDS from Gk that is codon optimized for C.c.). Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Yellow boxes indicate PseI functional orthologs. Immunoblots are representative of at least two independent experiments.


( A ) and ( B ) Motility assays of WT , ΔlegI ( A ) and ΔflmG ( B ) B . s . cells . Overnight cultures of WT and mutants harboring the empty pSRK - Gm vector ( + Plac ) or the corresponding complementing plasmid were spotted on PYE soft agar plates with gentamycin and IPTG ( 0 . 5 mM ) and incubated for 7 days at 30 ° C . Motility assays are representative of three independent experiments . The same original picture was used to generate Figs 3B and EV2C .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) and (B) Motility assays of WT, ΔlegI (A) and ΔflmG (B) B.s. cells. Overnight cultures of WT and mutants harboring the empty pSRK-Gm vector (+Plac) or the corresponding complementing plasmid were spotted on PYE soft agar plates with gentamycin and IPTG (0.5 mM) and incubated for 7 days at 30°C. Motility assays are representative of three independent experiments. The same original picture was used to generate Figs 3B and EV2C.


( C ) and ( D ) Immunoblots of cell extracts ( cells ) and supernatants ( SN ) of cultures from WT and mutant cells probed with antibodies to FljKCc , that detect the B . s . flagellins . The estimated molecular mass ( in kDa ) are indicated by the blue lines on the left . Empty carets indicate the position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Immunoblots are representative of at least two 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) and (D) Immunoblots of cell extracts (cells) and supernatants (SN) of cultures from WT and mutant cells probed with antibodies to FljKCc, that detect the B.s. flagellins. The estimated molecular mass (in kDa) are indicated by the blue lines on the left. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Immunoblots are representative of at least two independent experiments.


( F ) Flagellum length measurements determined by TEM of WT and mutants B . s . cells : WT , ΔlegX ( ΔBresu _ 3267 ) , ΔlegB ( ΔBresu _ 3266 ) expressing legX in trans from Plac on a plasmid , ΔlegH ( ΔBresu _ 0506 ) , ΔlegI ( ΔBresu _ 0507 ) , ΔflmG ( ΔBresu _ 2406 ) and ΔlegI ΔflmG . Box plots represent the distribution of the flagellum lengths . The cross indicates the average length , the central band indicates the median , the upper and lower sides of the box indicate the upper and lower quartiles , respectively , and the whiskers indicate the minimum and the maximum length measured . Twenty - five independent flagella were measured different cells ( biological replicates ) for each strain from TEM images .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Flagellum length measurements determined by TEM of WT and mutants B.s. cells: WT, ΔlegX (ΔBresu_3267), ΔlegB (ΔBresu_3266) expressing legX in trans from Plac on a plasmid, ΔlegH (ΔBresu_0506), ΔlegI (ΔBresu_0507), ΔflmG (ΔBresu_2406) and ΔlegI ΔflmG. Box plots represent the distribution of the flagellum lengths. The cross indicates the average length, the central band indicates the median, the upper and lower sides of the box indicate the upper and lower quartiles, respectively, and the whiskers indicate the minimum and the maximum length measured. Twenty-five independent flagella were measured different cells (biological replicates) for each strain from TEM images.


( A ) Motility assays of B . s . ΔlegI cells complemented with LegI - type ( yellow ) or PseI - type ( red ) synthases expressed from Plac on pSRK - Gm . Motility assays 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 O O O O O O O O O O O O O O O O O O O O O

(A) Motility assays of B.s. ΔlegI cells complemented with LegI-type (yellow) or PseI-type (red) synthases expressed from Plac on pSRK-Gm. Motility assays are representative of three independent experiments.


( B ) Immunoblots probed with antibodies to FljKCc , revealing the cell extracts levels of flagellin in B . s . ΔlegI derivatives shown in ( A ) . The blue lines represent the migration of the molecular size standards ( in kDa ) . Bl : Brevundimonas lutea , Bv : Brevundimonas viscosa , Dv : Dermabacter vaginalis , Li : Leptospira interrogans , Tp : Treponema pallidum , Td : Treponema denticola , AbACICU : Acinetobacter baumannii ACICU , AbLUH : Acinetobacter baumannii LUH , AbLAC : Acinetobacter baumannii LAC - 4 , Lp : Legionella pneumophila , PaY82 : Pseudomonas aeruginosa Y82 , PaPAPS475 : Pseudomonas aeruginosa PAPS475 , Sj : Shewanella japonicum , Basu : Bacillus subtilis , Ks : Kurthia sibirica , Cb : Clostridium botulinum , Gk : Geobacillus kaustophilus , Mh : Moorella humiferrea , Myco : Mycobacterium sp . KS0706 , HrrPV6 : Halorubrum sp . PV6 , Ms : Methanobrevibacter smithii . All the genes come from synthetic fragments codon optimized for E . coli ( except the synthase CDS from Gk that is codon optimized for C . c . ) . Empty carets indicate the position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Red boxes indicate LegI functional orthologs . Immunoblots are representative of at least two 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblots probed with antibodies to FljKCc, revealing the cell extracts levels of flagellin in B.s. ΔlegI derivatives shown in (A). The blue lines represent the migration of the molecular size standards (in kDa). Bl: Brevundimonas lutea, Bv: Brevundimonas viscosa, Dv: Dermabacter vaginalis, Li: Leptospira interrogans, Tp: Treponema pallidum, Td: Treponema denticola, AbACICU: Acinetobacter baumannii ACICU, AbLUH: Acinetobacter baumannii LUH, AbLAC: Acinetobacter baumannii LAC-4, Lp: Legionella pneumophila, PaY82: Pseudomonas aeruginosa Y82, PaPAPS475: Pseudomonas aeruginosa PAPS475, Sj: Shewanella japonicum, Basu: Bacillus subtilis, Ks: Kurthia sibirica, Cb: Clostridium botulinum, Gk: Geobacillus kaustophilus, Mh: Moorella humiferrea, Myco: Mycobacterium sp. KS0706, HrrPV6: Halorubrum sp. PV6, Ms: Methanobrevibacter smithii. All the genes come from synthetic fragments codon optimized for E. coli (except the synthase CDS from Gk that is codon optimized for C.c.). Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Red boxes indicate LegI functional orthologs. Immunoblots are representative of at least two independent experiments.


( A ) Motility assays of WT B . s . and ΔBresu _ 3266 ( ΔlegB ) cells expressing Bresu _ 3266 ( LegB ) , Bresu _ 3267 ( LegX ) or Bresu _ 3266 - Bresu _ 3267 ( LegB - LegX , Bresu _ 3266 - 67 ) from Plac on plasmid pSRK - Gm . Motility assays are representative of three independent experiments . ( D ) Motility assay of WT B . s . and ΔBresu _ 0506 ( ΔlegH ) cells complemented with a plasmid expressing Bresu _ 0506 ( LegH ) from Plac on pSRK - Gm . Motility assays 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Motility assays of WT B.s. and ΔBresu_3266 (ΔlegB) cells expressing Bresu_3266 (LegB), Bresu_3267 (LegX) or Bresu_3266-Bresu_3267 (LegB-LegX, Bresu_3266-67) from Plac on plasmid pSRK-Gm. Motility assays are representative of three independent experiments. (D) Motility assay of WT B.s. and ΔBresu_0506 (ΔlegH) cells complemented with a plasmid expressing Bresu_0506 (LegH) from Plac on pSRK-Gm. Motility assays are representative of three independent experiments.


( B ) Immunoblots probed with anti - FljKCc antibodies to reveal flagellins in cell extracts ( cells ) and supernatants ( SN ) of the strains described in ( A ) . ( E ) Immunoblots probed with anti - FljKCc antibodies to reveal flagellins in cell extracts ( cells ) and supernatants ( SN ) of the strains described in ( D ) . Immunoblots are representative of two 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblots probed with anti-FljKCc antibodies to reveal flagellins in cell extracts (cells) and supernatants (SN) of the strains described in (A). (E) Immunoblots probed with anti-FljKCc antibodies to reveal flagellins in cell extracts (cells) and supernatants (SN) of the strains described in (D). Immunoblots are representative of two independent experiments.


( A ) Motility assay of ΔBresu _ 3267 ( ΔlegX ) B . s . cells compared to WT B . s . cells harbouring the empty pSRK - Gm vector ( + Plac ) or a complementing derivative with PtmE / LegX orthologs from C . jejuni ( Cj ) , Tanerella forsythia ( Tf ) or P . aeruginosa NMI1897 ( PaNMI ) . Motility assays 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Motility assay of ΔBresu_3267 (ΔlegX) B.s. cells compared to WT B.s. cells harbouring the empty pSRK-Gm vector (+Plac) or a complementing derivative with PtmE/LegX orthologs from C. jejuni (Cj), Tanerella forsythia (Tf) or P. aeruginosa NMI1897 (PaNMI). Motility assays are representative of three independent experiments.


( B ) Immunoblots probed with anti - FljKCc antibodies from cell lysates ( cells ) and supernatants ( SN ) of WT B . s . and ΔBresu _ 3267 ( ΔlegX ) cultures harbouring either the empty pSRK - Gm vector ( + Plac ) or a complementing plasmid expressing different PtmE / LegX orthologs from C . jejuni ( Cj ) , Tanerella forsythia ( Tf ) or P . aeruginosa NMI1897 ( PaNMI ) . Molecular size standards are indicated by the blue lines with the corresponding value in kDa . Empty carets indicate the position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Immunoblots are representative of two 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblots probed with anti-FljKCc antibodies from cell lysates (cells) and supernatants (SN) of WT B.s. and ΔBresu_3267 (ΔlegX) cultures harbouring either the empty pSRK-Gm vector (+Plac) or a complementing plasmid expressing different PtmE/LegX orthologs from C. jejuni (Cj), Tanerella forsythia (Tf) or P. aeruginosa NMI1897 (PaNMI). Molecular size standards are indicated by the blue lines with the corresponding value in kDa. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Immunoblots are representative of two independent experiments.


( B ) Anti - FljKCc immunoblot analyses of whole cell lysates ( cells ) from C . crescentus ( C . c . ) mutant cultures expressing fljKBssyn ( codon optimized for E . coli ) and flmGBs from the replicative pMT463 plasmid , in the presence or absence of a compatible integrative plasmid carrying a legBs synthetic operon ( pXGFP4 - legBs ) and in the presence or absence of an additional compatible replicative plasmid carrying Bresu _ 3267 ( pMT375 ) . In the presence of pXGFP4 - legBs ( integrated at the xylX locus ) and pMT375 - Bresu _ 3267 , FljKBssyn migration is shifted toward higher molecular mass , indicative of glycosylation . Note that in the C . c . Δfljx6 background , the six flagellin - encoding genes have been deleted and the protein detected by the antibodies only corresponds to FljKBssyn . The legBs synthetic operon ( in red ) is composed of Bresu _ 3266 , Bresu _ 0765 , Bresu _ 0506 , Bresu _ 3264 , Bresu _ 0507 ( legI ) and Bresu _ 3265 . Molecular masses are indicated in kDa by the blue lines . Empty carets indicate the position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Asterisk indicates a modification of flagellin by FlmGBs that does not require Pse or Leg . Immunoblots are representative of at least two 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Anti-FljKCc immunoblot analyses of whole cell lysates (cells) from C. crescentus (C.c.) mutant cultures expressing fljKBssyn (codon optimized for E. coli) and flmGBs from the replicative pMT463 plasmid, in the presence or absence of a compatible integrative plasmid carrying a legBs synthetic operon (pXGFP4-legBs) and in the presence or absence of an additional compatible replicative plasmid carrying Bresu_3267 (pMT375). In the presence of pXGFP4-legBs (integrated at the xylX locus) and pMT375-Bresu_3267, FljKBssyn migration is shifted toward higher molecular mass, indicative of glycosylation. Note that in the C.c. Δfljx6 background, the six flagellin-encoding genes have been deleted and the protein detected by the antibodies only corresponds to FljKBssyn. The legBs synthetic operon (in red) is composed of Bresu_3266, Bresu_0765, Bresu_0506, Bresu_3264, Bresu_0507 (legI) and Bresu_3265. Molecular masses are indicated in kDa by the blue lines. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Asterisk indicates a modification of flagellin by FlmGBs that does not require Pse or Leg. Immunoblots are representative of at least two independent experiments.


( C ) Anti - FljKCc immunoblot analyses of whole cell lysates from C . crescentus mutant cultures expressing FlmGCc , FlmGBs and FlmGCc - Bschim chimera from the replicative pSRK - Gm plasmid ( Plac ) in the presence of a compatible integrative plasmid carrying a legBs synthetic operon ( pXGFP4 - legBs ) and in the presence or absence of an additional compatible replicative plasmid carrying Bresu _ 3267 ( pMT375 ) . Molecular masses are indicated in kDa by the blue lines . Empty carets indicate the position of modified ( glycosylated ) flagellin , whereas filled carets mark unmodified flagellin . Immunoblots are representative of at least two independent experiments .

(C) Anti-FljKCc immunoblot analyses of whole cell lysates from C. crescentus mutant cultures expressing FlmGCc, FlmGBs and FlmGCc-Bschim chimera from the replicative pSRK-Gm plasmid (Plac) in the presence of a compatible integrative plasmid carrying a legBs synthetic operon (pXGFP4-legBs) and in the presence or absence of an additional compatible replicative plasmid carrying Bresu_3267 (pMT375). Molecular masses are indicated in kDa by the blue lines. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Immunoblots are representative of at least two independent experiments.


Three independent samples ( Exp 1 to 3 ) of EV secreted by labeled 3T3 - F442A cells were analyzed by mass spectrometry ( in duplicate injections , Inj1 / 2 ) . The percentage of proteins bearing at least one peptide containing a heavy amino acid is 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 O O O O O O O O O O O O O O O O O O O O O O O

Three independent samples (Exp 1 to 3) of EV secreted by labeled 3T3-F442A cells were analyzed by mass spectrometry (in duplicate injections, Inj1/2). The percentage of proteins bearing at least one peptide containing a heavy amino acid is indicated.


Two human ( SKMEL28 and 1205Lu ) and a murine ( B16BL6 ) melanoma cell lines were exposed , or not , to the indicated EV from primary murine adipocytes obtained from lean mice fed a normal diet ( ND ) or obese mice fed a high fat diet ( HFD ) , then FAO was measured ( n = 5 for B16BL6 , SKMEL28 HFD EV and 1205Lu ND EV and n = 6 for other conditions ) .

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

Two human (SKMEL28 and 1205Lu) and a murine (B16BL6) melanoma cell lines were exposed, or not, to the indicated EV from primary murine adipocytes obtained from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD), then FAO was measured (n =5 for B16BL6, SKMEL28 HFD EV and 1205Lu ND EV and n=6 for other conditions).


Volcano plot of mass spectrometry - based quantitative proteomics results showing relative abundance of proteins in primary murine adipocyte EV from obese mice ( HFD ) , as compared to those from lean mice ( ND ) . The dashed lines indicate cut - off values and points colored in grey indicate proteins that display non - significant fold - change by Welch t - test between both conditions ( n = 3 for ND , 4 for HFD ) . Proteins involved in FAO are indicated by yellow dots .

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

Volcano plot of mass spectrometry-based quantitative proteomics results showing relative abundance of proteins in primary murine adipocyte EV from obese mice (HFD), as compared to those from lean mice (ND). The dashed lines indicate cut-off values and points colored in grey indicate proteins that display non-significant fold-change by Welch t-test between both conditions (n=3 for ND, 4 for HFD). Proteins involved in FAO are indicated by yellow dots.


Western blot analysis of the indicated FAO enzymes in the EV secreted by primary adipocytes from lean ( ND ) and obese ( HFD ) mice ( top panel ) and from human individuals with varying BMI ( normal weight , NW ; overweight , OW and obese , OB ) ( bottom panel ) . For each blot , extracts from 3 independent batches of murine samples or 3 independent individuals for human samples ( 1 - 3 ) are shown . Flotillin 1 ( FLOT1 ) is used as a loading control .

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

Western blot analysis of the indicated FAO enzymes in the EV secreted by primary adipocytes from lean (ND) and obese (HFD) mice (top panel) and from human individuals with varying BMI (normal weight, NW; overweight, OW and obese, OB) (bottom panel). For each blot, extracts from 3 independent batches of murine samples or 3 independent individuals for human samples (1-3) are shown. Flotillin 1 (FLOT1) is used as a loading control.


Western blot analysis of the indicated FAO enzymes in melanoma cells treated , or not , with EV from lean ( ND ) and obese ( HFD ) mice . Tubulin ( TUB ) is used as a loading control .

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

Western blot analysis of the indicated FAO enzymes in melanoma cells treated, or not, with EV from lean (ND) and obese (HFD) mice. Tubulin (TUB) is used as a loading control.


RT - qPCR analysis of mRNAs for the indicated genes in 1205Lu cells treated or not with EV secreted by primary adipocytes from lean ( ND ) and obese ( HFD ) mice for 48 h . Results are expressed relative to the corresponding value for control cells ( n = 3 ) .

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

RT-qPCR analysis of mRNAs for the indicated genes in 1205Lu cells treated or not with EV secreted by primary adipocytes from lean (ND) and obese (HFD) mice for 48 h. Results are expressed relative to the corresponding value for control cells (n=3).


Analysis of FAO levels in 1205Lu cells exposed to 3T3 - F442A EV and treated , or not , with cycloheximide ( CHX ) ( n = 5 ) .

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

Analysis of FAO levels in 1205Lu cells exposed to 3T3-F442A EV and treated, or not, with cycloheximide (CHX) (n=5).


Lipids were extracted from EV secreted by 3T3 - F442A pre - adipocytes and differentiated 3T3 - F442A adipocytes ( respectively preAd - 3T3 - EV and Ad - 3T3 - EV ) , and FA content was measured ( n = 6 ) .

O O O O O O O O O O O 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

Lipids were extracted from EV secreted by 3T3-F442A pre-adipocytes and differentiated 3T3-F442A adipocytes (respectively preAd-3T3-EV and Ad-3T3-EV), and FA content was measured (n=6).


Indicated melanoma cells were incubated with EV from 3T3 - F442A adipocytes previously loaded with BODIPY FL C16 ( 3T3 - FL C16 - EV ) and , 24h later , cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy .

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

Indicated melanoma cells were incubated with EV from 3T3-F442A adipocytes previously loaded with BODIPY FL C16 (3T3-FL C16-EV) and, 24h later, cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy.


Left panel , indicated cells were incubated with EV from 3T3 - F442A adipocytes previously loaded with BODIPY FL C16 and immediately treated , or not , with Etomoxir for 24h . Then , cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy . Right panel , quantification of BODIPY FL C16 staining area per cell ( n = 3 ) .

O O O O 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

Left panel, indicated cells were incubated with EV from 3T3-F442A adipocytes previously loaded with BODIPY FL C16 and immediately treated, or not, with Etomoxir for 24h. Then, cells were fixed and nuclei were counterstained with DAPI before observation by confocal microscopy. Right panel, quantification of BODIPY FL C16 staining area per cell (n=3).


Lipids were extracted from EV secreted by adipocytes from lean ( ND ) and obese ( HFD ) mice ( n = 5 ) ( left panel ) or from human adipose tissue samples from patients with varying BMI ( right panel ) and fatty acid content was measured ( n = 15 ) .

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

Lipids were extracted from EV secreted by adipocytes from lean (ND) and obese (HFD) mice (n=5) (left panel) or from human adipose tissue samples from patients with varying BMI (right panel) and fatty acid content was measured (n=15).


Indicated cells were exposed , or not , to adipocyte EV from primary adipocytes from lean mice fed a normal diet ( ND ) or obese mice fed a high fat diet ( HFD ) for 24h . Then , cells were fixed , stained with BODIPY and counterstained with DAPI . Left panel , confocal microscopy observation . Right panel , quantification of BODIPY staining area per cell ( n = 5 for SKMEL28 and n = 6 for 1205Lu ) .

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

Indicated cells were exposed, or not, to adipocyte EV from primary adipocytes from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) for 24h. Then, cells were fixed, stained with BODIPY and counterstained with DAPI. Left panel, confocal microscopy observation. Right panel, quantification of BODIPY staining area per cell (n=5 for SKMEL28 and n=6 for 1205Lu).


Transmission electron micrographs of 1205Lu cells exposed , or not , to 3T3 - F442A EV ( 3T3 - EV ) . Scale bar represents 1µm . A zoomed crop of the area with autophagic structures containing lipids ( indicated by an arrow ) is shown ( scale bar : 0 . 5µm ) .

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

Transmission electron micrographs of 1205Lu cells exposed, or not, to 3T3-F442A EV (3T3-EV). Scale bar represents 1µm. A zoomed crop of the area with autophagic structures containing lipids (indicated by an arrow) is shown (scale bar: 0.5µm).


1205Lu cells were incubated with EV from 3T3 - F442A adipocytes , previously loaded with BODIPY FL C16 in the presence , or not , of Lalistat 2 . Then , live cells were stained with the LysoTracker probe and observed by confocal microscopy . Arrows indicate colocalization .

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

1205Lu cells were incubated with EV from 3T3-F442A adipocytes, previously loaded with BODIPY FL C16 in the presence, or not, of Lalistat 2. Then, live cells were stained with the LysoTracker probe and observed by confocal microscopy. Arrows indicate colocalization.


1205Lu were incubated with EV from 3T3 - F442A adipocytes , previously loaded with BODIPY FL C16 and treated , or not , with Lalistat 2 . Then , cells were fixed and counterstained with DAPI before observation by confocal microscopy . Quantification of BOBIPY FL C16 staining per cell is shown beside ( n = 4 ) .

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

1205Lu were incubated with EV from 3T3-F442A adipocytes, previously loaded with BODIPY FL C16 and treated, or not, with Lalistat 2. Then, cells were fixed and counterstained with DAPI before observation by confocal microscopy. Quantification of BOBIPY FL C16 staining per cell is shown beside (n=4).


1205Lu cells were exposed to 3T3 - F442A EV ( 3T3 - EV ) and treated , or not , with Lalistat 2 ( Lal ) . Cell migration was then evaluated in Boyden chamber assays ( left panel ; n = 5 ) or cell motility was tracked by video microscopy ( right panel ; n = 40 cells per group ) .

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 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

1205Lu cells were exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Lalistat 2 (Lal). Cell migration was then evaluated in Boyden chamber assays (left panel; n=5) or cell motility was tracked by video microscopy (right panel; n=40 cells per group).


1205Lu cells were exposed , or not , to adipocyte EV from lean mice fed a normal diet ( ND ) or obese mice fed a high fat diet ( HFD ) with , or without , Lalistat 2 ( Lal ) . Cells were then fixed , stained with BODIPY and counterstain with DAPI before observation by confocal microscopy . Quantification of BODIPY staining per cell is shown on the right ( n = 5 ) .

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

1205Lu cells were exposed, or not, to adipocyte EV from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) with, or without, Lalistat 2 (Lal). Cells were then fixed, stained with BODIPY and counterstain with DAPI before observation by confocal microscopy. Quantification of BODIPY staining per cell is shown on the right (n=5).


1205Lu cells were exposed , or not , to adipocyte EV from ND or HFD mice with , or without , Lalistat 2 ( Lal ) . Cell motility was then tracked by video microscopy ( n = 40 cells per group ) .

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

1205Lu cells were exposed, or not, to adipocyte EV from ND or HFD mice with, or without, Lalistat 2 (Lal). Cell motility was then tracked by video microscopy (n=40 cells per group).


1205Lu cells exposed to 3T3 - F442A EV ( 3T3 - EV ) and treated , or not , with Mdivi - 1 , were stained with a MitoTracker probe , fixed and observed by confocal microscopy . Arrows indicate the presence of mitochondria in membrane protrusions . On the images to the right , the outline of cells is shown in dotted lines . Scale bars : 20µm . Right panel , quantification of the distance between mitochondria and nuclei ( n = 15 ) .

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

1205Lu cells exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Mdivi-1, were stained with a MitoTracker probe, fixed and observed by confocal microscopy. Arrows indicate the presence of mitochondria in membrane protrusions. On the images to the right, the outline of cells is shown in dotted lines. Scale bars: 20µm. Right panel, quantification of the distance between mitochondria and nuclei (n=15).


Western blot analysis of the indicated mitochondrial fission proteins in melanoma cells treated , or not , with EV from 3T3 - F442A adipocytes ( 3T3 - EV ) . Tubulin ( TUB ) is used as a loading control .

Western blot analysis of the indicated mitochondrial fission proteins in melanoma cells treated, or not, with EV from 3T3-F442A adipocytes (3T3-EV). Tubulin (TUB) is used as a loading control.


1205Lu cells were exposed to 3T3 - F442A EV ( 3T3 - EV ) and treated , or not , with Mdivi - 1 . Cell migration was then evaluated in Boyden chamber assays ( left panel ; n = 6 ) . Right panel , 3T3 - F442A EV were added to 1205Lu cells , which were immediately treated , or not , with Mdivi - 1 . Cell motility was then tracked by video microscopy ( n = 40 cells per group ) ( right panel ) .

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

1205Lu cells were exposed to 3T3-F442A EV (3T3-EV) and treated, or not, with Mdivi-1. Cell migration was then evaluated in Boyden chamber assays (left panel; n=6). Right panel, 3T3-F442A EV were added to 1205Lu cells, which were immediately treated, or not, with Mdivi-1. Cell motility was then tracked by video microscopy (n = 40 cells per group) (right panel).


1205Lu cells were transfected , or not , with 2 different siRNA targeted against DRP1 ( siDRP1 # 1 or 2 ) or an untargeted siRNA ( siUT ) . Top , 48h after transfection , protein extracts were prepared and DRP1 expression was evaluated by western blot . Bottom , 36h post transfection , cells were exposed to 3T3 - F442A EV ( 3T3 - EV ) and cell motility was tracked by video microscopy ( n = 40 cells per group ) .

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1205Lu cells were transfected, or not, with 2 different siRNA targeted against DRP1 (siDRP1#1 or 2) or an untargeted siRNA (siUT). Top, 48h after transfection, protein extracts were prepared and DRP1 expression was evaluated by western blot. Bottom, 36h post transfection, cells were exposed to 3T3-F442A EV (3T3-EV) and cell motility was tracked by video microscopy (n = 40 cells per group).


1205Lu cells were exposed to adipocyte EV from lean mice fed a normal diet ( ND ) or obese mice fed a high fat diet ( HFD ) and immediately treated , or not , with Mdivi - 1 . Cell motility was then tracked by video microscopy ( n = 40 cells per group ) .

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

1205Lu cells were exposed to adipocyte EV from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) and immediately treated, or not, with Mdivi-1. Cell motility was then tracked by video microscopy (n = 40 cells per group).


Left panel , indicated melanoma cells exposed to 3T3 - F442A EV were fixed , stained with BODIPY and Phalloidin before observation by confocal microscopy . Right panel , quantification of the percentage of cells presenting lipid droplets ( LD ) within membrane protrusions ( n = 6 ) .

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

Left panel, indicated melanoma cells exposed to 3T3-F442A EV were fixed, stained with BODIPY and Phalloidin before observation by confocal microscopy. Right panel, quantification of the percentage of cells presenting lipid droplets (LD) within membrane protrusions (n=6).


Left panel , melanoma cells exposed to EV secreted by adipocytes from lean mice fed a normal diet ( ND ) or obese mice fed a high fat diet ( HFD ) were fixed , stained with BODIPY and Phalloidin before observation by confocal microscopy . Right panel , quantification of the percentage of cells presenting lipid droplets ( LD ) within membrane protrusions ( n = 5 for SKMEL28 and n = 6 for 1205Lu ) .

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

Left panel, melanoma cells exposed to EV secreted by adipocytes from lean mice fed a normal diet (ND) or obese mice fed a high fat diet (HFD) were fixed, stained with BODIPY and Phalloidin before observation by confocal microscopy. Right panel, quantification of the percentage of cells presenting lipid droplets (LD) within membrane protrusions (n=5 for SKMEL28 and n=6 for 1205Lu).


1205Lu cells exposed to 3T3 - F442A EV were stained with a MitoTracker probe . Then , cells were fixed and stained with BODIPY before observation by confocal microscopy . A zoomed crop of the area containing mitochondria and lipid droplets in a membrane protrusion is shown ( scale bar : 5µm ) .

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

1205Lu cells exposed to 3T3-F442A EV were stained with a MitoTracker probe. Then, cells were fixed and stained with BODIPY before observation by confocal microscopy. A zoomed crop of the area containing mitochondria and lipid droplets in a membrane protrusion is shown (scale bar: 5µm).


Transmission electron microscope observations of 1205Lu cells exposed , or not , to 3T3 - F442A EV . Mitochondria are colored in pink and lipid droplets are colored in yellow on images on the right .

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Transmission electron microscope observations of 1205Lu cells exposed, or not, to 3T3-F442A EV. Mitochondria are colored in pink and lipid droplets are colored in yellow on images on the right.


Number of lipid droplets ( LD ) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed , or not , to 3T3 - F442A EV ( 3T3 - EV ) ( n = 28 for control and n = 27 for 3T3 - EV ) . Area of lipid droplets ( LD ) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed , or not , to 3T3 - F442A EV ( n = 19 for control and n = 25 for 3T3 - EV ) .

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Number of lipid droplets (LD) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed, or not, to 3T3-F442A EV (3T3-EV) (n=28 for control and n=27 for 3T3-EV). Area of lipid droplets (LD) found within membrane protrusions on transmission electron microscopy images of 1205Lu cells exposed, or not, to 3T3-F442A EV (n=19 for control and n=25 for 3T3-EV).


Left panel , 1205Lu melanoma cells were exposed , or not , to 3T3 - F442A EV . Then , live cells were stained with LysoTracker and BODIPY probes and observed by confocal microscopy . Right panel , quantification of the percentage of cells presenting lysosome within membrane protrusions ( n = 3 ) .

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

Left panel, 1205Lu melanoma cells were exposed, or not, to 3T3-F442A EV. Then, live cells were stained with LysoTracker and BODIPY probes and observed by confocal microscopy. Right panel, quantification of the percentage of cells presenting lysosome within membrane protrusions (n=3).


TCGA data were analyzed to reveal the effect of high mRNA levels of key FAO enzymes on the overall survival of patients with melanoma . Statistical significance of survival was evaluated with a Log - rank ( Mantel - Cox ) Test ( n = 449 , 450 and 459 for HADHA , HADHB and ECHS1 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

TCGA data were analyzed to reveal the effect of high mRNA levels of key FAO enzymes on the overall survival of patients with melanoma. Statistical significance of survival was evaluated with a Log-rank (Mantel-Cox) Test (n=449, 450 and 459 for HADHA, HADHB and ECHS1 respectively).


BODIPY staining of neutral lipid stores in the indicated melanoma cell lines , which present increasing degrees of aggressiveness ( from left to right ) . DAPI was used to counterstain nuclei . Scale bar : 20µm .

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BODIPY staining of neutral lipid stores in the indicated melanoma cell lines, which present increasing degrees of aggressiveness (from left to right). DAPI was used to counterstain nuclei. Scale bar: 20µm.


FAO in the indicated melanoma cell lines was measured . Bars and error bars represent means ± SEM ( n = 5 ) ; statistically significant by one - way ANOVA with post hoc Tukey ' s test , * * * P < 0 . 001 . If not indicated , non - significant .

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

FAO in the indicated melanoma cell lines was measured. Bars and error bars represent means ± SEM (n=5); statistically significant by one-way ANOVA with post hoc Tukey's test, ***P < 0.001. If not indicated, non-significant.


TCGA data were analyzed to reveal the effect of high mRNA levels of key actors in mitochondrial dynamics on the overall survival of patients with melanoma . Statistical significance of survival was evaluated with a Log - rank ( Mantel - Cox ) Test ( n = 459 ) .

TCGA data were analyzed to reveal the effect of high mRNA levels of key actors in mitochondrial dynamics on the overall survival of patients with melanoma. Statistical significance of survival was evaluated with a Log-rank (Mantel-Cox) Test (n=459).


A Immortalized Brca1F / − MEFs were infected with retroviruses expressing the indicated shRNAs and / or Cre recombinase , followed by selection with puromycin for 72 h . Cell extracts were prepared 48 h later and analysed by Western blotting as indicated . SMC1 and tubulin were used as loading controls . * non ‐ specific band .

A Immortalized Brca1F/−MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 and tubulin were used as loading controls. *non‐specific band.


B Cells treated as in ( A ) were fixed 48 h after selection and stained with an anti ‐ 53BP1 antibody ( green ) . Telomeres were visualized with a Cy3 ‐ conjugated ( CCCTAA ) 3 ‐ PNA probe ( red ) . Yellow arrowheads point to 53BP1 foci that co ‐ localize with telomeres . C Quantification of TIFs in cells treated as in ( B ) . A minimum of 200 nuclei were scored for each sample . Error bars represent SD of two independent experiments . P ‐ values were calculated using an unpaired two ‐ tailed t ‐ test . * P ≤ 0 . 05 ; NS , P > 0 . 05 .

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B Cells treated as in (A) were fixed 48 h after selection and stained with an anti‐53BP1 antibody (green). Telomeres were visualized with a Cy3‐conjugated (CCCTAA)3‐PNA probe (red). Yellow arrowheads point to 53BP1 foci that co‐localize with telomeres.C Quantification of TIFs in cells treated as in (B). A minimum of 200 nuclei were scored for each sample. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; NS, P > 0.05.


A Immortalized Brca1F / − MEFs were infected with retroviruses expressing the indicated shRNAs and / or Cre recombinase , followed by selection with puromycin for 72 h . Cells were arrested in mitosis with colcemid and mitotic chromosomes were processed for CO ‐ FISH analysis . Metaphase chromosome spreads were stained with Cy3 ‐ conjugated leading strand telomeric PNA probe ( red ) and FITC ‐ conjugated lagging strand telomeric PNA probe ( green ) . DNA was counter ‐ stained with DAPI ( blue ) . B The frequency of chromosome ‐ type telomeric fusions in cells treated as in ( A ) was quantified as a percentage of total number of chromosomes ( illustrated in Supplementary Fig S2A ) . A minimum of 2 , 000 chromosomes were scored for each treatment . Error bars represent SD of at least two independent experiments . P ‐ values were calculated using an unpaired two ‐ tailed t ‐ test . * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; NS , P > 0 . 05 .

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

A Immortalized Brca1F/−MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Cells were arrested in mitosis with colcemid and mitotic chromosomes were processed for CO‐FISH analysis. Metaphase chromosome spreads were stained with Cy3‐conjugated leading strand telomeric PNA probe (red) and FITC‐conjugated lagging strand telomeric PNA probe (green). DNA was counter‐stained with DAPI (blue).B The frequency of chromosome‐type telomeric fusions in cells treated as in (A) was quantified as a percentage of total number of chromosomes (illustrated in Supplementary Fig S2A). A minimum of 2,000 chromosomes were scored for each treatment. Error bars represent SD of at least two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; **P ≤ 0.01; NS, P > 0.05.


A Immortalized Brca1F / − MEFs were infected with retroviruses expressing the indicated shRNAs and / or Cre recombinase , followed by selection with puromycin for 72 h . Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3 ‐ conjugated ( CCCTAA ) 3 ‐ PNA probe . The frequency of end ‐ to ‐ end chromosome ‐ type fusions is represented as a percentage of fusions observed after TRF2 depletion . A minimum of 2 , 000 chromosomes were scored for each sample . Error bars represent SD of three independent experiments . P ‐ values were calculated using an unpaired two ‐ tailed t ‐ test . NS , P > 0 . 05 .

A Immortalized Brca1F/− MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion. A minimum of 2,000 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.


B Immortalized Brca1F / C61G MEFs were infected with retroviruses expressing the indicated shRNAs and / or Cre recombinase , followed by selection with puromycin for 72 h . The frequency of end ‐ to ‐ end chromosome ‐ type fusions was analysed as in ( A ) .

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B Immortalized Brca1F/C61G MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. The frequency of end‐to‐end chromosome‐type fusions was analysed as in (A).


C - F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and / or CtIP shRNAs , followed by selection with puromycin for 72 h . Cell extracts were prepared 48 h later and analysed by Western blotting as indicated . GAPDH was used as a loading control . * non ‐ specific band .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and/or CtIP shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. GAPDH was used as a loading control. *non‐specific band.


C - F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and / or CtIP shRNAs , followed by selection with puromycin for 72 h . Cells treated as in ( C ) and ( E ) were arrested in mitosis with colcemid , and mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3 ‐ conjugated ( CCCTAA ) 3 ‐ PNA probe ( D , F ) . The frequency of end ‐ to ‐ end chromosome ‐ type fusions is represented as a percentage of fusions observed after TRF2 depletion . Error bars represent SD of two independent experiments . P ‐ values were calculated using an unpaired two ‐ tailed t ‐ test . * P ≤ 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and/or CtIP shRNAs, followed by selection with puromycin for 72 h. Cells treated as in (C) and (E) were arrested in mitosis with colcemid, and mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe (D, F). The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05.


A Immortalized MEFs were infected with retroviruses expressing the indicated shRNAs , followed by selection with puromycin for 72 h . Cell extracts were prepared 48 h later and analysed by Western blotting as indicated . SMC1 was used as a loading control . * non ‐ specific band . B Quantification of the frequency of end ‐ to ‐ end chromosome ‐ type fusions of cells treated as in ( A ) represented as a percentage of fusions observed after TRF2 depletion . A minimum of 2 , 000 chromosomes were scored for each sample . Error bars represent SD of three independent experiments . The P ‐ value was calculated using an unpaired two ‐ tailed t ‐ test . NS , P > 0 . 05 .

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A Immortalized MEFs were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 was used as a loading control. *non‐specific band.B Quantification of the frequency of end‐to‐end chromosome‐type fusions of cells treated as in (A) represented as a percentage of fusions observed after TRF2 depletion. A minimum of 2,000 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. The P‐value was calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.


Immortalized Brca1F / − MEFs were infected with retroviruses expressing the indicated shRNAs and / or Cre recombinase . Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3 ‐ conjugated ( CCCTAA ) 3 ‐ PNA probe . The frequency of end ‐ to ‐ end chromosome ‐ type fusions is represented as a percentage of fusions observed after TRF2 depletion . A minimum of 2 , 000 chromosomes were scored for each sample . Error bars represent SD of three independent experiments . P ‐ values were calculated using an unpaired two ‐ tailed t ‐ test . NS , P > 0 . 05 .

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Immortalized Brca1F/− MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion. A minimum of 2,000 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.


D Immortalized Brca1F / − MEFs were infected with retroviruses expressing TRF2 shRNAs and / or Cre recombinase , together with a lentivirus expressing PARP1 shRNA , followed by selection with puromycin for 72 h . Cell extracts were prepared 48 h later and analysed by Western blotting as indicated . SMC1 was used as a loading control . * non ‐ specific band . E Quantification of the frequency of end ‐ to ‐ end chromosome ‐ type fusions of cells treated as in ( D ) represented as a percentage of fusions observed after TRF2 depletion . A minimum of 1 , 500 chromosomes were scored for each sample . Error bars represent SD of three independent experiments . The P ‐ value was calculated using an unpaired two ‐ tailed t ‐ test . NS , P > 0 . 05 .

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D Immortalized Brca1F/−MEFs were infected with retroviruses expressing TRF2 shRNAs and/or Cre recombinase, together with a lentivirus expressing PARP1 shRNA, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 was used as a loading control. *non‐specific band.E Quantification of the frequency of end‐to‐end chromosome‐type fusions of cells treated as in (D) represented as a percentage of fusions observed after TRF2 depletion. A minimum of 1,500 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. The P‐value was calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.


A Immortalized MEFs were infected with retroviruses expressing the indicated shRNAs , followed by selection with puromycin for 72 h . Cell extracts were prepared 48 h later and analysed by Western blotting as indicated . SMC1 and GAPDH were used as loading controls . * non ‐ specific band .

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A Immortalized MEFs were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 and GAPDH were used as loading controls. *non‐specific band.


B MboI ‐ and AluI ‐ digested DNA from cells treated as in ( A ) was resolved by pulsed ‐ field gel electrophoresis and probed with end ‐ labelled ( AACCCT ) 4 probe . Representative pulsed ‐ field gel samples run under native and denatured conditions are shown .

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

B MboI‐ and AluI‐digested DNA from cells treated as in (A) was resolved by pulsed‐field gel electrophoresis and probed with end‐labelled (AACCCT)4 probe. Representative pulsed‐field gel samples run under native and denatured conditions are shown.


C Quantification of the 3 ′ overhang in cells treated as in ( B ) . For each sample , the ss / total DNA ratios were expressed relative to the GFP shRNA ‐ treated control . Error bars represent SD of two independent experiments .

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C Quantification of the 3′ overhang in cells treated as in (B). For each sample, the ss/total DNA ratios were expressed relative to the GFP shRNA‐treated control. Error bars represent SD of two independent experiments.


A - C MEFs of the indicated genotypes were infected with retroviruses expressing the indicated shRNAs , followed by selection with puromycin for 72 h . Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3 ‐ conjugated ( CCCTAA ) 3 ‐ PNA probe . The frequency of end ‐ to ‐ end chromosome ‐ type fusions is represented as a percentage of fusions observed after TRF2 depletion in each experiment . A minimum of 1 , 200 chromosomes were scored for each sample . Error bars represent SD of two independent experiments . P ‐ values were calculated using an unpaired two ‐ tailed t ‐ test . * P ≤ 0 . 05 ; NS , P > 0 . 05 . Each graph represents a separate set of experiments .

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A-C MEFs of the indicated genotypes were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion in each experiment. A minimum of 1,200 chromosomes were scored for each sample. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; NS, P > 0.05. Each graph represents a separate set of experiments.


( C - G ) Resembling the phenotype caused by Notch overexpression ( D ) or dSir2 mutation ( E ) , HP1c knockdown ( F ) and mutant ( G ) flies also exhibit supernumerary anterior scutellar ( aSC ) bristles on the notum , as indicated by the enlarged views in the left bottom panes . Scale bars , 100 μm .

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(C-G) Resembling the phenotype caused by Notch overexpression (D) or dSir2 mutation (E), HP1c knockdown (F) and mutant (G) flies also exhibit supernumerary anterior scutellar (aSC) bristles on the notum, as indicated by the enlarged views in the left bottom panes. Scale bars, 100 μm.


( H - M ) Compared with control flies ( H and K ) , HP1c knockdown ( J ) and mutant ( M ) flies exhibit an ectopic vein at the distal of L4 and L5 ( arrows ) and a broken posterior cross vein ( arrowhead ) , which is similar to the phenotype caused by Notch overexpression ( I ) and dSir2 mutant ( L ) . Scale bars , 500 μm .

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(H-M) Compared with control flies (H and K), HP1c knockdown (J) and mutant (M) flies exhibit an ectopic vein at the distal of L4 and L5 (arrows) and a broken posterior cross vein (arrowhead), which is similar to the phenotype caused by Notch overexpression (I) and dSir2 mutant (L). Scale bars, 500 μm.


( A - D ) The posterior midguts of 10 - day - old control ( A ) , HP1c knockdown ( B ) , HP1c null mutant ( C ) and HP1c overexpression flies ( D ) . Arrows mark ISCs , arrowheads mark ee cells , circles mark ISC - EB , EB - EB or ISC - ISC nests . ISCs were visualized with esg - GFP alone , EBs were stained with both esg - GFP and Su ( H ) - lacZ , while ee cells were labeled by Pros and DNA was marked by DAPI . Scale bars , 30 μm .

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(A-D) The posterior midguts of 10-day-old control (A), HP1c knockdown (B), HP1c null mutant (C) and HP1c overexpression flies (D). Arrows mark ISCs, arrowheads mark ee cells, circles mark ISC-EB, EB-EB or ISC-ISC nests. ISCs were visualized with esg-GFP alone, EBs were stained with both esg-GFP and Su(H)-lacZ, while ee cells were labeled by Pros and DNA was marked by DAPI. Scale bars, 30 μm.


( E - I ) Confocal images of the posterior midgut clones of the indicated genotypes , marked by GFP ( dotted lines ) . Arrows mark ISCs / EBs , arrowheads mark ee cells , and asterisks mark ECs . DNA was visualized with DAPI and ee cells were stained with Pros . Scale bars , 10 μm .

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(E-I) Confocal images of the posterior midgut clones of the indicated genotypes, marked by GFP (dotted lines). Arrows mark ISCs/EBs, arrowheads mark ee cells, and asterisks mark ECs. DNA was visualized with DAPI and ee cells were stained with Pros. Scale bars, 10 μm.


( A - D ) Compared with the control ( A ) flies , posterior midguts of Notch OE flies exhibit decreased ISCs and ee cells ( B ) , while HP1c OE flies show increased ISCs and ee cells ( C ) , and the phenotype of Notch OE can be rescued by HP1c OE ( D ) . Arrows mark ISCs , arrowheads mark ee cells , circles mark ISC - ISC , ISC - EB or EB - EB nests . ISCs were visualized with esg - GFP alone , EBs were stained with both esg - GFP and Su ( H ) - lacZ , while ee cells were labeled by Pros and DNA was marked by DAPI . Scale bars , 30 μm .

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(A-D) Compared with the control (A) flies, posterior midguts of Notch OE flies exhibit decreased ISCs and ee cells (B), while HP1c OE flies show increased ISCs and ee cells (C), and the phenotype of Notch OE can be rescued by HP1c OE (D). Arrows mark ISCs, arrowheads mark ee cells, circles mark ISC-ISC, ISC-EB or EB-EB nests. ISCs were visualized with esg-GFP alone, EBs were stained with both esg-GFP and Su(H)-lacZ, while ee cells were labeled by Pros and DNA was marked by DAPI. Scale bars, 30 μm.


ChIP - qPCR results show the association of HP1c with Notch target genes compared to RFP control . The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin ( equivalent to 1 ) . Error bars indicate SEM . n = 3 technical replicates . Data are evaluated with two - tailed Student ' s t - test ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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ChIP-qPCR results show the association of HP1c with Notch target genes compared to RFP control. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to 1). Error bars indicate SEM. n=3 technical replicates. Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001).


( B ) RT - qPCR analysis determined the expression of Notch target genes when HP1c was depleted ( n = 3 technical replicates , , mean ± s . d . ) . Data are evaluated with two - tailed Student ' s t - test ( * p < 0 . 05 , * * * p < 0 . 001 ) .

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(B) RT-qPCR analysis determined the expression of Notch target genes when HP1c was depleted (n=3 technical replicates,, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (*p < 0.05, ***p < 0.001).


NRE - GFP signals of S2 cells carrying NICD OE , Hairless KD , Su ( H ) KD , dSir2 KD and HP1c KD , and the statistical results ( F ) ( n = 4 biological replicates , mean ± s . d . ) . Data are evaluated with two - tailed Student ' s t - test ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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NRE-GFP signals of S2 cells carrying NICD OE, Hairless KD, Su(H) KD, dSir2 KD and HP1c KD , and the statistical results (F) (n=4 biological replicates, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001).


( D , E ) The interaction between HP1c and Su ( H ) is further confirmed through co - immunoprecipitation ( Co - IP ) . Plasmids expressing HP1c - eGFP and Su ( H ) - flag were transfected into S2 cells . Immunoprecipitation and western blotting were performed with the indicated antibodies .

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(D, E) The interaction between HP1c and Su(H) is further confirmed through co-immunoprecipitation (Co-IP). Plasmids expressing HP1c-eGFP and Su(H)-flag were transfected into S2 cells. Immunoprecipitation and western blotting were performed with the indicated antibodies.


( B ) The relative binding of HP1c with Notch target genes when Su ( H ) KD using ChIP - qPCR analysis ( n = 3 technical replicates , mean ± s . d . ) . Data are evaluated with two - tailed Student ' s t - test ( * p < 0 . 05 , * * p < 0 . 01 ) . ( C ) The relative binding of HP1c on Notch target genes when NICD OE using ChIP - qPCR analysis . ( n = 3 technical replicates , mean ± s . d . ) . Data are evaluated with two - tailed Student ' s t - test ( * * * p < 0 . 001 ) .

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(B) The relative binding of HP1c with Notch target genes when Su(H) KD using ChIP-qPCR analysis (n=3 technical replicates, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01). (C) The relative binding of HP1c on Notch target genes when NICD OE using ChIP-qPCR analysis. (n=3 technical replicates, mean ± s.d.). Data are evaluated with two-tailed Student's t-test (***p < 0.001).


( M ) Statistical results of the number of ISCs and ee cells per unit area ( 200 μm × 200 μm ) in ( D - F ) . More than twenty - five midguts were examined for each group . Error bars indicate SEM . Data are evaluated with two - tailed Student ' s t - test ( * p < 0 . 05 , * * p < 0 . 01 ) .

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(M) Statistical results of the number of ISCs and ee cells per unit area (200 μm × 200 μm) in (D-F). More than twenty-five midguts were examined for each group. Error bars indicate SEM. Data are evaluated with two-tailed Student's t-test (*p < 0.05, **p < 0.01).


A HeLa cells expressing LAMP1 - RFP were treated with vehicle alone ( control ) or LLOMe for 1 h to induce lysosome damage . Cells were fixed 2 h after washout , stained with p62 and galectin - 3 ( Gal3 ) antibodies , and analyzed by confocal microscopy .

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A HeLa cells expressing LAMP1-RFP were treated with vehicle alone (control) or LLOMe for 1 h to induce lysosome damage. Cells were fixed 2 h after washout, stained with p62 and galectin-3 (Gal3) antibodies, and analyzed by confocal microscopy.


B p97 translocates to ruptured lysosomes . Cells co - expressing LAMP1 - RFP and GFP - p97 were treated as in ( A ) and stained with the pan - ubiquitin antibody FK2 ( Ub ) . Percentage of cells showing recruitment of p97 in three independent experiments ( mean ± SD ) .

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B p97 translocates to ruptured lysosomes. Cells co-expressing LAMP1-RFP and GFP-p97 were treated as in (A) and stained with the pan-ubiquitin antibody FK2 (Ub). Percentage of cells showing recruitment of p97 in three independent experiments (mean ± SD).


C Immunodetection of endogenous p97 after LLOMe treatment as in ( A ) . Percentage of cells showing recruitment of p97 in three independent experiments ( mean ± SD ) .

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C Immunodetection of endogenous p97 after LLOMe treatment as in (A). Percentage of cells showing recruitment of p97 in three independent experiments (mean ± SD).


D Clearance of damaged lysosomes depends on p97 . Cells were transfected with control ( Ctrl ) or p97 siRNA oligos , fixed at indicated time points after LLOMe washout and stained for endogenous Gal3 . Quantification of the number of Gal3 - positive vesicles per cell . Data represent mean ± SD of four independent experiments with ≥ 30 cells quantified per condition ( one - sided Welch ' s unequal variances t - tests comparing the siCtrl with sip97 ) .

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D Clearance of damaged lysosomes depends on p97. Cells were transfected with control (Ctrl) or p97 siRNA oligos, fixed at indicated time points after LLOMe washout and stained for endogenous Gal3. Quantification of the number of Gal3-positive vesicles per cell. Data represent mean ± SD of four independent experiments with ≥30 cells quantified per condition (one-sided Welch's unequal variances t-tests comparing the siCtrl with sip97).


E Cells were chased after LLOMe treatment for 12 h with vehicle alone ( DMSO ) , bortezomib ( Btz , 10 nM ) , NH4Cl ( 20 mM ) or the p97 inhibitor NMS - 873 ( 5 µM ) , and Gal3 vesicles quantified . Data represent mean ± SEM of three independent experiments with ≥ 30 cells quantified per condition ( one - sided Welch ' s t - tests ) .

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E Cells were chased after LLOMe treatment for 12 h with vehicle alone (DMSO), bortezomib (Btz, 10 nM), NH4Cl (20 mM) or the p97 inhibitor NMS-873 (5 µM), and Gal3 vesicles quantified. Data represent mean ± SEM of three independent experiments with ≥30 cells quantified per condition (one-sided Welch's t-tests).


F p97 is essential for cell survival after lysosome rupture . Cells were transfected with control ( Ctrl ) , p97 or SEL1L siRNA oligos and treated with indicated concentrations of LLOMe for 12 h . Cell viability was measured using the MTS assay . Data represent mean ± SD of three independent experiments .

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F p97 is essential for cell survival after lysosome rupture. Cells were transfected with control (Ctrl), p97 or SEL1L siRNA oligos and treated with indicated concentrations of LLOMe for 12 h. Cell viability was measured using the MTS assay. Data represent mean ± SD of three independent experiments.


G Taufibrils are endocytosed and induce lysosomal damage . Taufibrils were generated in vitro , fluorescently labelled by Dylight488 and fragmented by sonication ( 488 - labelled tau ) . HeLa cells expressing mCherry - Gal3 were incubated with 300 nM taufibrils for 24 h . Arrows indicate compartments that contain 488 - labelled tau and are positive for mCherry - Gal3 indicating lysosome rupture . Percentage of cells with Gal3 - positive vesicles was quantified from three independent experiments ( mean ± SD ) .

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G Taufibrils are endocytosed and induce lysosomal damage. Taufibrils were generated in vitro, fluorescently labelled by Dylight488 and fragmented by sonication (488-labelled tau). HeLa cells expressing mCherry-Gal3 were incubated with 300 nM taufibrils for 24 h. Arrows indicate compartments that contain 488-labelled tau and are positive for mCherry-Gal3 indicating lysosome rupture. Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments (mean ± SD).


H Accumulation of tau - induced lysosomal damage upon p97 depletion . Cells transfected with control ( Ctrl ) or p97 siRNA oligos were treated as in ( G ) . Percentage of cells with Gal3 - positive vesicles was quantified from three independent experiments ( mean ± SD ) . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 . Scale bars , 10 µm .

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H Accumulation of tau-induced lysosomal damage upon p97 depletion. Cells transfected with control (Ctrl) or p97 siRNA oligos were treated as in (G). Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments (mean ± SD). * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Scale bars, 10 µm.


A Impaired clearance of damaged lysosomes in cells expressing a p97 disease - mutant . Wild - type and p97R155H / + MEFs were LLOMe - treated for 1 h and released for indicated times . Percentage of cells with Gal3 - positive vesicles was quantified from three independent experiments ( mean ± SD , Student ' s unpaired t - test ) . Scale bar , 25 µm .

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A Impaired clearance of damaged lysosomes in cells expressing a p97 disease-mutant. Wild-type and p97R155H/+MEFs were LLOMe-treated for 1 h and released for indicated times. Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments (mean ± SD, Student's unpaired t-test). Scale bar, 25 µm.


B Stable U2OS cell lines were doxycycline - induced to express myc - tagged p97 wild - type or disease - mutants as indicated . Induced parental cells served as control ( U2OS - ) . Cells were treated with LLOMe for 1h and chased for indicated time before fixation . Percentage of cells with Gal3 - positive vesicles was quantified from three independent experiments . Comparable expression levels were verified ( see Figure EV2A ) .

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B Stable U2OS cell lines were doxycycline-induced to express myc-tagged p97 wild-type or disease-mutants as indicated. Induced parental cells served as control (U2OS -). Cells were treated with LLOMe for 1h and chased for indicated time before fixation. Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments. Comparable expression levels were verified (see Figure EV2A).


C Stable U2OS cell lines described in ( B ) were treated with LLOMe for 1 h and chased for indicated time . LC3 and GAPDH levels were analyzed by immunoblotting . Quantification is shown in Figure EV2B .

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C Stable U2OS cell lines described in (B) were treated with LLOMe for 1 h and chased for indicated time. LC3 and GAPDH levels were analyzed by immunoblotting. Quantification is shown in Figure EV2B.