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 B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O

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 B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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

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

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

(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-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

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

(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 .

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

(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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 B-GENEPROD B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR 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 B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM B-TISSUE O O O O O B-TISSUE O O O O O B-TISSUE O O O O O B-TISSUE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-TISSUE I-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-ORGANISM O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-SUBCELLULAR O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O

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

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

(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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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

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 .

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

(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 ) .

B-ORGANISM I-ORGANISM O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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


( 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 .

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

(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 B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

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

(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 .

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

(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 .

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

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

(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 B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 .

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

(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 B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-ORGANISM B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O 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).


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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O B-CELL_TYPE O O O O O B-TISSUE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE O O O O O O O O O

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 ) .

O O B-EXP_ASSAY O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O B-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O B-TISSUE O O O B-TISSUE O O O O O O O O B-TISSUE O O B-TISSUE O O O O O O O O O O O O B-TISSUE O O O O

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 .

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


F X - Gal and H & amp ; amp ; E staining on artificial muscle section revealing vessel connection with host vasculature by Indian ink , injected into the femoral artery immediately before sacrifice . Scale bar : 20 μm .

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F X-Gal and H&amp;E staining on artificial muscle section revealing vessel connection with host vasculature by Indian ink, injected into the femoral artery immediately before sacrifice. Scale bar: 20 μm.


G Western blot analysis of protein extracts from artificial muscles ( PF ) and host TA ( Ctrl ) reveals remarkable expression of muscle - and vessel - specific proteins ( n = 5 ) .

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G Western blot analysis of protein extracts from artificial muscles (PF) and host TA (Ctrl) reveals remarkable expression of muscle- and vessel-specific proteins (n = 5).


I - L X - Gal and H & amp ; amp ; E staining of artificial muscle sections at 4 ( I , K ) and 8 weeks ( J , L ) after implantation , showing donor origin of muscle cells ( nLacZ + nuclei ) and correlation between implant duration and myofibre maturation ( I , J images are the results of the collage of several photos ) . Scale bar : ( I , J ) 200 μm , ( K , L ) 100 μm .

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I-L X-Gal and H&amp;E staining of artificial muscle sections at 4 (I, K) and 8 weeks (J, L) after implantation, showing donor origin of muscle cells (nLacZ+ nuclei) and correlation between implant duration and myofibre maturation (I, J images are the results of the collage of several photos). Scale bar: (I, J) 200 μm, (K, L) 100 μm.


M - P Immunofluorescence analysis on adjacent sections revealing laminin organization ( green ) and myosin heavy chain ( MyHC ) expression ( red ) at 4 weeks ( M ) and 8 weeks ( N ) after grafting ( M , N displayed items are obtained from a multi - photo collage ) ; DAPI staining ( blue ) shows centrally located nuclei in developing fibres ( O ) and peripheral nuclei in mature myofibres ( P ) . Scale bar : ( M , N ) 200 μm , ( O , P ) 50 μm .

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M-P Immunofluorescence analysis on adjacent sections revealing laminin organization (green) and myosin heavy chain (MyHC) expression (red) at 4 weeks (M) and 8 weeks (N) after grafting (M, N displayed items are obtained from a multi-photo collage); DAPI staining (blue) shows centrally located nuclei in developing fibres (O) and peripheral nuclei in mature myofibres (P). Scale bar: (M, N) 200 μm, (O, P) 50 μm.


Q Confocal isosurface image of neurofilament ( green ) and bungarotoxin ( red ) immunofluorescence in the artificial muscle section shows a mature neuromuscular plaque . Scale bar : 10 μm .

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Q Confocal isosurface image of neurofilament (green) and bungarotoxin (red) immunofluorescence in the artificial muscle section shows a mature neuromuscular plaque. Scale bar: 10 μm.


R Immunofluorescence against smooth muscle actin ( blue ) , laminin ( green ) and VE - Cad ( red ) superimposed on phase contrast image shows blood vessels ( arrows ) adjacent to the myofibres in the artificial muscle section . Scale bar : 20 μm .

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R Immunofluorescence against smooth muscle actin (blue), laminin (green) and VE-Cad (red) superimposed on phase contrast image shows blood vessels (arrows) adjacent to the myofibres in the artificial muscle section. Scale bar: 20 μm.


S X - Gal staining on mature artificial muscle ( arrowhead ) and the underneath host TA ( arrow ) revealing no tendon development at the extremity of the artificial tissue ; implanted LacZ - positive Mabs are present within the host TA . Scale bar : 200 μm .

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S X-Gal staining on mature artificial muscle (arrowhead) and the underneath host TA (arrow) revealing no tendon development at the extremity of the artificial tissue; implanted LacZ-positive Mabs are present within the host TA. Scale bar: 200 μm.


A Electron microscopy with Bluo - Gal staining reveals a LacZ - positive donor nucleus ( arrow ) peripheral to transversely sectioned sarcomeres of the artificial muscle . Scale bar : 1 μm .

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A Electron microscopy with Bluo-Gal staining reveals a LacZ-positive donor nucleus (arrow) peripheral to transversely sectioned sarcomeres of the artificial muscle. Scale bar: 1 μm.


B Longitudinal EM section of an artificial muscle showing mature sarcomere myofibril organization . Scale bar : 0 . 5 μm .

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B Longitudinal EM section of an artificial muscle showing mature sarcomere myofibril organization. Scale bar: 0.5 μm.


C A single fibre from an artificial muscle showing a striation pattern typical of a mature myofibre . Scale bar : 10 μm .

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C A single fibre from an artificial muscle showing a striation pattern typical of a mature myofibre. Scale bar: 10 μm.


D X - Gal staining of freshly dissected artificial muscle bundle at 8 weeks showing abundant LacZ - positive donor nuclei . Scale bar : 100 μm .

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D X-Gal staining of freshly dissected artificial muscle bundle at 8 weeks showing abundant LacZ-positive donor nuclei. Scale bar: 100 μm.


E Immunofluorescence analysis of LacZ - positive nuclei ( green ) and dystrophin ( red ) in an individual muscle fibre after functional analysis . Scale bar : 10 μm . F The same fibre shown in ( E ) is counterstained with DAPI to show that the interstitial nuclei ( blue ) are of donor origin . Scale bar : 10 μm .

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E Immunofluorescence analysis of LacZ-positive nuclei (green) and dystrophin (red) in an individual muscle fibre after functional analysis. Scale bar: 10 μm.F The same fibre shown in (E) is counterstained with DAPI to show that the interstitial nuclei (blue) are of donor origin. Scale bar: 10 μm.


G - I Specific force ( Po / CSA ) ( G ) , maximum shortening velocity ( Vo ) ( H ) , and cross - sectional area ( CSA ) ( I ) of single fibres ( n = 153 ) from artificial muscles and control ( CTRL ) muscles ( n = 103 ) .

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G-I Specific force (Po/CSA) (G), maximum shortening velocity (Vo) (H), and cross-sectional area (CSA) (I) of single fibres (n = 153) from artificial muscles and control (CTRL) muscles (n = 103).


J Silver - stained SDS - PAGE ( 8 % ) gel revealing myosin heavy chain ( MyHC ) - 2B isoform expression in two single fibres of artificial muscles ( lanes 1 , 2 ) and a bulk muscle control sample from soleus muscle containing all four adult MyHC isoforms indicated in ( K ) ( lane 3 ) .

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J Silver-stained SDS-PAGE (8%) gel revealing myosin heavy chain (MyHC)-2B isoform expression in two single fibres of artificial muscles (lanes 1, 2) and a bulk muscle control sample from soleus muscle containing all four adult MyHC isoforms indicated in (K) (lane 3).


K Comassie blue - stained SDS - PAGE ( 8 % ) of bulk samples of two artificial muscles ( lanes 1 , 2 ) and of a bulk control muscle sample ( lane 3 ) , as in ( J ) , showing different MyHC isoform expression .

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K Comassie blue-stained SDS-PAGE (8%) of bulk samples of two artificial muscles (lanes 1, 2) and of a bulk control muscle sample (lane 3), as in (J), showing different MyHC isoform expression.


A , B Gross morphology of the TA injury at 6 months after massive muscle ablation , revealing the new artificial TA regeneration ( arrow ) when grafted with PF - embedded Mabs - PlGF ( B ) and absence of TA muscle ( arrow ) when treated with empty PF as a control ( A ) .

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A, B Gross morphology of the TA injury at 6 months after massive muscle ablation, revealing the new artificial TA regeneration (arrow) when grafted with PF-embedded Mabs-PlGF (B) and absence of TA muscle (arrow) when treated with empty PF as a control (A).


C PF - embedded Mabs - PlGF - derived artificial TA ( right ) showing comparable size to the wild - type normal TA ( left ) .

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C PF-embedded Mabs-PlGF-derived artificial TA (right) showing comparable size to the wild-type normal TA (left).


D - O Immunofluorescence against myosin heavy chain ( MyHC ) ( red ) and laminin ( green ) on mice TA cross sections at early ( 10 days ) ( D - I ) and later stage ( 6 months ) ( J - O ) after massive removal of the muscle ( D , G , J , M are the results of a collage of several photos ) . TA regeneration at early stages showed very few residual myofibres after implantation of acellular PF in place of the ablated TA ( white box in D ) , an enlarged view ( E ) , and DAPI - counterstained view ( F ) reveals the infiltration of undifferentiated mono - nucleated cells . Conversely , a PF - embedded Mabs - PlGF graft ( G ) demonstrated early - stage regeneration with several MyHC - positive ( red ) regenerating myofibres surrounded by laminin ( green ) ( H ) , most of these ( arrows ) being centre - nucleated ( DAPI labelled in I ) . At the later stages , acellular PF implanted mice ( J ) showed a very small portion of muscle tissue likely derived from residual myofibres from TA removal , higher magnification ( K ) revealed very few mature myofibres with peripheral nuclei ( DAPI labelled in L ) . Contrary , the PF - embedded Mabs - PlGF treatment ( M ) showed a complete replacement of the ablated TA with the recovered native structure of MyHC - positive myofibres surrounded by laminin ( N ) , some of these ( arrows ) still being centre nucleate ( DAPI labelled in O ) . Scale bar : ( D , G , J , M ) 500 μm , ( E , F , H , I , K , L , N , O ) 100 μm .

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

D-O Immunofluorescence against myosin heavy chain (MyHC) (red) and laminin (green) on mice TA cross sections at early (10 days) (D-I) and later stage (6 months) (J-O) after massive removal of the muscle (D, G, J, M are the results of a collage of several photos). TA regeneration at early stages showed very few residual myofibres after implantation of acellular PF in place of the ablated TA (white box in D), an enlarged view (E), and DAPI-counterstained view (F) reveals the infiltration of undifferentiated mono-nucleated cells. Conversely, a PF-embedded Mabs-PlGF graft (G) demonstrated early-stage regeneration with several MyHC-positive (red) regenerating myofibres surrounded by laminin (green) (H), most of these (arrows) being centre-nucleated (DAPI labelled in I). At the later stages, acellular PF implanted mice (J) showed a very small portion of muscle tissue likely derived from residual myofibres from TA removal, higher magnification (K) revealed very few mature myofibres with peripheral nuclei (DAPI labelled in L). Contrary, the PF-embedded Mabs-PlGF treatment (M) showed a complete replacement of the ablated TA with the recovered native structure of MyHC-positive myofibres surrounded by laminin (N), some of these (arrows) still being centre nucleate (DAPI labelled in O). Scale bar: (D, G, J, M) 500 μm, (E, F, H, I, K, L, N, O) 100 μm.


P - R Functional analysis demonstrated the effective ability of the PF and Mabs - PlGF combination to generate a complete and functional artificial TA replacing the ablated TA injury . ( P ) The fore - hind limb grip test diagram reveals functional grab force recovery for mice treated with PF - embedded Mabs - PlGF ( TA ABL - PF + MABS ) after only 4 weeks post - TA injury , being comparable with not - ablated wild - type ( WT ) mice and significantly higher than control ablated mice ( TA ABL - CONT ) or mice implanted with void PF ( TA ABL - PF CONT ) ( n = 10 mice per group ) . ( Q , R ) The treadmill analysis of the treated mice relative to wild - type not - ablated ( wt ) shows both exhaustion time and distance indicative of a full recovery of motor functionality already at 3 weeks after TA ablation with an increase in time and distance run for mice treated with PF - embedded Mabs - PlGF , while control mice ( TA ABL - CONT or TA ABL - PF CONT ) showed a much lower resistance ( n = 10 mice per group ) . The values shown in the diagrams are expressed as means ± standard error and statistical significance was tested using Student ' s t - test ( P < 0 . 05 was considered significant ) .

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P-R Functional analysis demonstrated the effective ability of the PF and Mabs-PlGF combination to generate a complete and functional artificial TA replacing the ablated TA injury. (P) The fore-hind limb grip test diagram reveals functional grab force recovery for mice treated with PF-embedded Mabs-PlGF (TA ABL-PF+MABS) after only 4 weeks post-TA injury, being comparable with not-ablated wild-type (WT) mice and significantly higher than control ablated mice (TA ABL-CONT) or mice implanted with void PF (TA ABL-PF CONT) (n = 10 mice per group). (Q, R) The treadmill analysis of the treated mice relative to wild-type not-ablated (wt) shows both exhaustion time and distance indicative of a full recovery of motor functionality already at 3 weeks after TA ablation with an increase in time and distance run for mice treated with PF-embedded Mabs-PlGF, while control mice (TA ABL-CONT or TA ABL-PF CONT) showed a much lower resistance (n = 10 mice per group). The values shown in the diagrams are expressed as means ± standard error and statistical significance was tested using Student's t-test (P < 0.05 was considered significant).


S - Z Morphological analysis demonstrated the effective ability of the PF and Mabs - PlGF combination to generate a complete and functional artificial TA replacing the ablated TA injury . H & amp ; amp ; E staining of TA injury in mice by cross section at an early ( 10 days ) ( S - V ) and late ( 6 months ) ( W - Z ) time point after ablation ( in S , U , W , Y displayed items are obtained from a multi - photo collage ) . At the early time point , the acellular PF - treated mice revealed few mature muscle fibres that survive the massive ablation ( box in S ) signs of continued muscle degeneration ( arrows in T ) , whereas PF - embedded Mabs implanted mice showed remarkable muscle regeneration indicated by centre - nucleated myofibres ( arrows in V ) . At later stages , the control mice grafted with acellular PF showed an almost devoid TA ( W ) with very few muscle fibres ( X ) , in contrast to PF - embedded Mabs , which exhibited a complete muscle replacement generating an artificial TA ( Y ) , enlarged view ( Z ) proved the complete and mature organization of the newly formed muscle showing capillary and blood vessel around myofibres ( arrows ) . Scale bar : ( S , U , W , Y ) 500 μm , ( T , V , X , Z ) 50 μm .

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S-Z Morphological analysis demonstrated the effective ability of the PF and Mabs-PlGF combination to generate a complete and functional artificial TA replacing the ablated TA injury. H&amp;E staining of TA injury in mice by cross section at an early (10 days) (S-V) and late (6 months) (W-Z) time point after ablation (in S, U, W, Y displayed items are obtained from a multi-photo collage). At the early time point, the acellular PF-treated mice revealed few mature muscle fibres that survive the massive ablation (box in S) signs of continued muscle degeneration (arrows in T), whereas PF-embedded Mabs implanted mice showed remarkable muscle regeneration indicated by centre-nucleated myofibres (arrows in V). At later stages, the control mice grafted with acellular PF showed an almost devoid TA (W) with very few muscle fibres (X), in contrast to PF-embedded Mabs, which exhibited a complete muscle replacement generating an artificial TA (Y), enlarged view (Z) proved the complete and mature organization of the newly formed muscle showing capillary and blood vessel around myofibres (arrows). Scale bar: (S, U, W, Y) 500 μm, (T, V, X, Z) 50 μm.


B WT and SirT7 - / - mice at 10 days ( top ) and 2 months ( bottom ) of age .

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B WT and SirT7-/- mice at 10 days (top) and 2 months (bottom) of age.


C Weight distribution of WT and SirT7 - / - mice ( n = 3 - 10 female mice per time point and genotype ; P value = 0 . 003 by unpaired t - Test ) .

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C Weight distribution of WT and SirT7-/- mice (n=3-10 female mice per time point and genotype; P value = 0.003 by unpaired t-Test).


Kaplan Meier survival curves ( n = 170 WT and n = 58 SirT7 - / - mice ; Log - rank Test P value < 0 . 0001 ) .

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Kaplan Meier survival curves (n=170 WT and n=58 SirT7-/- mice; Log-rank Test P value <0.0001).


A Representative 3D reconstructed CT scans showing increased kyphosis in 16 month old SirT7 - / - mice compared with WT .

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A Representative 3D reconstructed CT scans showing increased kyphosis in 16 month old SirT7-/- mice compared with WT.


B Representative gonadal fat pads from WT and SirT7 - / - 16 month old mice .

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B Representative gonadal fat pads from WT and SirT7-/- 16 month old mice.


C Quantitation of body weight ( left ) and gonadal fat pad mass normalized to total body weight ( right ) ( mean ± SEM ; 4 samples per genotype ) .

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C Quantitation of body weight (left) and gonadal fat pad mass normalized to total body weight (right) (mean ± SEM; 4 samples per genotype).


D IGF - 1 protein levels in serum measured by ELISA ( mean ± SEM ; 3 - 8 mice per genotype and age group ) .

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D IGF-1 protein levels in serum measured by ELISA (mean ± SEM; 3-8 mice per genotype and age group).


E , F Dot plots of Lin - Sca1 + cKit + ( LSK ) cells ( middle and right ) from WT and SirT7 - / - bone marrow cells . Cells were gated for negative staining of lineage markers B220 , CD3 , CD11b , CD19 , Gr - 1 , and Ter - 119 ( Left ) , and analyzed for Sca1 and cKit expression ( middle and right ) . ( F ) Quantitation of ( E ) ( mean ± SEM ; 4 mice per genotype ) .

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E, F Dot plots of Lin-Sca1+cKit+ (LSK) cells (middle and right) from WT and SirT7-/- bone marrow cells. Cells were gated for negative staining of lineage markers B220, CD3, CD11b, CD19, Gr-1, and Ter-119 (Left), and analyzed for Sca1 and cKit expression (middle and right). (F) Quantitation of (E) (mean ± SEM; 4 mice per genotype).


G Bone marrow , thymus , and spleen cell number in young WT and SirT7 - / - mice ( mean ± SEM ; 3 - 5 mice per genotype ) .

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G Bone marrow, thymus, and spleen cell number in young WT and SirT7-/- mice (mean ± SEM; 3-5 mice per genotype).


H mRNA expression of p16 gene normalized to GAPDH measured by RT - PCR from young and old WT and SirT7 - / - fibroblasts ( mean ± SEM ; 3 samples per genotype ) .

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H mRNA expression of p16 gene normalized to GAPDH measured by RT-PCR from young and old WT and SirT7-/- fibroblasts (mean ± SEM; 3 samples per genotype).


I , J Senescence - associated β - galactosidasestaining ( I , blue ) in HT1080 cells transfected with Scramble Control or SirT7 Knockdown and grown for 7 days in selection media . ( J ) Quantitation of the number of senescent cells shown in ( I ) ( mean ± SEM ; 3 independent cell lines per genotype ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 by ANOVA Single Factor .

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I, J Senescence-associated β-galactosidasestaining (I, blue) in HT1080 cells transfected with Scramble Control or SirT7 Knockdown and grown for 7 days in selection media. (J) Quantitation of the number of senescent cells shown in (I) (mean ± SEM; 3 independent cell lines per genotype).*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by ANOVA Single Factor.


A , B Dot plots of FACS cell cycle analyses of WT and SirT7 - / - MEFs in passage 3 ( P3 ) and 6 ( P6 ) using EdU incorporation and 7AAD ( A ) . Percentages of cells in Sub G1 ( apoptotic cells , left square ) and cells with DNA content above 4N ( polyploid , right square ) . ( B ) Quantitation of experiment shown in ( A ) ( mean ± SEM ; 3 samples per genotype ) .

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A, B Dot plots of FACS cell cycle analyses of WT and SirT7-/- MEFs in passage 3 (P3) and 6 (P6) using EdU incorporation and 7AAD (A). Percentages of cells in Sub G1 (apoptotic cells, left square) and cells with DNA content above 4N (polyploid, right square). (B) Quantitation of experiment shown in (A) (mean ± SEM; 3 samples per genotype).


C Survival curve for WT and SirT7 - / - thymocytes after X - Ray irradiation ( IR ) at the indicated doses . Cell death was quantified by FACS using AnexinV and 7AAD staining 18 hours post insult ( mean ± SEM ; 3 samples per genotype from one of two independent experiments ) .

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C Survival curve for WT and SirT7-/- thymocytes after X-Ray irradiation (IR) at the indicated doses. Cell death was quantified by FACS using AnexinV and 7AAD staining 18 hours post insult (mean ± SEM; 3 samples per genotype from one of two independent experiments).


D Clonogenic assays in HT1080 cells transfected with Scramble Control or SirT7 Knockdown and irradiated at the indicated X - Ray doses , then plated at low density . After 9 days , colonies were stained with crystal violet and counted ( mean ± SEM ; 3 independent cell lines per genotype ) .

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D Clonogenic assays in HT1080 cells transfected with Scramble Control or SirT7 Knockdown and irradiated at the indicated X-Ray doses, then plated at low density. After 9 days, colonies were stained with crystal violet and counted (mean ± SEM; 3 independent cell lines per genotype).


F Quantitation of experiment shown in ( E ) ( mean ± SEM ; 9 WT and 3 SirT7 - / - samples ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by ANOVA Single Factor .

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F Quantitation of experiment shown in (E) (mean ± SEM; 9 WT and 3 SirT7-/- samples).*P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA Single Factor.


A Quantitation of neutral comet assays , using passage 3 WT and SirT7 - / - primary MEFs , showing ( left ) the amount ( % ) of DNA in the tail and ( right ) the tail moment ( see Fig S3A for representative images ) .

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A Quantitation of neutral comet assays, using passage 3 WT and SirT7-/- primary MEFs, showing (left) the amount (%) of DNA in the tail and (right) the tail moment (see Fig S3A for representative images).


B Western blot showing ATM and KAP1 phosphorylation and total protein levels in WT and SirT7 - / - primary fibroblasts after IR ( 8Gy ) . ATM inhibitor ( ATMi ) KU - 55933 was added 30 min prior to irradiation where indicated . One representative blot from 4 independent experiments is shown .

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B Western blot showing ATM and KAP1 phosphorylation and total protein levels in WT and SirT7-/- primary fibroblasts after IR (8Gy). ATM inhibitor (ATMi) KU-55933 was added 30 min prior to irradiation where indicated. One representative blot from 4 independent experiments is shown.


C - F IF analysis of WT and SirT7 - / - primary fibroblasts showing γH2AX dynamics after DNA damage induction . Cells were untreated ( NoIR ) or treated with 1Gy of X - rays ( IR ) and fixed at the indicated times post insult . Cells were pulsed with EdU ( green ) 30 min prior to fixation , then stained for γH2AX ( red ) and counterstained with DAPI ( blue ) ( n > 30 cells per group / mice ; 3 mice per genotype ) . ( C ) Representative images of untreated S - phase ( NoIR , EdU positive ) WT and SirT7 - / - nuclei ( scale bar 5µm ) . ( D ) Quantitation of the number of γH2AX foci per nucleus at the indicated time points post IR ( 1Gy ) , and indicated cell cycle phases . ( E ) Quantitation of experiment described in ( C ) showing mean number of γH2AX foci per nucleus in euchromatic and heterochromatic regions at the indicated time points before and after IR in G2 ( mean ± SEM from 3 independent experiments ) . Total γH2AX foci and γH2AX foci overlapping with or at the periphery of heterochromatic regions were enumerated . Nuclei and pericentric heterochromatin ( chromocenters ) were segmented by DAPI staining . Euchromatic numbers were estimated by subtracting the heterochromatic number of foci from the total foci number .

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C-F IF analysis of WT and SirT7-/- primary fibroblasts showing γH2AX dynamics after DNA damage induction. Cells were untreated (NoIR) or treated with 1Gy of X-rays (IR) and fixed at the indicated times post insult. Cells were pulsed with EdU (green) 30 min prior to fixation, then stained for γH2AX (red) and counterstained with DAPI (blue) (n>30 cells per group/mice; 3 mice per genotype). (C) Representative images of untreated S-phase (NoIR, EdU positive) WT and SirT7-/- nuclei (scale bar 5µm). (D) Quantitation of the number of γH2AX foci per nucleus at the indicated time points post IR (1Gy), and indicated cell cycle phases. (E) Quantitation of experiment described in (C) showing mean number of γH2AX foci per nucleus in euchromatic and heterochromatic regions at the indicated time points before and after IR in G2 (mean ± SEM from 3 independent experiments). Total γH2AX foci and γH2AX foci overlapping with or at the periphery of heterochromatic regions were enumerated. Nuclei and pericentric heterochromatin (chromocenters) were segmented by DAPI staining. Euchromatic numbers were estimated by subtracting the heterochromatic number of foci from the total foci number.


( F ) Representative images of WT and SirT7 - / - primary fibroblasts in G2 phase showing γH2AX ( red ) and DAPI ( blue ) at the indicated period of time after IR . ( Right ) 3D rendering of the IF segmentation depicting nuclei ( pale blue ) , chromocenters ( darker blue ) , and γH2AX ( yellow denotes foci associated with pericentric heterochromatin , otherwise foci are red ) . Scale bar 2 µm .

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(F) Representative images of WT and SirT7-/- primary fibroblasts in G2 phase showing γH2AX (red) and DAPI (blue) at the indicated period of time after IR. (Right) 3D rendering of the IF segmentation depicting nuclei (pale blue), chromocenters (darker blue), and γH2AX (yellow denotes foci associated with pericentric heterochromatin, otherwise foci are red). Scale bar 2 µm.


G FACS quantitation of WT and SirT7 - / - MEF cells in S - phase after insult with 10 mM hydroxyurea ( HU ) for 24 hours . Cells were fixed and stained with 7AAD and cell cycle was monitored by FACS ( mean ± SEM ; 5 samples per genotype ) .

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G FACS quantitation of WT and SirT7-/- MEF cells in S-phase after insult with 10 mM hydroxyurea (HU) for 24 hours. Cells were fixed and stained with 7AAD and cell cycle was monitored by FACS (mean ± SEM; 5 samples per genotype).


H , I DNA fiber labeling analysis was used to assess DNA replication fork progression in passage 3 and passage 6 primary WT and SirT7 - / - MEFs . ( H ) Representative images from cells labeled for 20 min with IdU ( green ) followed by 20 min of CldU ( red ) . ( I ) Quantitation of fork velocity ( fiber length / labeling time ; mean ± SEM ; 3 samples per genotype per condition ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by ANOVA Single Factor .

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H, I DNA fiber labeling analysis was used to assess DNA replication fork progression in passage 3 and passage 6 primary WT and SirT7-/- MEFs. (H) Representative images from cells labeled for 20 min with IdU (green) followed by 20 min of CldU (red). (I) Quantitation of fork velocity (fiber length/labeling time; mean ± SEM; 3 samples per genotype per condition).*P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA Single Factor.


A - E IF analysis of WT and SirT7 - / - primary fibroblasts after IR ( 1Gy ) and 1 hour chase . Cells were pulsed with EdU 30 min prior to fixation , stained with antibodies against γH2AX and 53BP1 and then counterstained with DAPI ( scale bar 5µm ) . ( A ) Representative images from each cell cycle stage showing merge ( top - left ) including EdU ( green ) , 53BP1 ( top - right , magenta ) , γH2AX ( bottom - left , red ) , and 3D rendering of reconstructed Z - stacks with 53BP1 foci modeled as spheres ( bottom - right ; pale - blue : nucleus ; magenta spheres : 53BP1 foci ) . ( B ) Quantitation of the number of 53BP1 foci per nucleus , and ( C ) mean volume of 53BP1 foci ( n > 30 cells per group / mice ; 3 mice per genotype ) . ( D - E ) Same as in ( B - C ) , upon overexpression of SIRT7 WT or catalytically inactive point mutant SIRT7 - H188Y in WT and SirT7 - / - primary fibroblasts .

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A-E IF analysis of WT and SirT7-/- primary fibroblasts after IR (1Gy) and 1 hour chase. Cells were pulsed with EdU 30 min prior to fixation, stained with antibodies against γH2AX and 53BP1 and then counterstained with DAPI (scale bar 5µm). (A) Representative images from each cell cycle stage showing merge (top-left) including EdU (green), 53BP1 (top-right, magenta), γH2AX (bottom-left, red), and 3D rendering of reconstructed Z-stacks with 53BP1 foci modeled as spheres (bottom-right; pale-blue: nucleus; magenta spheres: 53BP1 foci). (B) Quantitation of the number of 53BP1 foci per nucleus, and (C) mean volume of 53BP1 foci (n>30 cells per group/mice; 3 mice per genotype). (D-E) Same as in (B-C), upon overexpression of SIRT7 WT or catalytically inactive point mutant SIRT7-H188Y in WT and SirT7-/- primary fibroblasts.


F Detection of endogenous chromatin - bound and nucleoplasmic 53BP1 protein from WT and SirT7 - / - MEFs before and after IR ( 8Gy ) by western blot . Histone H3 was used as loading control , and tubulin as control for fractionation .

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F Detection of endogenous chromatin-bound and nucleoplasmic 53BP1 protein from WT and SirT7-/- MEFs before and after IR (8Gy) by western blot. Histone H3 was used as loading control, and tubulin as control for fractionation.


G Same as ( F ) , using 293T - REX cells treated with doxycycline or vehicle to induce SIRT7 - HA expression .

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G Same as (F), using 293T-REX cells treated with doxycycline or vehicle to induce SIRT7-HA expression.


H ChIP - on break assay . ( Top ) Schematic of I - SceI substrate construct introduced into HT1080 cells , which contains a single I - SceI site located within a puromycin resistance cassette . Shown along the top are the coordinates for amplicons probed by Q - PCR . ( Bottom ) 53BP1 enrichment at the indicated loci in Scramble control or SirT7 knockdown cells . Q - PCR measurements were normalized to input DNA and non - I - SceI treated samples ( No Cut ) ( mean ± SEM ; one of two independent experiments shown ) . ( Bottom Right ) western blot demonstrating efficient knockdown of SirT7 , with Histone H3 as a loading control .

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H ChIP-on break assay. (Top) Schematic of I-SceI substrate construct introduced into HT1080 cells, which contains a single I-SceI site located within a puromycin resistance cassette. Shown along the top are the coordinates for amplicons probed by Q-PCR. (Bottom) 53BP1 enrichment at the indicated loci in Scramble control or SirT7 knockdown cells. Q-PCR measurements were normalized to input DNA and non-I-SceI treated samples (No Cut) (mean ± SEM; one of two independent experiments shown). (Bottom Right) western blot demonstrating efficient knockdown of SirT7, with Histone H3 as a loading control.


I , J NHEJ repair assay using GFP expression - based reporter system in SirT7 - depleted HT1080 cells ( SirT7 Knockdown ) versus control ( Scramble Control ) . ( I ) Representative FACS dot plots . ( J ) Quantitation of ( I ) ; mean ± SEM of 3 independent clones per condition . Values are normalized to control ( Scramble Control + I - SceI ) mean .

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I, J NHEJ repair assay using GFP expression-based reporter system in SirT7-depleted HT1080 cells (SirT7 Knockdown) versus control (Scramble Control). (I) Representative FACS dot plots. (J) Quantitation of (I); mean ± SEM of 3 independent clones per condition. Values are normalized to control (Scramble Control + I-SceI) mean.


K , L Class - switch recombination in splenic B cells from WT and SirT7 - / - mice stimulated with lipopolysaccharides ( LPS ) and interleukin 4 ( IL4 ) . ( K ) The switching from IgM to IgG1 ( left ) and to IgG3 ( right ) was measured by FACS . ( L ) Quantitation of ( K ) showing mean ± SEM of 3 - 4 samples per genotype . One representative experiment from two is shown . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by ANOVA Single Factor .

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K, L Class-switch recombination in splenic B cells from WT and SirT7-/- mice stimulated with lipopolysaccharides (LPS) and interleukin 4 (IL4). (K) The switching from IgM to IgG1 (left) and to IgG3 (right) was measured by FACS. (L) Quantitation of (K) showing mean ± SEM of 3-4 samples per genotype. One representative experiment from two is shown.*P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA Single Factor.


A , B H3K18Ac IF in WT and SirT7 - / - fibroblasts . Cells were pulsed with EdU ( green ) , stained with antibody against H3K18Ac ( magenta ) and then counterstained with DAPI ( blue ) . ( A ) Representative IF images of late S - phase nuclei ( EdU positive ; scale bar 5µm ) . ( B ) Quantitation of ( A ) throughout the cell cycle .

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A, B H3K18Ac IF in WT and SirT7-/- fibroblasts. Cells were pulsed with EdU (green), stained with antibody against H3K18Ac (magenta) and then counterstained with DAPI (blue). (A) Representative IF images of late S-phase nuclei (EdU positive; scale bar 5µm). (B) Quantitation of (A) throughout the cell cycle.


C Same as in ( B ) , in S - phase cells at the indicated times after IR ( 1Gy ) ( mean ± SEM of > 30 cells per cell cycle , time point , and genotype ) .

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C Same as in (B), in S-phase cells at the indicated times after IR (1Gy) (mean ± SEM of >30 cells per cell cycle, time point, and genotype).


D Laser - induced DNA damage in HT1080 cells expressing HA - tagged SirT7 stained with antibodies against γH2AX ( red ) and HA ( green ) , then counterstained with DAPI ( blue ) 30 min post damage ( scale bar 3 µm ) .

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D Laser-induced DNA damage in HT1080 cells expressing HA-tagged SirT7 stained with antibodies against γH2AX (red) and HA (green), then counterstained with DAPI (blue) 30 min post damage (scale bar 3 µm).


E ChIP assays of SIRT7 enrichment at the indicated loci as described in Figure 5H ( mean ± SEM ; one of two independent experiments is shown ) .

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E ChIP assays of SIRT7 enrichment at the indicated loci as described in Figure 5H (mean ± SEM; one of two independent experiments is shown).


F Recruitment kinetics of GFP - tagged SirT7 after laser - induced microirradiation . ( Top ) Representative cell at the indicated times after induction of DNA damage ( white circle ; scale bar 2µm ) . Images were adjusted to account for photobleaching by normalizing to nucleoplasmic background signal . ( Middle ) Quantitation of recruitment kinetics at the site of induced damage in the presence of 5M KU - 55933 ATM inhibitor ( ATMi ) , 10M Olaparib PARP inhibitor ( PARPi ) , or DMSO . KU - 55933 and Olaparib were added 30 min and 1 hour respectively prior to DNA damage ( mean ± SEM ; sample size : SIRT7 - GFP , n = 34 ; SIRT7 - GFP + ATMi , n = 36 ; SIRT7 - GFP + PARPi = 18 ) . ( Bottom ) Same as ( middle ) except quantitation of SIRT7 - GFP relative intensity within the nucleolus . Data was acquired at five second intervals over a span of five min .

O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-GENEPROD O O B-GENEPROD O O O B-SMALL_MOLECULE B-GENEPROD O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O

F Recruitment kinetics of GFP-tagged SirT7 after laser-induced microirradiation. (Top) Representative cell at the indicated times after induction of DNA damage (white circle; scale bar 2µm). Images were adjusted to account for photobleaching by normalizing to nucleoplasmic background signal. (Middle) Quantitation of recruitment kinetics at the site of induced damage in the presence of 5M KU-55933 ATM inhibitor (ATMi), 10M Olaparib PARP inhibitor (PARPi), or DMSO. KU-55933 and Olaparib were added 30 min and 1 hour respectively prior to DNA damage (mean ± SEM; sample size: SIRT7-GFP, n=34; SIRT7-GFP + ATMi, n=36; SIRT7-GFP + PARPi=18). (Bottom) Same as (middle) except quantitation of SIRT7-GFP relative intensity within the nucleolus. Data was acquired at five second intervals over a span of five min.


G ChIP assays of H3K18Ac enrichment at the indicated loci as described in Figure 5H . H3K18Ac enrichment adjacent to the induced DSB in control and SIRT7 depleted cells ( bottom left ) , or in cells overexpressing SIRT7 in the presence or absence of PARP inhibitors ( PARPi ) ( bottom right ) ( mean ± SEM ; one of two independent experiments is shown ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 by ANOVA Single Factor .

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G ChIP assays of H3K18Ac enrichment at the indicated loci as described in Figure 5H. H3K18Ac enrichment adjacent to the induced DSB in control and SIRT7 depleted cells (bottom left), or in cells overexpressing SIRT7 in the presence or absence of PARP inhibitors (PARPi) (bottom right) (mean ± SEM; one of two independent experiments is shown).*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by ANOVA Single Factor.


A - H NHEJ activity in HT1080 or NIH3T3 cells stably overexpressing H3 - WT , H3 - K18Q or H3 - K18R ( naive , acetylated or deacetylated H3K18 residues , respectively ) . A Random integration assay in HT1080 cells using a linearized pSMCV vector containing a puromycin resistance cassette ( mean number of puromycin resistant colonies normalized by plating efficiency ± SEM ; One representative transfection from 3 is shown ) .

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A-H NHEJ activity in HT1080 or NIH3T3 cells stably overexpressing H3-WT, H3-K18Q or H3-K18R (naive, acetylated or deacetylated H3K18 residues, respectively).A Random integration assay in HT1080 cells using a linearized pSMCV vector containing a puromycin resistance cassette (mean number of puromycin resistant colonies normalized by plating efficiency ± SEM; One representative transfection from 3 is shown).


B NHEJ repair assay using GFP expression - based reporter system described in Fig 5I ( mean ± SEM of 3 independent experiments . See Fig EV5B for representative FACS dot plots ) .

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B NHEJ repair assay using GFP expression-based reporter system described in Fig 5I (mean ± SEM of 3 independent experiments. See Fig EV5B for representative FACS dot plots).