D Representative microscopy images showing Fab - PLA results measuring the BCR proximity for the IgM - BCR ( upper ) and the IgD - BCR ( lower ) on untreated or treated B1 - 8 splenic B cells . PLA signals are shown as red dots and the nuclei are visualized by DAPI staining . Scale bar : 5 μm . E The Fab - PLA results are quantified by Blobfinder software and presented as box plots . The median values are highlighted as thick lines and the whiskers represent the minimum and maximum value . PLA signals ( dots / cells ) were counted from at least 100 cells for each sample . Data from treated samples were compared to data from resting cells , p - values were calculated by Kruskal - Wallis one - way analysis of variance ( ANOVA ) .

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

D Representative microscopy images showing Fab-PLA results measuring the BCR proximity for the IgM-BCR (upper) and the IgD-BCR (lower) on untreated or treated B1-8 splenic B cells. PLA signals are shown as red dots and the nuclei are visualized by DAPI staining. Scale bar: 5 μm.E The Fab-PLA results are quantified by Blobfinder software and presented as box plots. The median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. Data from treated samples were compared to data from resting cells, p-values were calculated by Kruskal-Wallis one-way analysis of variance (ANOVA).


A - C Calcium flux measured by FACScan for splenic B cells isolated from Lyn - deficient B1 - 8 transgenic mice after stimulation with ( A ) NIP15 - BSA ( 30 pM ) or 1NIP - pep ( 80 nM ) ; ( B ) Ac146 antibody ( 12 . 5 nM ) or Ac146Fab ( 25 nM ) ; ( C ) Ac38 antibody ( 12 . 5 nM ) or Ac38Fab ( 25 nM ) . Arrows indicate the addition of the stimuli to the cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C Calcium flux measured by FACScan for splenic B cells isolated from Lyn-deficient B1-8 transgenic mice after stimulation with (A) NIP15-BSA (30 pM) or 1NIP-pep (80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.


D Representative microscopy images showing Fab - PLA results monitoring the BCR proximity for the IgM - BCR ( upper ) and the IgD - BCR ( lower ) on untreated or treated B1 - 8 splenic B cells . Scale bar : 5 μm . E Quantified Fab - PLA results are presented as box plots , where the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value . PLA signals ( dots / cells ) were counted from at least 100 cells for each sample . p - values were calculated by Kruskal - Wallis one - way ANOVA .

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

D Representative microscopy images showing Fab-PLA results monitoring the BCR proximity for the IgM-BCR (upper) and the IgD-BCR (lower) on untreated or treated B1-8 splenic B cells. Scale bar: 5 μm.E Quantified Fab-PLA results are presented as box plots, where the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. p-values were calculated by Kruskal-Wallis one-way ANOVA.


A - D Proximity between IgM - BCR on the surface of 3046SM ( A , C ) or 3046M ( B , D ) cells before and after a 1 - min stimulation with the indicated reagents , assayed by Fab - PLA . Results are presented as representative microscopy images ( A , B ) and box plots after quantification ( C , D ) . Scale bar : 5 μm . In the box plots , the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value . PLA signals ( dots / cells ) were counted from at least 100 cells for each sample and p - values were calculated by Kruskal - Wallis one - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Proximity between IgM-BCR on the surface of 3046SM (A, C) or 3046M (B, D) cells before and after a 1-min stimulation with the indicated reagents, assayed by Fab-PLA. Results are presented as representative microscopy images (A, B) and box plots after quantification (C, D). Scale bar: 5 μm. In the box plots, the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample and p-values were calculated by Kruskal-Wallis one-way ANOVA.


E - H Calcium flux measured by FACScan for 3046SM ( E , F ) and 3046M ( G , H ) cells after stimulation with NIP15 - BSA ( 30 pM ) , 1NIP - pep ( 80 nM ) , Ac146Fab ( 25 nM ) or Ac38Fab ( 25 nM ) . Arrows indicate the addition of the stimuli to the cells .

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

E-H Calcium flux measured by FACScan for 3046SM (E, F) and 3046M (G, H) cells after stimulation with NIP15-BSA (30 pM), 1NIP-pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.


A - D Proximity between IgD - BCRs on the surface of 3046SD ( A , C ) or 3046D ( B , D ) cells before and after a 1 - min stimulation , assayed by Fab - PLA . Results are presented as representative microscopy images ( A , B ) and box plots after quantification ( C , D ) . PLA signals are shown as red dots and nuclei were visualized by DAPI staining . Scale bar : 5 μm . In the box plots , the median values were highlighted as thick lines and the whiskers represent the minimum and maximum value . PLA signals ( dots / cells ) were counted from at least 100 cells for each sample and p - values were calculated by Kruskal - Wallis one - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Proximity between IgD-BCRs on the surface of 3046SD (A, C) or 3046D (B, D) cells before and after a 1-min stimulation, assayed by Fab-PLA. Results are presented as representative microscopy images (A, B) and box plots after quantification (C, D). PLA signals are shown as red dots and nuclei were visualized by DAPI staining. Scale bar: 5 μm. In the box plots, the median values were highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample and p-values were calculated by Kruskal-Wallis one-way ANOVA.


E - H Calcium flux measured by FACScan for 3046SD ( E , F ) and 3046D ( G , H ) cells after stimulation with NIP15 - BSA ( 30 pM ) , 1NIP - pep ( 80 nM ) , Ac146Fab ( 25 nM ) or Ac38Fab ( 25 nM ) . Arrows indicate the addition of the stimuli to the cells .

E-H Calcium flux measured by FACScan for 3046SD (E, F) and 3046D (G, H) cells after stimulation with NIP15-BSA (30 pM), 1NIP-pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.


Calcium flux measured by FACScan for 3046M ( A , D ) , 3046SD ( B , E ) and 3046D ( C , F ) cells stimulated with anti - HC ( 2 μl / ml ) ( A , B , C ) , or anti - λ ( 2 μl / ml ) ( D , E , F ) antibodies with or without 1 min prestimulation with 1NIP - pep ( 80 nM ) . The addition of antibodies is indicated by black arrows . The 1NIP - pep was added immediately before recording . Data 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

Calcium flux measured by FACScan for 3046M (A, D), 3046SD (B, E) and 3046D (C, F) cells stimulated with anti-HC (2 μl/ml) (A, B, C), or anti-λ (2 μl/ml) (D, E, F) antibodies with or without 1 min prestimulation with 1NIP-pep (80 nM). The addition of antibodies is indicated by black arrows. The 1NIP-pep was added immediately before recording. Data are representative of three independent experiments.


A - E B1 - 8 B cells , 3046SM cells , 3046M cells , 3046SD cells , or 3046D cells were stimulated with 1NIP - pep ( 80 nM ) , or anti - CD19 antibody ( 62 nM ) , or costimulated with 1NIP - pep and anti - CD19 antibody . The calcium fluxes were measured by FACScan .

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A-E B1-8 B cells, 3046SM cells, 3046M cells, 3046SD cells, or 3046D cells were stimulated with 1NIP-pep (80 nM), or anti-CD19 antibody (62 nM), or costimulated with 1NIP-pep and anti-CD19 antibody. The calcium fluxes were measured by FACScan.


F - J B1 - 8 B cells , 3046SM cells , 3046M cells , 3046SD cells , or 3046D cells were stimulated with 1NIP - pep ( 80 nM ) , or anti - CD20 antibody ( 62 nM ) , or costimulated with 1NIP - pep and anti - CD20 antibody . The calcium fluxes were measured by FACScan .

F-J B1-8 B cells, 3046SM cells, 3046M cells, 3046SD cells, or 3046D cells were stimulated with 1NIP-pep (80 nM), or anti-CD20 antibody (62 nM), or costimulated with 1NIP-pep and anti-CD20 antibody. The calcium fluxes were measured by FACScan.


API delivery is blocked at low temperatures . SEY6210 cells were pulse labeled for 5 min and chased for the times indicated . Identical experiments were performed at 10 ° , 14 ° , and 30 ° C . API was recovered by immunoprecipitation followed by SDS - PAGE and detected by a STORM phosphorimager ( Molecular Dynamics , Sunnyvale , CA ) . The positions of prAPI and mAPI are indicated .

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API delivery is blocked at low temperatures. SEY6210 cells were pulse labeled for 5 min and chased for the times indicated. Identical experiments were performed at 10°, 14°, and 30°C. API was recovered by immunoprecipitation followed by SDS-PAGE and detected by a STORM phosphorimager (Molecular Dynamics, Sunnyvale, CA). The positions of prAPI and mAPI are indicated.


Precursor API is trapped in a prevacuolar compartment in vps18 cells . ( A ) Yeast strain JSR18Δ1 with plasmid pJSR9 ( vps18 ts ) was grown at the permissive temperature of 26 ° C . Before labeling , the cells were incubated at either 26 ° or 38 ° C for 10 min . Pulse labeling was for 5 min , followed by the indicated chase times .

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Precursor API is trapped in a prevacuolar compartment in vps18 cells. (A) Yeast strain JSR18Δ1 with plasmid pJSR9 (vps18 ts) was grown at the permissive temperature of 26°C. Before labeling, the cells were incubated at either 26° or 38°C for 10 min. Pulse labeling was for 5 min, followed by the indicated chase times.


( B ) vps18 spheroplasts were shifted to 38 ° C for 5 min , pulse labeled for 5 min , and then chased for either 0 or 60 min . At each time point the spheroplasts were collected by centrifugation and resuspended in PS0 buffer . The Total ( T ) fraction either received no treatment , was treated with 50 μg / ml proteinase K , or received proteinase K in the presence of 0 . 2 % Triton X - 100 . The supernatant ( S0 ) and pellet ( P0 ) fractions were collected after centrifugation at 10 , 000 g for 5 min .

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

(B) vps18 spheroplasts were shifted to 38°C for 5 min, pulse labeled for 5 min, and then chased for either 0 or 60 min. At each time point the spheroplasts were collected by centrifugation and resuspended in PS0 buffer. The Total (T) fraction either received no treatment, was treated with 50 μg/ml proteinase K, or received proteinase K in the presence of 0.2% Triton X-100. The supernatant (S0) and pellet (P0) fractions were collected after centrifugation at 10,000 g for 5 min.


A mutant in the API propeptide causes accumulation of prAPI on a nonvacuolar compartment . ( A ) Spheroplasts from an ape1Δ strain containing a plasmid bearing P22L API were subjected to differential lysis in PS200 buffer . The Total ( T ) fraction ( after differential lysis ) either received no treatment , was treated with 50 μg / ml proteinase K , or received proteinase K in the presence of 0 . 2 % Triton X - 100 .

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

A mutant in the API propeptide causes accumulation of prAPI on a nonvacuolar compartment. (A) Spheroplasts from an ape1Δ strain containing a plasmid bearing P22L API were subjected to differential lysis in PS200 buffer. The Total (T) fraction (after differential lysis) either received no treatment, was treated with 50 μg/ ml proteinase K, or received proteinase K in the presence of 0.2% Triton X-100.


( B ) The supernatant ( S200 ) and pellet ( P200 ) fractions were collected after centrifugation at 5 , 000 g for 5 min . The P200 fraction was resuspended in 10 % Ficoll , and vacuoles were isolated by flotation through 4 % Ficoll . V , vacuole ; I , 4 / 10 % Ficoll interface ; P , gradient pellet . Proteins were detected by Western blotting . The positions of prAPI , mAPI , and mPrA are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The supernatant (S200) and pellet (P200) fractions were collected after centrifugation at 5,000 g for 5 min. The P200 fraction was resuspended in 10% Ficoll, and vacuoles were isolated by flotation through 4% Ficoll. V, vacuole; I, 4/10% Ficoll interface; P, gradient pellet. Proteins were detected by Western blotting. The positions of prAPI, mAPI, and mPrA are indicated.


( C ) ape1Δ cells containing P22L API on a plasmid were pulse labeled for 5 min , harvested by centrifugation , and subjected to a nonradioactive chase in either nitrogen - containing ( SMD ) or nitrogen starvation ( SD - N ) medium . Samples were collected at the times indicated and immunoprecipitated with API antiserum . The resulting SDS - PAGE gels were quantified using a Molecular Dynamics STORM phosphorimager .

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(C) ape1Δ cells containing P22L API on a plasmid were pulse labeled for 5 min, harvested by centrifugation, and subjected to a nonradioactive chase in either nitrogen-containing (SMD) or nitrogen starvation (SD-N) medium. Samples were collected at the times indicated and immunoprecipitated with API antiserum. The resulting SDS-PAGE gels were quantified using a Molecular Dynamics STORM phosphorimager.


Precursor API is trapped within a vesicle in cvt17 mutants . ( A ) Precursor API is in a protease - protected compartment in cvt17 mutants . THY32 ( cvt17 ) spheroplasts were subjected to differential lysis and protease treatment in PS200 buffer as in Fig

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Precursor API is trapped within a vesicle in cvt17 mutants. (A) Precursor API is in a protease-protected compartment in cvt17 mutants. THY32 (cvt17) spheroplasts were subjected to differential lysis and protease treatment in PS200 buffer as in Fig 3


B ) Precursor API is not free in the vacuolar lumen in cvt17 mutants . Differential fractionation was continued by resuspending the P200 fraction in PS0 buffer and separating the supernatant ( S0 ) and pellet ( P0 ) fractions by centrifugation at 10 , 000 g . The S0 and P0 fractions were treated with proteinase K in the presence and absence of Triton X - 100 as indicated . All fractions were precipitated with 10 % TCA and resolved by SDS PAGE ; proteins of interest were detected by Western blotting . The positions of prAPI , mAPI , and mPrA are indicated .

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

B) Precursor API is not free in the vacuolar lumen in cvt17 mutants. Differential fractionation was continued by resuspending the P200 fraction in PS0 buffer and separating the supernatant (S0) and pellet (P0) fractions by centrifugation at 10,000 g. The S0 and P0 fractions were treated with proteinase K in the presence and absence of Triton X-100 as indicated. All fractions were precipitated with 10% TCA and resolved by SDS PAGE; proteins of interest were detected by Western blotting. The positions of prAPI, mAPI, and mPrA are indicated.


Precursor API is contained within subvacuolar vesicles in cvt17 mutants . ( A ) Isolated vacuoles ( V ) from cvt17 cells were lysed in PS0 buffer , loaded on top of an Optiprep step gradient , and centrifuged at 170 , 000 g for 60 min as described in Materials and Methods . Fractions VV , 0 / 1 . 06 interface ; LD , 1 . 06 region ; SV , 1 . 06 / 1 . 12 interface ; HD , 1 . 12 region .

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Precursor API is contained within subvacuolar vesicles in cvt17 mutants. (A) Isolated vacuoles (V) from cvt17 cells were lysed in PS0 buffer, loaded on top of an Optiprep step gradient, and centrifuged at 170,000 g for 60 min as described in Materials and Methods. Fractions VV, 0/1.06 interface; LD, 1.06 region; SV, 1.06/1.12 interface; HD, 1.12 region.


( B ) An aliquot of fractions VV and SV was treated with proteinase K either in the presence or absence of Triton X - 100 as indicated . Because of the presence of protease inhibitors in the vesicle isolation procedure , an intermediate - sized API degradation product results from proteinase K treatment . The V fraction represents one - tenth of the load of the Optiprep gradient . Proteins were detected by Western blotting . The positions of prAPI and mALP are indicated .

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

(B) An aliquot of fractions VV and SV was treated with proteinase K either in the presence or absence of Triton X-100 as indicated. Because of the presence of protease inhibitors in the vesicle isolation procedure, an intermediate-sized API degradation product results from proteinase K treatment. The V fraction represents one-tenth of the load of the Optiprep gradient. Proteins were detected by Western blotting. The positions of prAPI and mALP are indicated.


Immuno - EM of cvt17 cells . Yeast strains SEY6210 ( parental wild type ) and THY32 ( cvt17 ) were grown in YPD and prepared for microscopy as described in Materials and Methods . ( A ) Wild - type cells probed with antibody against mature API . ( B ) A cvt17 section showing a cytosolic vesicle containing prAPI probed with antibody against the API proregion . ( C ) A cvt17 section showing subvacuolar vesicles containing API probed with antibody against mature API . N , nucleus ; V , vacuole ; ( arrows ) API containing vesicles . Bars , 0 . 5 μm .

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

Immuno-EM of cvt17 cells. Yeast strains SEY6210 (parental wild type) and THY32 (cvt17) were grown in YPD and prepared for microscopy as described in Materials and Methods. (A) Wild-type cells probed with antibody against mature API. (B) A cvt17 section showing a cytosolic vesicle containing prAPI probed with antibody against the API proregion. (C) A cvt17 section showing subvacuolar vesicles containing API probed with antibody against mature API. N, nucleus; V, vacuole; (arrows) API containing vesicles. Bars, 0.5 μm.


Characterization of API - containing vesicles . ( A ) Fractionations were performed as in Fig . 5 . The total ( T ) , vacuole ( V ) , vacuolar vesicle ( VV ) , and subvacuolar vesicle ( SV ) fractions are shown . Proteins were detected by immunoblotting as indicated in the figure .

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Characterization of API-containing vesicles. (A) Fractionations were performed as in Fig. 5. The total (T), vacuole (V), vacuolar vesicle (VV), and subvacuolar vesicle (SV) fractions are shown. Proteins were detected by immunoblotting as indicated in the figure.


( B ) SYPRO orange staining detected by fluorescent scanning mode on a Molecular Dynamics STORM phosphorimager . The relative mobility of the molecular mass standards are indicated on the left . Polypeptides enriched in the subvacuolar vesicles are indicated on the right .

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(B) SYPRO orange staining detected by fluorescent scanning mode on a Molecular Dynamics STORM phosphorimager. The relative mobility of the molecular mass standards are indicated on the left. Polypeptides enriched in the subvacuolar vesicles are indicated on the right.


C Maximal cytochrome c release for all DX / REL pairs with ≥ 3 biological replicates ) ; box - whisker plots summarize data ( box 25 % - 75 % ; belt = median ; dot = mean ; whiskers = minimum and maximum values ) . Statistical analyses were performed using an unpaired two - sided Student ' s t - test , with significance p < 0 . 05 ( trend p < 0 . 10 ) . Cyto c , cytochrome

C Maximal cytochrome c release for all DX/REL pairs with ≥3 biological replicates); box-whisker plots summarize data (box 25%-75%; belt=median; dot=mean; whiskers=minimum and maximum values). Statistical analyses were performed using an unpaired two-sided Student's t-test, with significance p<0.05 (trend p<0.10). Cyto c, cytochrome c


D Bak oligomerization in response to escalating tBid concentration for DX / REL pairs . Relative mitochondrial protein loading per lane is assessed by densitometry , showing ration of loading control in REL lane with patient - matched DX lane at same tBid exposure ( no REL lane is underloaded compared to DX lane ) .

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D Bak oligomerization in response to escalating tBid concentration for DX/REL pairs. Relative mitochondrial protein loading per lane is assessed by densitometry, showing ration of loading control in REL lane with patient-matched DX lane at same tBid exposure (no REL lane is underloaded compared to DX lane).


A In vitro viability curves after 72hr exposure to mafosfamide or doxorubicin . Results are shown for three DX / REL neuroblastoma cell line pairs , two with attenuated cytochrome c release ( CHLA15 / CHLA20 and SKNBE1 / SKNBE2C ) and one without ( CHLA122 / CHLA136 ) . Data information : data points are mean and SD from triplicate wells , experiments are representative of at least three biological replicates dotted - line represents 50 % viability .

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A In vitro viability curves after 72hr exposure to mafosfamide or doxorubicin. Results are shown for three DX/REL neuroblastoma cell line pairs, two with attenuated cytochrome c release (CHLA15/CHLA20 and SKNBE1/SKNBE2C) and one without (CHLA122/CHLA136). Data information: data points are mean and SD from triplicate wells, experiments are representative of at least three biological replicates dotted-line represents 50% viability.


C , In vitro viability for CHLA15 ( DX ) and CHLA20 ( REL ) following ionizing radiation at 7 days Data information : data points are mean and SD from triplicate wells , experiments are representative of at least three biological replicates , dotted - line represents 50 % viability .

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C, In vitro viability for CHLA15 (DX) and CHLA20 (REL) following ionizing radiation at 7 days Data information: data points are mean and SD from triplicate wells, experiments are representative of at least three biological replicates, dotted-line represents 50% viability.


F Cytochrome c release from mitochondria after exposure to tBid or BimBH3 peptide for parental NB1643 and SY5Y cells , in comparison to cells cultured in escalating concentrations of crizotinib until resistant ( NB1643 - ALKR and SY5Y - ALKR ) . Data information : data points are mean of duplicate wells ( SD < 0 . 05 at all points ) in a representative experiment from at least two biological replicates .

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F Cytochrome c release from mitochondria after exposure to tBid or BimBH3 peptide for parental NB1643 and SY5Y cells, in comparison to cells cultured in escalating concentrations of crizotinib until resistant (NB1643-ALKR and SY5Y-ALKR). Data information: data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates.


A , B , C Transmission electron microscopy image analysis was used to quantify mitochondrial size , circularity and roundness in DX / REL neuroblastoma pairs ( mean + / - SD shown ) . Data information : , n = 137 - 241 mitochondria per cell line statistical analyses were performed using a two - tailed Mann - Whitney U test , with significance p < 0 . 05 ( trend p < 0 . 10 ) .

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A, B, C Transmission electron microscopy image analysis was used to quantify mitochondrial size, circularity and roundness in DX/REL neuroblastoma pairs (mean +/- SD shown). Data information: , n=137-241 mitochondria per cell line statistical analyses were performed using a two-tailed Mann-Whitney U test, with significance p<0.05 (trend p<0.10).


H In vitro viability of CHLA15 - ctrl , CHLA15 - shMFN2 , CHLA15 - shPACS2 , and CHLA20 cells following 72hr exposure to ABT - 737 ; dotted - line represents 50 % viability . Data information : data points are mean and SD from triplicate wells , experiments are representative of at least two biological replicates .

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H In vitro viability of CHLA15-ctrl, CHLA15-shMFN2, CHLA15-shPACS2, and CHLA20 cells following 72hr exposure to ABT-737; dotted-line represents 50% viability. Data information: data points are mean and SD from triplicate wells, experiments are representative of at least two biological replicates.


I Mitochondrial cytochrome c release in response to tBid and BimBH3 peptide in CHLA15 - Ctrl , CHLA15 - shMFN2 and CHLA15 - shPACS2 cells . Data information : , data points are mean of duplicate wells ( SD < 0 . 05 at all points ) in a representative experiment from at least two biological

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I Mitochondrial cytochrome c release in response to tBid and BimBH3 peptide in CHLA15-Ctrl, CHLA15-shMFN2 and CHLA15-shPACS2 cells. Data information: , data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates


A , B Cytosolic ( black - tracing ) and mitochondrial ( colored - tracing ) Ca2 + concentration measured using fluorescent reporters in the CHLA15 ( n = 29 cells ) / CHLA20 ( n = 65 cells ) and SKNBE1 ( n = 37 cells ) / SKNBE2C ( n = 52 cells ) pairs . ER Ca2 + release was induced by 100μM carbachol , an IP3R - agonist ; time added indicated by arrow ; tracings are mean + / - SD error bars . Coupling time ( time between achieving 50 % of maximal cytosolic and mitochondrial concentrations ) is an index of ER - mitochondrial proximity and transfer efficiency ; plotted below . Data information : data is derived from at least 3 separate cell transfections ( biological replicates ) . statistical analyses were performed using an unpaired two - sided Student ' s t - test , with significance p < 0 . 05 .

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A, B Cytosolic (black-tracing) and mitochondrial (colored-tracing) Ca2+ concentration measured using fluorescent reporters in the CHLA15 (n=29 cells)/CHLA20 (n=65 cells) and SKNBE1 (n=37 cells)/SKNBE2C (n=52 cells) pairs. ER Ca2+ release was induced by 100μM carbachol, an IP3R-agonist; time added indicated by arrow; tracings are mean +/- SD error bars. Coupling time (time between achieving 50% of maximal cytosolic and mitochondrial concentrations) is an index of ER-mitochondrial proximity and transfer efficiency; plotted below. Data information: data is derived from at least 3 separate cell transfections (biological replicates). statistical analyses were performed using an unpaired two-sided Student's t-test, with significance p<0.05.


F Cytochrome c release in response to tBid and BimBH3 assessed . Data information : data points are mean of duplicate wells ( SD < 0 . 05 at all points ) in a representative experiment from at least two biological replicates .

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F Cytochrome c release in response to tBid and BimBH3 assessed. Data information: data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates.


A , B , C Concentration of ceramide species as measured by LC / MS from DX and REL pair whole cell pellets . Data information : show mean of 3 - 4 biological replicates plotted ; 3 technical replicates

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A, B, C Concentration of ceramide species as measured by LC/MS from DX and REL pair whole cell pellets. Data information: show mean of 3-4 biological replicates plotted; 3 technical replicates each


D , E Cumulative ceramides and sphingomyelins ( of C32 - C36 chain length ) for DX / REL pairs . Data information : show mean of 3 - 4 biological replicates plotted ; 3 technical replicates each error bars are + / - SD .

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D, E Cumulative ceramides and sphingomyelins (of C32-C36 chain length) for DX/REL pairs. Data information: show mean of 3-4 biological replicates plotted; 3 technical replicates each error bars are +/- SD.


H Summary data for CHLA15 / CHLA20 and SKNBE1 / SKNBE2C showing relative cytochrome c release when pre - incubated with GW4869 to inhibit ceramide generation , compared with untreated cells . Data information : All data points from two ( CHLA15 ) or three ( SKNBE1 ) biological replicates are depicted in H . Statistical analyses were performed using an unpaired two - sided Student ' s t - test , with significance p < 0 . 05 ( trend , p < 0 . 10 ) .

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H Summary data for CHLA15/CHLA20 and SKNBE1/SKNBE2C showing relative cytochrome c release when pre-incubated with GW4869 to inhibit ceramide generation, compared with untreated cells. Data information: All data points from two (CHLA15) or three (SKNBE1) biological replicates are depicted in H. Statistical analyses were performed using an unpaired two-sided Student's t-test, with significance p<0.05 (trend, p<0.10).


A - D Mesenteric blood ( PECAM1 , red ) and lymphatic vessels ( LYVE1 , green ; PROX1 , gray ) . Asterisks indicate lymphatic valves , arrow indicates a lymphatic vessel stub , and arrowhead indicates an isolated lymphatic vessel fragmentE - H Blood vessels ( PECAM1 , green ) and lymphatic vessels ( VEGFR - 3 , red ) in the small intestinal wall .

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A-D Mesenteric blood (PECAM1, red) and lymphatic vessels (LYVE1, green; PROX1, gray). Asterisks indicate lymphatic valves, arrow indicates a lymphatic vessel stub, and arrowhead indicates an isolated lymphatic vessel fragmentE-H Blood vessels (PECAM1, green) and lymphatic vessels (VEGFR-3, red) in the small intestinal wall.


I , J Detection of chylous ascites ( arrows ) in the VCiΔR26 mice at P6 .

I, J Detection of chylous ascites (arrows) in the VCiΔR26 mice at P6.


K , L LYVE1 staining of intestinal lymphatic vessels at P6 .

K, L LYVE1 staining of intestinal lymphatic vessels at P6.


M - T LYVE1 staining of lymphatic vessels in the intestinal wall in adult mice . Genotypes and deletion lengths are indicated in ( U ) . U Quantification of LYVE1 areas in ( M - T ) . Length of the i ∆ Vegfc gene deletion is indicated in months ( mo ) , and Vegfd indicates the VEGF - D genotype . Data are represented as mean ± SEM . Significant differences were determined using one - way ANOVA and Bonferroni post hoc analysis compared to WT intestine represented in ( M ) . * P = 0 . 003 , * * P = 0 . 001 , * * * P = 0 . 0008 , ♯ P = 0 . 0002 , § P = 0 . 0001 .

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M-T LYVE1 staining of lymphatic vessels in the intestinal wall in adult mice. Genotypes and deletion lengths are indicated in (U).U Quantification of LYVE1 areas in (M-T). Length of the i∆Vegfc gene deletion is indicated in months (mo), and Vegfd indicates the VEGF-D genotype. Data are represented as mean ± SEM. Significant differences were determined using one-way ANOVA and Bonferroni post hoc analysis compared to WT intestine represented in (M). *P = 0.003, **P = 0.001, ***P = 0.0008, ♯P = 0.0002, §P = 0.0001.


Overview of the small intestine cross section stained with nuclear red and highlighting the location of higher magnification images in ( B - F ) ; ( I ) for the entire villus and ( II ) for the villus base . β - Gal staining pattern of the villus in wild - type ( Ctrl ) , Vegfc / LacZ ( VC ) , Vegfr3 / LacZ ( VR - 3 ) , and Vegfr2 / LacZ ( VR - 2 ) mice . Higher magnification images representing β - Gal staining of the villus base in Vegfc / LacZmice . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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Overview of the small intestine cross section stained with nuclear red and highlighting the location of higher magnification images in (B-F); (I) for the entire villus and (II) for the villus base. β-Gal staining pattern of the villus in wild-type (Ctrl), Vegfc/LacZ (VC), Vegfr3/LacZ (VR-3), and Vegfr2/LacZ (VR-2) mice.Higher magnification images representing β-Gal staining of the villus base in Vegfc/LacZmice.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Vegfc / LacZβ - Gal staining reaction with smooth muscle actin ( SMA ) peroxidase staining . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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Vegfc/LacZβ-Gal staining reaction with smooth muscle actin (SMA) peroxidase staining.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Vegfc / LacZβ - Gal staining and LYVE1 peroxidase staining . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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Vegfc/LacZβ-Gal staining and LYVE1 peroxidase staining.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Surface image of Vegfc / LacZβ - Gal - stained intestine ( left ) and cross section counterstaining with PECAM1 ( right ) . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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Surface image of Vegfc/LacZβ-Gal-stained intestine (left) and cross section counterstaining with PECAM1 (right).Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Immunofluorescence staining of lacteal lymphatic vessel ( LYVE1 ) , blood capillaries ( PECAM1 ) , and smooth muscle cells ( SMA ) . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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Immunofluorescence staining of lacteal lymphatic vessel (LYVE1), blood capillaries (PECAM1), and smooth muscle cells (SMA).Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Body weight change during seven weeks of HFD , expressed as average fold change in comparison with the starting weight . n = 16 , WT ; n = 6 , Vegfd − / − ; n = 16 , VCiΔR26 ; n = 5 , Vegfd − / − ; VCiΔR26 . Body weight comparisons at seven weeks of HFD . Significant differences were determined using one - way ANOVA and Bonferroni post hoc analysis compared to WT . * P = 0 . 004 ; * * P = 0 . 003 . n = 16 , WT ; n = 6 , Vegfd − / − ; n = 16 , VCiΔR26 ; n = 5 , Vegfd − / − ; VCiΔR26 .

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Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis.Body weight change during seven weeks of HFD, expressed as average fold change in comparison with the starting weight. n = 16, WT; n = 6, Vegfd−/−; n = 16, VCiΔR26; n = 5, Vegfd−/−; VCiΔR26.Body weight comparisons at seven weeks of HFD. Significant differences were determined using one-way ANOVA and Bonferroni post hoc analysis compared to WT. *P = 0.004; **P = 0.003. n = 16, WT; n = 6, Vegfd−/−; n = 16, VCiΔR26; n = 5, Vegfd−/−; VCiΔR26.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Glucose tolerance test ( GTT ) after six weeks of HFD . Significant differences were determined using unpaired two - tailed t - test . * P = 0 . 014 ; * * P = 0 . 041 . n = 5 , WT ; n = 6 , VCiΔR26 .

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Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Glucose tolerance test (GTT) after six weeks of HFD. Significant differences were determined using unpaired two-tailed t-test. *P = 0.014; **P = 0.041. n = 5, WT; n = 6, VCiΔR26.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Total fatweight , fat percentage from body composition measurements after six weeks of HFD , and weights of visceral fat ( VF ) and subcutaneous fat ( SF ) at the time of necropsy . Significant differences were determined using unpaired two - tailed t - test . * P = 0 . 006 ; * * P = 0 . 001 ; # P = 0 . 008 ; § P = 0 . 006 . n = 4 in each group .

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Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Total fatweight, fat percentage from body composition measurements after six weeks of HFD, and weights of visceral fat (VF) and subcutaneous fat (SF) at the time of necropsy. Significant differences were determined using unpaired two-tailed t-test. *P = 0.006; **P = 0.001; #P = 0.008; §P = 0.006. n = 4 in each group.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Food consumption during the fifth week of HFD . n = 9 , WT ; n = 10 , VCiΔR26 .

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Food consumption during the fifth week of HFD. n = 9, WT; n = 10, VCiΔR26.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Whole - mount immunofluorescence staining of blood ( PECAM1 , red ) and lymphatic vessels ( LYVE1 , green ) in intestinal villi and intestinal wall . Quantification of the lacteal and villus length ( solid and striped color bars , respectively ) and the intestinal wallLYVE1 + area percentage from images represented in ( F ) . Significant differences were determined using unpaired two - tailed t - test . * P = 0 . 0002 ; * * P = 0 . 00007 . n = 5 , WT ; n = 6 , VCiΔR26 .

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Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis.Whole-mount immunofluorescence staining of blood (PECAM1, red) and lymphatic vessels (LYVE1, green) in intestinal villi and intestinal wall.Quantification of the lacteal and villus length (solid and striped color bars, respectively) and the intestinal wallLYVE1+ area percentage from images represented in (F). Significant differences were determined using unpaired two-tailed t-test. *P = 0.0002; **P = 0.00007. n = 5, WT; n = 6, VCiΔR26.


Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Free fatty acid ( FFA ) and cholesterol measurements from the feces after six weeks of HFD . Significant differences were determined using unpaired two - tailed t - test . * P = 0 . 001 ; * * P = 0 . 007 . n = 5 , WT ; n = 6 , VCiΔR26 .

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Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Free fatty acid (FFA) and cholesterol measurements from the feces after six weeks of HFD. Significant differences were determined using unpaired two-tailed t-test. *P = 0.001; **P = 0.007. n = 5, WT; n = 6, VCiΔR26.


D , RT - qPCR time courses . Promoters of the indicated oscillating genes were used to drive expression of a destabilized , nuclear GFP protein from a single copy integrated transgene ; gfp mRNA and the endogenous transcript driven by the same promoter were quantified from the same RNA samples . Relative expression was calculated as - dCT = - ( target CT values - actin CT values ) and then mean normalized for each trace individually . Peak phases ( ϕPeak ) and amplitudes ( A ) for the endogenous transcripts are from ( Meeuse et al . , 2020 ) . qPCR was performed in technical replicate , shown are averages .

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D, RT-qPCR time courses. Promoters of the indicated oscillating genes were used to drive expression of a destabilized, nuclear GFP protein from a single copy integrated transgene; gfp mRNA and the endogenous transcript driven by the same promoter were quantified from the same RNA samples. Relative expression was calculated as -dCT = - (target CT values - actin CT values) and then mean normalized for each trace individually. Peak phases (ϕPeak) and amplitudes (A) for the endogenous transcripts are from (Meeuse et al., 2020). qPCR was performed in technical replicate, shown are averages.


B , C Heatmap showing trend - corrected luminescence as in ( A ) , for indicated RNAi conditions causing altered numbers ( B ) or durations ( C ) of molts , respectively .

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B, C Heatmap showing trend-corrected luminescence as in (A), for indicated RNAi conditions causing altered numbers (B) or durations (C) of molts, respectively.


C , Image sequence of an L1 synchronized grh - 1 : : aid animal ( HW2418 ) grown at 20 ° C on a 250 μM auxin - containing plates . A lethargic animal was transferred to an agar - pad containing microscopy slide and images were collected every 1 sec , using DIC , 100x magnification . Selected images of Movie EV1 are shown . Time stamp ( min : sec ) is indicated . Arrows indicate phenotypic features : loosening of the cuticle ( 0 : 00 ) ; back - and - forth movements ( 0 : 05 ) ; inflation of the cuticle ( 2 : 46 ) ; vesicles underneath loosened cuticle ( 6 : 11 ) ; rupturing of the cuticle ( 6 : 20 , 6 : 40 ) .

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C, Image sequence of an L1 synchronized grh-1::aid animal (HW2418) grown at 20°C on a 250 μM auxin-containing plates. A lethargic animal was transferred to an agar-pad containing microscopy slide and images were collected every 1 sec, using DIC, 100x magnification. Selected images of Movie EV1 are shown. Time stamp (min:sec) is indicated. Arrows indicate phenotypic features: loosening of the cuticle (0:00); back-and-forth movements (0:05); inflation of the cuticle (2:46); vesicles underneath loosened cuticle (6:11); rupturing of the cuticle (6:20, 6:40).


A , Quantification of the percentage of grh - 1 : : aid animals constitutively expressing luciferase ( HW2434 ) that enter each of four molts molt upon hatching into increasing concentrations of auxin as indicated .

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A, Quantification of the percentage of grh-1::aid animals constitutively expressing luciferase (HW2434) that enter each of four molts molt upon hatching into increasing concentrations of auxin as indicated.


B , Boxplot showing the duration of M1 , M2 and M3 of animals treated with indicated concentrations of auxin . Animals that failed to develop beyond M3 in ( A ) were excluded . The horizontal line represents the median , hinges extend to first and third quartiles , and the whiskers extend up to the most extreme value within 1 . 5 * IQR ( interquartile range ) of the median . Significant differences relative to 0 nM auxin are indicated . P - values were determined by a non - parametric Wilcoxon rank sum test that does not assume normal distribution .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Boxplot showing the duration of M1, M2 and M3 of animals treated with indicated concentrations of auxin. Animals that failed to develop beyond M3 in (A) were excluded. The horizontal line represents the median, hinges extend to first and third quartiles, and the whiskers extend up to the most extreme value within 1.5*IQR (interquartile range) of the median. Significant differences relative to 0 nM auxin are indicated. P-values were determined by a non-parametric Wilcoxon rank sum test that does not assume normal distribution.


A - D , Heatmaps showing trend - corrected luminescence ( Lum , arbitrary units ) of grh - 1 : : aid animals constitutively expressing luciferase ( HW2434 ) . T = 0h corresponds to time of plating embryos , which subsequently hatch at different times . Arrow indicates time point when 250 μM auxin was added , i . e . prior to the first molt ( A ; M1 ) , M2 ( B ) , M3 ( C ) or M4 ( D ) larval stage . Note that for technical convenience in ( A ) , auxin was provided at time of plating . Animals are sorted by entry into M1 ( A ) , M2 ( B ) , M3 ( C ) , M4 ( D ) , respectively .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D, Heatmaps showing trend-corrected luminescence (Lum, arbitrary units) of grh-1::aid animals constitutively expressing luciferase (HW2434). T = 0h corresponds to time of plating embryos, which subsequently hatch at different times. Arrow indicates time point when 250 μM auxin was added, i.e. prior to the first molt (A; M1), M2 (B), M3 (C) or M4 (D) larval stage. Note that for technical convenience in (A), auxin was provided at time of plating. Animals are sorted by entry into M1 (A), M2 (B), M3 (C), M4 (D), respectively.


Time - lapse imaging of grh - 1 : : gfp : : 3xflag animals producing endogenously GFP - tagged GRH - 1 protein . Average + / - 95 % confidence interval ( cyan shading ) are shown ; gray boxes indicate average time of molts .

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Time-lapse imaging of grh-1::gfp::3xflag animals producing endogenously GFP-tagged GRH-1 protein. Average +/- 95% confidence interval (cyan shading) are shown; gray boxes indicate average time of molts.


D , Western blot revealing rapid GRH - 1 depletion in the grh - 1 : : aid strain ( HW2434 ) upon addition of 250 µM auxin . A synchronized culture of animals was grown in liquid at 20 ° C . After 21 h ( denoted t = 0 h in the figure ) , the culture was split in two and either auxin or vehicle were added as indicated . Cultures were sampled hourly and protein lysates probed by Western blotting using anti - FLAG and anti - actin antibodies as indicated .

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

D, Western blot revealing rapid GRH-1 depletion in the grh-1::aid strain (HW2434) upon addition of 250 µM auxin. A synchronized culture of animals was grown in liquid at 20°C. After 21 h (denoted t=0 h in the figure), the culture was split in two and either auxin or vehicle were added as indicated. Cultures were sampled hourly and protein lysates probed by Western blotting using anti-FLAG and anti-actin antibodies as indicated.


A , Scatter plot of GRH - 1 : : GFP : : 3xFLAG ChIP - seq peak enrichments across two biological replicates , using an anti - GFP antibody to immunoprecipitate the endogenously tagged protein from L2 stage larval lysates . Peaks assigned to genes encoding rhythmically expressed transcription factors are plotted in red , all others in blue . Peaks assigned to candidate core clock genes identified in this study are labeled .

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

A, Scatter plot of GRH-1::GFP::3xFLAG ChIP-seq peak enrichments across two biological replicates, using an anti-GFP antibody to immunoprecipitate the endogenously tagged protein from L2 stage larval lysates. Peaks assigned to genes encoding rhythmically expressed transcription factors are plotted in red, all others in blue. Peaks assigned to candidate core clock genes identified in this study are labeled.


Dose - dependent increase of C . albicans ( SC5314 ) aggregation after 4 - h exposure ( a ) and filamentation after 24 - h exposure ( b ) to different concentrations of IL - 17A in RPMI - 1640 liquid medium . Cells aliquots were imaged by microscopy and magnified in the insets . Also note the increased turbidity of cell suspension at 24 h . Scale bars , 100 μm .

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

Dose-dependent increase of C. albicans (SC5314) aggregation after 4-h exposure (a) and filamentation after 24-h exposure (b) to different concentrations of IL-17A in RPMI-1640 liquid medium. Cells aliquots were imaged by microscopy and magnified in the insets. Also note the increased turbidity of cell suspension at 24 h. Scale bars, 100 μm.


( c ) Percentages of germination of C . albicans untreated ( vehicle - treated , open circle ) or treated with IL - 17A at 1 ( black triangle ) or 10 ng ml − 1 ( black square ) for 4 or 24 h . Data are mean ± s . d . P 0 . 05 , IL - 17A - treated versus vehicle - treated cells , at 4 h ; P 0 . 01 ( 1 ng ) and P 0 . 001 ( 10 ng ) at 24 h ( one - way analysis of variance Bonferroni post - test ) ( n = 3 ) .

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 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

(c) Percentages of germination of C. albicans untreated (vehicle-treated, open circle) or treated with IL-17A at 1 (black triangle) or 10 ng ml−1 (black square) for 4 or 24 h. Data are mean±s.d. P 0.05, IL-17A-treated versus vehicle-treated cells, at 4 h; P 0.01 (1 ng) and P 0.001 (10 ng) at 24 h (one-way analysis of variance Bonferroni post-test) (n=3).


( d , e ) IL - 17A promotes biofilm formation . SEM images of C . albicans SC5314 untreated ( d ) or exposed to 10 ng ml − 1 IL - 17A ( e ) for 4 h and subsequently cultured onto plastic discs for 24 h . Scale bars , 10 μm .

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 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

(d,e) IL-17A promotes biofilm formation. SEM images of C. albicans SC5314 untreated (d) or exposed to 10 ng ml−1 IL-17A (e) for 4 h and subsequently cultured onto plastic discs for 24 h. Scale bars, 10 μm.


( f ) IL - 17A increases resistance to nitrogen starvation . Cells were first grown in YPD medium overnight and then switched to nitrogen - free SLAD medium in the presence of 10 ng ml − 1 IL - 17A . Viability , expressed as a percentage of colony - forming units at zero time , was measured over a 15 - day period . Cells were exposed to 0 . 1 ( black circle ) , 1 ( black triangle ) or 10 ng ml − 1 ( black square ) IL - 17A or drug vehicle ( open circle ) at the beginning of the cultures or , in the case of 1 ng ml − 1 ( open triangle ) , also added every two other days . Data are mean ± s . d . P - values ranged from 0 . 05 to 0 . 001 for all samples treated with 10 ng IL - 17A ; P0 . 05 , on days 3 and 5 for samples treated 1 ng IL - 17A every two other days ( one - way analysis of variance Bonferroni post - test ) ( n = 3 ) .

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 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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

(f) IL-17A increases resistance to nitrogen starvation. Cells were first grown in YPD medium overnight and then switched to nitrogen-free SLAD medium in the presence of 10 ng ml−1 IL-17A. Viability, expressed as a percentage of colony-forming units at zero time, was measured over a 15-day period. Cells were exposed to 0.1 (black circle), 1 (black triangle) or 10 ng ml−1 (black square) IL-17A or drug vehicle (open circle) at the beginning of the cultures or, in the case of 1 ng ml−1 (open triangle), also added every two other days. Data are mean±s.d. P-values ranged from 0.05 to 0.001 for all samples treated with 10 ng IL-17A; P0.05, on days 3 and 5 for samples treated 1 ng IL-17A every two other days (one-way analysis of variance Bonferroni post-test) (n=3).


( g , l ) C . albicans cells are metabolically active . Untreated ( g - i ) or IL - 17A - treated ( 10 ng ml − 1 in RPMI - 1640 liquid media for 24 h ) ( j - l ) cells were red after staining with FUN - 1 ( g , j ) , green with Con A ( h , k ) and yellow in the merged images ( i , l ) . Metabolically active cells are shown in red , and cell wall polysaccharides are shown in green . Scale bars , 5 μm . Images from one representative experiment out of two are shown .

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

(g,l) C. albicans cells are metabolically active. Untreated (g-i) or IL-17A-treated (10 ng ml−1 in RPMI-1640 liquid media for 24 h) (j-l) cells were red after staining with FUN-1 (g,j), green with Con A (h,k) and yellow in the merged images (i,l). Metabolically active cells are shown in red, and cell wall polysaccharides are shown in green. Scale bars, 5 μm. Images from one representative experiment out of two are shown.


( g , h ) TOR inhibition in cells treated with 10 ng ml − 1 IL - 17A , 15 nM rapamycin or vehicle ( none ) by western blotting after 4 - h ( g ) or 24 - h ( h ) exposure .

O O O O O B-MEASURED_VAR 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

(g,h) TOR inhibition in cells treated with 10 ng ml−1 IL-17A, 15 nM rapamycin or vehicle (none) by western blotting after 4-h (g) or 24-h (h) exposure.


i - n ) Immunofluorescence of ATG8 in untreated ( i - k ) or IL - 17A - treated ( 10 ng ml − 1 for 4 h ) ( l - n ) cells . Cells were red after TRITC - ATG8 staining ( i , l ) , green after ConA staining ( j , m ) , merged images ( k , n ) . Mean values ( % ATG8 + cells ( merge ) ; mean ± s . d . ) : ATG8 / none , 8 . 2 ± 1 . 6 ; ATG8 / IL - 17A , 58 . 0 ± 11 . 3 ( P = 0 . 002 ) ( n = 3 ) . Scale bars , 10 μm .

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

i-n) Immunofluorescence of ATG8 in untreated (i-k) or IL-17A-treated (10 ng ml−1 for 4 h) (l-n) cells. Cells were red after TRITC-ATG8 staining (i,l), green after ConA staining (j,m), merged images (k,n). Mean values (% ATG8+ cells (merge); mean±s.d.): ATG8/none, 8.2±1.6; ATG8/IL-17A, 58.0±11.3 (P=0.002) (n=3). Scale bars, 10 μm.


( o - r ) Monodansilcadaverine ( MDC ) labelling performed on cells unexposed ( o ) or exposed to 1 ( p ) or 10 ng ml − 1 ( q ) IL - 17A for 4 h . Cells were magnified in the inset ( r ) . Representative results from three independent experiments . The mean values ( % hyphal cells with MDC - stained organelles ( merge ) ; mean ± s . d . ) were as follows : none , 9 . 5 ± 3 . 2 ; 1 ng ml − 1 IL - 17A , 14 . 0 ± 4 . 7 ; 10 ng ml − 1 IL - 17A , 56 . 2 ± 18 . 71 ( P = 0 . 004 , IL - 17A 10 ng ml − 1 versus none ) . Scale bars , 10 μm .

O O O O O O O O O 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 O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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-r) Monodansilcadaverine (MDC) labelling performed on cells unexposed (o) or exposed to 1 (p) or 10 ng ml−1 (q) IL-17A for 4 h. Cells were magnified in the inset (r). Representative results from three independent experiments. The mean values (% hyphal cells with MDC-stained organelles (merge); mean±s.d.) were as follows: none, 9.5±3.2; 1 ng ml−1 IL-17A, 14.0±4.7; 10 ng ml−1 IL-17A, 56.2±18.71 (P=0.004, IL-17A 10 ng ml−1 versus none). Scale bars, 10 μm.


( s , t ) Quantitative real - time PCR of orfs . 19 . 4325 , 19 . 130 , RIC1 on cDNA from Candida SC5314 ( s ) or the TOR1 - 1 / TOR1 mutant ( t ) strains exposed to vehicle ( open column ) , 10 ng ml − 1 IL - 17A ( black column ) or 15 nM rapamycin ( grey column ) for 4 h . The actual P - values are indicated ( two - tailed t - test ) ( n = 3 ) .

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

(s,t) Quantitative real-time PCR of orfs. 19.4325, 19.130, RIC1 on cDNA from Candida SC5314 (s) or the TOR1-1/TOR1 mutant (t) strains exposed to vehicle (open column), 10 ng ml−1 IL-17A (black column) or 15 nM rapamycin (grey column) for 4 h. The actual P-values are indicated (two-tailed t-test) (n=3).


( u , v ) Intracellular ( u ) or extracellular ( v ) cAMP levels , vehicle ( open circle ) , 10 ng ml − 1 IL - 17A ( black square ) or rapamycin ( grey square ) . P0 . 05 , IL - 17A - treated versus untreated at 1 h and P0 . 01 , IL - 17A - treated versus untreated at 24 h ( u ) , and P0 . 05 , rapamycin - treated versus untreated at 6 h ( v ) ( one - way analysis of variance Bonferroni post - test ) ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(u,v) Intracellular (u) or extracellular (v) cAMP levels, vehicle (open circle), 10 ng ml−1 IL-17A (black square) or rapamycin (grey square). P0.05, IL-17A-treated versus untreated at 1 h and P0.01, IL-17A-treated versus untreated at 24 h (u), and P0.05, rapamycin-treated versus untreated at 6 h (v) (one-way analysis of variance Bonferroni post-test) (n=3).


( w ) orf . 19 . 5258 expression after 24 - h Candida SC5314 exposure as in ( n , o ) . Data are mean ± s . d . ( n = 3 ) . The actual P - values are indicated ( two - tailed t - test ) .

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

(w) orf. 19.5258 expression after 24-h Candida SC5314 exposure as in (n,o). Data are mean±s.d. (n=3). The actual P-values are indicated (two-tailed t-test).


( a - g ) GFP C . albicans cells were untreated ( a - c ) or treated ( d - g ) for 4 h with 10 ng ml − 1 IL - 17A followed by anti - IL17A - PE and examined by fluorescence microscopy ( scale bars , 5 μm ) . Cells were green in ( a , d ) , red upon staining with anti - IL17A - PE ( b , e ) and double stained in the merged images ( c , f ) . Note the binding of IL - 17A to hyphal tips ( arrows and magnified in g ) ( n = 3 ) . Images are from one representative experiment out of three .

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

(a-g) GFP C. albicans cells were untreated (a-c) or treated (d-g) for 4 h with 10 ng ml−1 IL-17A followed by anti-IL17A-PE and examined by fluorescence microscopy (scale bars, 5 μm). Cells were green in (a,d), red upon staining with anti-IL17A-PE (b,e) and double stained in the merged images (c,f). Note the binding of IL-17A to hyphal tips (arrows and magnified in g) (n=3). Images are from one representative experiment out of three.


( h , i ) Binding of murine ( h ) or human ( i ) IL - 17A to clinical isolates of C . albicans from human urine ( yellow triangle ) , blood ( green triangle ) , vagina ( red triangle ) and gut ( blue triangle ) . IL - 17A was added to paraformaldehyde - fixed fungal cells followed by biotinylated secondary anti - IL - 17A antibody and streptavidin - horse radish peroxidase . The optical density at 450 nm was corrected by subtracting the optical density at 570 nm ( normalized OD ) in the enzyme - linked immunosorbent assay . P - values of samples exposed to 0 . 1 and 1 ng IL - 17A ranged from 0 . 05 to 0 . 001 ( one - way analysis of variance Bonferroni post - test ) . All samples exposed to 10 ng IL - 17A displayed significant ( P0 . 001 , one - way analysis of variance Bonferroni post - test ) OD values relative to unexposed samples ( 0 ) .

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

(h,i) Binding of murine (h) or human (i) IL-17A to clinical isolates of C. albicans from human urine (yellow triangle), blood (green triangle), vagina (red triangle) and gut (blue triangle). IL-17A was added to paraformaldehyde-fixed fungal cells followed by biotinylated secondary anti-IL-17A antibody and streptavidin-horse radish peroxidase. The optical density at 450 nm was corrected by subtracting the optical density at 570 nm (normalized OD) in the enzyme-linked immunosorbent assay. P-values of samples exposed to 0.1 and 1 ng IL-17A ranged from 0.05 to 0.001 (one-way analysis of variance Bonferroni post-test). All samples exposed to 10 ng IL-17A displayed significant (P0.001, one-way analysis of variance Bonferroni post-test) OD values relative to unexposed samples (0).


( j - o ) Direct visualization of IL - 17A on Candida freshly harvested from human vagina . Same experiment as in ( a - g ) on cells previously stained with Con A ( j , m , green ) and incubated with PE - anti - human IL - 17A ( k , n , red ) and double stained in the merged images ( l , o ) . Scale bars , 10 μm . Shown is one representative experiment out of three .

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

(j-o) Direct visualization of IL-17A on Candida freshly harvested from human vagina. Same experiment as in (a-g) on cells previously stained with Con A (j,m, green) and incubated with PE-anti-human IL-17A (k,n, red) and double stained in the merged images (l,o). Scale bars, 10 μm. Shown is one representative experiment out of three.


( p - r ) Cytospins of vaginal fluids from IL - 17F - deficient ( p ) , IL - 17A - deficient ( q ) and C57BL / 6 ( r ) mice intravaginally infected two days before with C . albicans SC5314 blastospores . Note the presence of hyphae in the vagina of IL - 17F - deficient mice after May - Grünwald - Giemsa staining ( scale bars , 20 μm ) . Shown is one representative experiment out of three .

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 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 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

(p-r) Cytospins of vaginal fluids from IL-17F-deficient (p), IL-17A-deficient (q) and C57BL/6 (r) mice intravaginally infected two days before with C.albicans SC5314 blastospores. Note the presence of hyphae in the vagina of IL-17F-deficient mice after May-Grünwald-Giemsa staining (scale bars, 20 μm). Shown is one representative experiment out of three.


( s ) Binding of IL - 17A to clinical isolates of C . krusei ( red square ) , C . glabrata ( green square ) and S . cerevisiae ( yellow square ) done as in ( h , i ) . IL - 17A was added to paraformaldehyde - fixed fungal cells followed by biotinylated secondary anti - IL - 17A antibody and streptavidin - horse radish peroxidase . The optical density at 450 nm was corrected by subtracting the optical density at 570 nm ( normalized OD ) in the enzyme - linked immunosorbent assay . P - values of samples exposed to 0 . 1 and 1 ng IL - 17A ranged from 0 . 05 to 0 . 001 ( one - way analysis of variance Bonferroni post - test ) . All samples exposed to 10 ng IL - 17A displayed significant ( P0 . 001 , one - way analysis of variance Bonferroni post - test ) OD values relative to unexposed samples ( 0 ) .

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

(s) Binding of IL-17A to clinical isolates of C. krusei (red square), C. glabrata (green square) and S. cerevisiae (yellow square) done as in (h,i). IL-17A was added to paraformaldehyde-fixed fungal cells followed by biotinylated secondary anti-IL-17A antibody and streptavidin-horse radish peroxidase. The optical density at 450 nm was corrected by subtracting the optical density at 570 nm (normalized OD) in the enzyme-linked immunosorbent assay. P-values of samples exposed to 0.1 and 1 ng IL-17A ranged from 0.05 to 0.001 (one-way analysis of variance Bonferroni post-test). All samples exposed to 10 ng IL-17A displayed significant (P0.001, one-way analysis of variance Bonferroni post-test) OD values relative to unexposed samples (0).


( a ) Dose - dependent binding of IL - 17A to fungal Crh11p ( red circle ) and Gas1p ( light blue circle ) proteins , but not to the fungal polysaccharides β - 1 , 3 - D - glucan ( open circle ) , mannan ( blue circle ) and zymosan ( grey circle ) . The lack of binding of IL - 17E to Crh11p ( orange circle ) serves as a negative control . The optical density at 450 nm was corrected by subtracting the optical density at 570 nm ( normalized OD ) in the enzyme - linked immunosorbent assay . Data are mean ± s . d . , n = 3 . P0 . 001 , Crh11p and Gas1p without versus with IL - 17A ( one - way analysis of variance Bonferroni post - test ) .

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

(a) Dose-dependent binding of IL-17A to fungal Crh11p (red circle) and Gas1p (light blue circle) proteins, but not to the fungal polysaccharides β-1,3-D-glucan (open circle), mannan (blue circle) and zymosan (grey circle). The lack of binding of IL-17E to Crh11p (orange circle) serves as a negative control. The optical density at 450 nm was corrected by subtracting the optical density at 570 nm (normalized OD) in the enzyme-linked immunosorbent assay. Data are mean±s.d., n=3. P0.001, Crh11p and Gas1p without versus with IL-17A (one-way analysis of variance Bonferroni post-test).


( b ) Co - immunoprecipitation assay of fungal Crh11p and murine IL - 17A proteins . Immunoprecipitates were probed with antibodies to the corresponding antigens and secondary HRP - conjugated antibody . C , irrelevant antibody .

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(b) Co-immunoprecipitation assay of fungal Crh11p and murine IL-17A proteins. Immunoprecipitates were probed with antibodies to the corresponding antigens and secondary HRP-conjugated antibody. C, irrelevant antibody.


( c ) Failure of IL - 17A to bind to wild - type ( red square ) or mutant C . albicans strains in which the three genes of the CRH family ( UTR2 , open light blue , CRH1 , open black and CRH12 , open blue square ) had been deleted , and restoration of binding by CRH1 ( black square ) and CRH12 ( blue square ) , but not UTR2 ( light blue square ) , reconstitution . Data are mean ± s . d . ( n = 3 ) . P0 . 001 for red square , open light blue , black square and blue square ( one - way analysis of variance Bonferroni post - test ) .

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

(c) Failure of IL-17A to bind to wild-type (red square) or mutant C. albicans strains in which the three genes of the CRH family (UTR2, open light blue, CRH1, open black and CRH12, open blue square) had been deleted, and restoration of binding by CRH1 (black square) and CRH12 (blue square), but not UTR2 (light blue square), reconstitution. Data are mean±s.d. (n=3). P0.001 for red square, open light blue, black square and blue square (one-way analysis of variance Bonferroni post-test).


( d , e ) Cellular localization of bound IL - 17A . Electron micrographs of ultrafine sections of C . albicans MKY 378 wild - type ( d ) or the crh11Δ mutant ( e ) incubated with biotinylated IL - 17A and streptavidin gold - conjugated particles . Arrows indicate immunogold particles . Scale bars , 200 nm .

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(d,e) Cellular localization of bound IL-17A. Electron micrographs of ultrafine sections of C. albicans MKY 378 wild-type (d) or the crh11Δ mutant (e) incubated with biotinylated IL-17A and streptavidin gold-conjugated particles. Arrows indicate immunogold particles. Scale bars, 200 nm.


Fungi were harvested from the stomachs of C57BL / 6 ( a - c ) , IL - 17F - deficient ( d - f ) or IL - 17A - deficient ( g - i ) mice infected with GFP - Candida for 2 days , and stained using PE - labeled antibody to IL - 17A . Cells were green in ( a , d and g ) , red upon staining with anti - IL17A - PE ( b , e and h ) and double stained in the merged images ( c , f and i ) . Scale bars , 10 μm . Images are from one representative experiment out of two . ( j - l ) SEM images showing fungal germination in the stomachs of C57BL / 6 mice ( j ) , filamentation in IL - 17F - deficient mice ( k ) and neither one in IL - 17A - deficient ( l ) mice intragastrically infected 2 days before . Scale bars , 10 μm . ( a - l ) Images are from one representative experiment out of two .

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

Fungi were harvested from the stomachs of C57BL/6 (a-c), IL-17F-deficient (d-f) or IL-17A-deficient (g-i) mice infected with GFP-Candida for 2 days, and stained using PE-labeled antibody to IL-17A. Cells were green in (a,d and g), red upon staining with anti-IL17A-PE (b,e and h) and double stained in the merged images (c,f and i). Scale bars, 10 μm. Images are from one representative experiment out of two. (j-l) SEM images showing fungal germination in the stomachs of C57BL/6 mice (j), filamentation in IL-17F-deficient mice (k) and neither one in IL-17A-deficient (l) mice intragastrically infected 2 days before. Scale bars, 10 μm. (a-l) Images are from one representative experiment out of two.


( m - o ) C57BL / 6 mice were infected intragastrically with C . albicans MKY 378 strain ( m ) or the crh11Δ ( n ) and crh12Δ ( o ) mutants , and were monitored for fungal growth ( log10 colony - forming units ( CFU ) at 2 ( open circle ) or 8 ( black circle ) days post infection ) . Data are mean ± s . d . ( n = 3 ) . The actual P - values are indicated ( two - tailed t - test ) for 2 and 8 days ( m ) and for 2 days ( o ) . Fungal cells were untreated ( none ) or exposed to 10 ng ml − 1 IL - 17A for 4 h before injection .

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(m-o) C57BL/6 mice were infected intragastrically with C. albicans MKY 378 strain (m) or the crh11Δ (n) and crh12Δ (o) mutants, and were monitored for fungal growth (log10 colony-forming units (CFU) at 2 (open circle) or 8 (black circle) days post infection). Data are mean±s.d. (n=3). The actual P-values are indicated (two-tailed t-test) for 2 and 8 days (m) and for 2 days (o). Fungal cells were untreated (none) or exposed to 10 ng ml−1 IL-17A for 4 h before injection.


Candida SC5314 ( a ) , TOR1 - 1 / TOR1 ( b ) and atg9Δ / atg9Δ ( c ) mutant strains were untreated ( open column ) or pre - incubated with IL - 17A ( filled column ) for 4 h before exposure to neutrophils for additional 2 h before the assessment of adhesion , phagocytosis and killing . Error bars , mean ± s . d . ( n = 3 ) . The actual P - values are indicated ( two - tailed t - test ) .

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Candida SC5314 (a), TOR1-1/TOR1(b) and atg9Δ/atg9Δ (c) mutant strains were untreated (open column) or pre-incubated with IL-17A (filled column) for 4 h before exposure to neutrophils for additional 2 h before the assessment of adhesion, phagocytosis and killing. Error bars, mean±s.d. (n=3). The actual P-values are indicated (two-tailed t-test).


i - n ) Immunofluorescence of ATG8 in untreated ( i - k ) or IL - 17A - treated ( 10 ng ml − 1 for 4 h ) ( l - n ) cells . Cells were red after TRITC - ATG8 staining ( i , l ) , green after ConA staining ( j , m ) , merged images ( k , n ) . Mean values ( % ATG8 + cells ( merge ) ; mean ± s . d . ) : ATG8 / none , 8 . 2 ± 1 . 6 ; ATG8 / IL - 17A , 58 . 0 ± 11 . 3 ( P = 0 . 002 ) ( n = 3 ) . Scale bars , 10 μm .

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i-n) Immunofluorescence of ATG8 in untreated (i-k) or IL-17A-treated (10 ng ml−1 for 4 h) (l-n) cells. Cells were red after TRITC-ATG8 staining (i,l), green after ConA staining (j,m), merged images (k,n). Mean values (% ATG8+ cells (merge); mean±s.d.): ATG8/none, 8.2±1.6; ATG8/IL-17A, 58.0±11.3 (P=0.002) (n=3). Scale bars, 10 μm.


( g ) Fungi purified from the stomachs of mice infected as in ( d - f ) were subjected to RT - PCR for autophagy genes expression . C + , fungal cells grown on SPIDER solid medium . C - , no cells .

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(g) Fungi purified from the stomachs of mice infected as in (d-f) were subjected to RT-PCR for autophagy genes expression. C+, fungal cells grown on SPIDER solid medium. C-, no cells.


( h - j ) Log10 colony - forming units ( CFU ) at 2 days post infection with C . albicans SC5314 ( wild - type ) or different mutant strains in C57BL / 6 ( h ) , IL - 17F - deficient ( i ) or IL - 17A - deficient ( j ) mice . Data are mean ± s . d . ( n = 3 ) . 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

(h-j) Log10 colony-forming units (CFU) at 2 days post infection with C. albicans SC5314 (wild-type) or different mutant strains in C57BL/6 (h), IL-17F-deficient (i) or IL-17A-deficient (j) mice. Data are mean±s.d. (n=3). The actual P-values are indicated (two-tailed t-test).


( 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 .

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

(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 ) .

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(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 ) .

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(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).


A Phase contrast images of Mabs cultured into PF hydrogels ( 5 days ) : cells formed a thick three - dimensional myotube network with numerous thick myofibres ( arrows ) . Scale bar : 50 μm .

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A Phase contrast images of Mabs cultured into PF hydrogels (5 days): cells formed a thick three-dimensional myotube network with numerous thick myofibres (arrows). Scale bar: 50 μm.


B Confocalimmunofluorescence analysis with an antibody against myosin heavy chain ( MyHC ) ( red ) and SYTOX green which labels nuclei ( green ) , showing multi - nucleated , mature musclefibres ( arrows ) developed from Mabs in PF ( 5 days ) . Scale bar : 50 μm .

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B Confocalimmunofluorescence analysis with an antibody against myosin heavy chain (MyHC) (red) and SYTOX green which labels nuclei (green), showing multi-nucleated, mature musclefibres (arrows) developed from Mabs in PF (5 days). Scale bar: 50 μm.


C , D Western blot and densitometry analysis of different protein extracts of Mabs differentiated inside PF constructs ( PF ) showing expression levels comparable to adult TA ( Ctrl ) . Group of n = 6 of Mabs differentiated inside PF and host TA has been tested , densitometric analysis from n = 3 different Western blots gauging the level of expression of muscle markers is presented as means ± standard error .

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C, D Western blot and densitometry analysis of different protein extracts of Mabs differentiated inside PF constructs (PF) showing expression levels comparable to adult TA (Ctrl). Group of n = 6 of Mabs differentiated inside PF and host TA has been tested, densitometric analysis from n = 3 different Western blots gauging the level of expression of muscle markers is presented as means ± standard error.


A Gross morphology of the PF implant containing Mabs - nLacZ / PlGF immediately after implantation on the surface of underlying TA ( arrow ) . B , C Artificial muscles ( arrows ) developed within 8 weeks over the host TA surface ( arrowheads ) , encapsulated by the host perimysium ( B ) and released after tendon resection of the TA ( C ) . D The isolated artificial muscle from the TA surface ( arrow ) is comparable in size to the underlying host TA ( arrowhead ) .

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A Gross morphology of the PF implant containing Mabs-nLacZ/PlGF immediately after implantation on the surface of underlying TA (arrow).B, C Artificial muscles (arrows) developed within 8 weeks over the host TA surface (arrowheads), encapsulated by the host perimysium (B) and released after tendon resection of the TA (C).D The isolated artificial muscle from the TA surface (arrow) is comparable in size to the underlying host TA (arrowhead).


E Stereomicroscopy images revealing blood vessel formation in the artificial muscle . Scale bar : 100 μm .

E Stereomicroscopy images revealing blood vessel formation in the artificial muscle. Scale bar: 100 μm.