( E ) Representative Gomori modified staining and quantification of myofiber area and relative frequency of the myofiber cross - sectional area ( CSA ) expressed as the frequency distribution of the TA muscles of 3m C57Bl ( n = 4 ) , 3m mdx ( n = 4 ) , mdx + ABX ( n = 4 ) and GFmdx mice ( n = 5 ) . Pooled samples for each group with n = 6240 for 3m C57Bl ; n = 6001 for 3m mdx ; n = 10556 for 3m mdx + ABX ; n = 23059 for GFmdx . For morphometric analysis , images were quantified with Image J software for each mouse . Scale bars : 50 μm . Data information : Data are presented as mean ± SD ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; * * * * p < 0 . 0001 , ordinary one - way

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

(E) Representative Gomori modified staining and quantification of myofiber area and relative frequency of the myofiber cross-sectional area (CSA) expressed as the frequency distribution of the TA muscles of 3m C57Bl (n=4), 3m mdx (n=4), mdx+ABX (n=4) and GFmdx mice (n=5). Pooled samples for each group with n=6240 for 3m C57Bl; n=6001 for 3m mdx; n=10556 for 3m mdx+ABX; n=23059 for GFmdx. For morphometric analysis, images were quantified with Image J software for each mouse. Scale bars: 50 μm. Data information: Data are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, ordinary one-way ANOVA


( G ) Representative images of skeletal muscle showed the distribution and composition of the myosin heavy chain ( MyHC ) isoforms ( Type IIa , Type IIx and Type IIb ) . ( H , I ) Graph portrays ( H ) the percentage of myofibers expressing different MyHC isoforms and ( I ) myofibers area per type of MyHC in TAs of 3m mdx ( n = 4 ) , mdx + ABX ( n = 4 ) and GFmdx ( n = 5 ) mice ( n = 12 images per animal ) . Data information : Data are presented as mean ± SD ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; * * * * p < 0 . 0001 , ordinary one - way

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

(G) Representative images of skeletal muscle showed the distribution and composition of the myosin heavy chain (MyHC) isoforms (Type IIa, Type IIx and Type IIb). (H, I) Graph portrays (H) the percentage of myofibers expressing different MyHC isoforms and (I) myofibers area per type of MyHC in TAs of 3m mdx (n=4), mdx+ABX (n=4) and GFmdx (n=5) mice (n=12 images per animal). Data information: Data are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, ordinary one-way ANOVA


( J ) Representative SDH staining and quantification of percentage of SDH + myofibers of TAs from 3m mdx ( n = 4 ) , mdx + ABX ( n = 4 ) and GFmdx mice ( n = 5 ) ( n = 12 images per animal ) . Scale bars : 50 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 O O O O O O O O O O O O O O O O O O O O O O

(J) Representative SDH staining and quantification of percentage of SDH+ myofibers of TAs from 3m mdx (n=4), mdx+ABX (n=4) and GFmdx mice (n=5) (n=12 images per animal). Scale bars: 50 μm.


( D , E ) Representative H & E staining ( D ) and quantification of myofiber area with Image J software ( E ) of TA muscles from 3m C57Bl ( n = 5 ) , mdx ( n = 5 ) and ABX - mdxFMT _ C57Bl ( n = 6 ) . Scale bars for H & E : 200 μm . Data information : Data for staining quantification are presented as mean ± SD ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; * * * * p < 0 . 0001 , one - way ANOVA , Tukey ' s multiple comparison test ) .

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

(D, E) Representative H&E staining (D) and quantification of myofiber area with Image J software (E) of TA muscles from 3m C57Bl (n=5), mdx (n=5) and ABX-mdxFMT_C57Bl (n=6). Scale bars for H&E: 200 μm. Data information: Data for staining quantification are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, one-way ANOVA, Tukey's multiple comparison test).


( H ) Representative images of skeletal muscle showed the distribution and composition of MyHC isoforms ( Type IIa , Type IIx and Type IIb ) . Scale bar : 50 μm . ( I ) Graph portrays the percentage of myofibers expressing different MyHC isoforms . n = 12 images were analysed for each mouse .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of skeletal muscle showed the distribution and composition of MyHC isoforms (Type IIa, Type IIx and Type IIb). Scale bar: 50 μm. (I) Graph portrays the percentage of myofibers expressing different MyHC isoforms. n=12 images were analysed for each mouse.


( J - K ) Representative SDH staining and quantification of percentage of SDH + myofibers of TA muscles from mdx ( n = 5 ) and ABX - mdxFMT _ C57Bl ( n = 6 ) ( n = 12 images per mouse ) . Scale bar : 200 μm . Data information : Data for staining quantification are presented as mean ± SD ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; * * * * p < 0 . 0001 , one - way ANOVA , Tukey ' s multiple comparison test ) .

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

(J-K) Representative SDH staining and quantification of percentage of SDH+ myofibers of TA muscles from mdx (n=5) and ABX-mdxFMT_C57Bl (n=6) (n=12 images per mouse). Scale bar: 200 μm. Data information: Data for staining quantification are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, one-way ANOVA, Tukey's multiple comparison test).


( L - M ) Representative image of CD31 ( in cyan ) , α - SMA ( in green ) and isolectin ( in red ) staining and their quantification in TA muscles from mdx ( n = 5 ) and ABX - mdxFMT _ C57Bl ( n = 6 ) mice . Scale bar : 500 μm . Data information : Data for staining quantification are presented as mean ± SD ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; * * * * p < 0 . 0001 , one - way ANOVA , Tukey ' s multiple comparison test ) .

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

(L-M) Representative image of CD31 (in cyan), α-SMA (in green) and isolectin (in red) staining and their quantification in TA muscles from mdx (n=5) and ABX-mdxFMT_C57Bl (n=6) mice. Scale bar: 500 μm. Data information: Data for staining quantification are presented as mean ± SD (*p<0.05, **p<0.01, ***p<0.001; ****p<0.0001, one-way ANOVA, Tukey's multiple comparison test).


( E ) Tadpoles at NF50 were amputated , fixed at the indicated time and then processed for whole mount in situ hybridisation using a probe specific for foxm1 . The two last panels show transverse section in the non - regenerating spinal cord ( nr ) and the regenerate at 3dpa . The red circle highlights the spinal cord and the asterisk the notochord .

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(E) Tadpoles at NF50 were amputated, fixed at the indicated time and then processed for whole mount in situ hybridisation using a probe specific for foxm1. The two last panels show transverse section in the non-regenerating spinal cord (nr) and the regenerate at 3dpa. The red circle highlights the spinal cord and the asterisk the notochord.


( F ) Total RNA was isolated from regenerating tails at indicated time points post amputation , reversed transcribed into cDNA and analysed for foxm1 expression by qPCR , using ef1α as a reference gene . The graph represents the mean + / - SD of three independent experiments . One - way ANOVA with Dunnett ' s multiple comparisons test was used . * p = 0 .

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

(F) Total RNA was isolated from regenerating tails at indicated time points post amputation, reversed transcribed into cDNA and analysed for foxm1 expression by qPCR, using ef1α as a reference gene. The graph represents the mean +/- SD of three independent experiments. One-way ANOVA with Dunnett's multiple comparisons test was used. * p=0.0149


( A ) NF40 tadpoles with the following genotypes foxm1 - / - ( mut ) , foxm1 + / - ( het ) and foxm1 + / + ( wt ) were amputated and left to regenerate for 7 days . The images show representative tails at 3 and 7 dpa . The white arrowheads indicate the amputation site . ( B ) Quantification of the rate of regeneration . The ratio of the length of the regenerate to the length that has originally be amputated was compared for the whole tail at 3 and 7 dpa . The graph represents the mean + / - SD of 5 independent experiments from 3 different clutches with at least 5 tadpoles in each experiment .

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(A) NF40 tadpoles with the following genotypes foxm1-/- (mut), foxm1+/- (het) and foxm1+/+ (wt) were amputated and left to regenerate for 7 days. The images show representative tails at 3 and 7 dpa. The white arrowheads indicate the amputation site. (B) Quantification of the rate of regeneration. The ratio of the length of the regenerate to the length that has originally be amputated was compared for the whole tail at 3 and 7 dpa. The graph represents the mean+/-SD of 5 independent experiments from 3 different clutches with at least 5 tadpoles in each experiment.


( D ) Representative images of EdU ( red ) and DAPI ( blue ) staining at 5 dpa . ( E ) The graph represents the mean + / - SD of EdU + cells over the total number of cells in the spinal cord of 5 to 12 tadpoles . Each data point represents a tadpole , with an average of 9 sections analysed per animal .

(D) Representative images of EdU (red) and DAPI (blue) staining at 5 dpa. (E) The graph represents the mean +/- SD of EdU+ cells over the total number of cells in the spinal cord of 5 to 12 tadpoles. Each data point represents a tadpole, with an average of 9 sections analysed per animal.


( G ) Representative images of EdU ( green ) , BrdU ( magenta ) , Sox3 ( white ) and DAPI ( blue ) staining at 3 dpa . ( H ) Quantification of images in ( G ) . The graph represents the mean + / - SD of 6 tadpoles with an average of 13 sections per tadpole analysed .

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

(G) Representative images of EdU (green), BrdU (magenta), Sox3 (white) and DAPI (blue) staining at 3 dpa. (H) Quantification of images in (G). The graph represents the mean +/- SD of 6 tadpoles with an average of 13 sections per tadpole analysed.


( C ) Representative sections labelled with an anti - PCNA antibody ( red ) and DAPI ( blue ) at the indicated day after amputation ( dpa ) . The white arrowheads point to cells in S phase in the spinal cord and the yellow arrowhead at cells in G1 , G2 and M phases . The right panels correspond to the inset indicated as a white box in the middle panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative sections labelled with an anti-PCNA antibody (red) and DAPI (blue) at the indicated day after amputation (dpa). The white arrowheads point to cells in S phase in the spinal cord and the yellow arrowhead at cells in G1, G2 and M phases. The right panels correspond to the inset indicated as a white box in the middle panels.


( A ) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa and fixed at 5 dpa . After sectioning , the samples were labelled with antibodies against Sox3 ( green ) , EdU ( magenta ) and DAPI ( blue ) . White arrowheads show Sox3 positive extensions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa and fixed at 5 dpa. After sectioning, the samples were labelled with antibodies against Sox3 (green), EdU (magenta) and DAPI (blue). White arrowheads show Sox3 positive extensions.


( B ) Sections of spinal cords at 5 dpa in the stump ( non - regen . ) or in the regenerate ( regen ) labelled with anti - Sox3 ( green ) , anti - Acetylated Tubulin ( AcTub , magenta ) and DAPI ( blue ) . White arrowheads show Sox3 positive extensions .

(B) Sections of spinal cords at 5 dpa in the stump (non-regen.) or in the regenerate (regen) labelled with anti-Sox3 (green), anti-Acetylated Tubulin (AcTub, magenta) and DAPI (blue). White arrowheads show Sox3 positive extensions.


( E ) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa and fixed at 5 dpa . After sectioning , the samples were labelled with antibodies against Myt1 ( green ) , EdU ( magenta ) and DAPI ( blue ) . ( F ) Quantification of images shown in ( E ) . The graph represents the mean + / - SD from 3 tadpoles with an average of 12 sections each .

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(E) Representative sections of spinal cords from tadpoles injected with EdU at 3 dpa and fixed at 5 dpa. After sectioning, the samples were labelled with antibodies against Myt1 (green), EdU (magenta) and DAPI (blue). (F) Quantification of images shown in (E). The graph represents the mean +/- SD from 3 tadpoles with an average of 12 sections each.


Gene - trap - based genetic screen in haploid human cells for regulators of p38 kinase activation ( p38 Thr180 / Tyr182 phosphorylation ) in response to hyperosmotic shock ( 500 mM Sorbitol , 1 h ) . Per gene ( dots ) , the frequency of gene - trap insertions in the ' high ' phospho - p38 population divided by the frequency of insertions in the ' low ' population is plotted as mutation index ( y - axis ) against the total number of insertions assigned to the gene ( x - axis ) . Significant negative and positive regulators are colored in orange and blue , respectively ( two - sided Fisher ' s exact test , false discovery rate - corrected , p ≤ 0 . 05 ) .

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Gene-trap-based genetic screen in haploid human cells for regulators of p38 kinase activation (p38 Thr180/Tyr182 phosphorylation) in response to hyperosmotic shock (500 mM Sorbitol, 1 h). Per gene (dots), the frequency of gene-trap insertions in the 'high' phospho-p38 population divided by the frequency of insertions in the 'low' population is plotted as mutation index (y-axis) against the total number of insertions assigned to the gene (x-axis). Significant negative and positive regulators are colored in orange and blue, respectively (two-sided Fisher's exact test, false discovery rate-corrected, p≤0.05).


Human U2OS cells and cells deleted for ZAK ( ∆ ZAK ) were treated with anisomycin ( 1 h ) or 500 mM sorbitol ( 1 h ) . Lysates were analyzed by immunoblotting as indicated .

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

Human U2OS cells and cells deleted for ZAK (∆ZAK) were treated with anisomycin (1 h) or 500 mM sorbitol (1 h). Lysates were analyzed by immunoblotting as indicated.


Unique gene - trap insertions ( dots ) mapped to the genomic ZAK locus ( x - axis ) identified in the low ( blue ) and high ( orange ) channel of two individual haploid genetic screens for stress - induced p38 activation and Anisomycin ( Brockmann et al . , 2017 ) ) . The total number of identified insertions was similar for each channel within the individual screens . For visual purposes , insertion dots were spread on the y - axis and exons in the ZAK gene body schematic have been scaled up ( compared to introns ) .

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Unique gene-trap insertions (dots) mapped to the genomic ZAK locus (x-axis) identified in the low (blue) and high (orange) channel of two individual haploid genetic screens for stress-induced p38 activation and Anisomycin(Brockmann et al., 2017)). The total number of identified insertions was similar for each channel within the individual screens. For visual purposes, insertion dots were spread on the y-axis and exons in the ZAK gene body schematic have been scaled up (compared to introns).


U2OS / S - HA - ZAKβ cells were induced for expression with doxycycline ( DOX ) and subjected to the indicated drugs and treatments ( 1 h ) . Lysates were analyzed by immunoblotting with the indicated antibodies . Sorb . , sorbitol ; Ani . , anisomycin ; Ars . , arsenite .

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

U2OS/S-HA-ZAKβ cells were induced for expression with doxycycline (DOX) and subjected to the indicated drugs and treatments (1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. Sorb., sorbitol; Ani., anisomycin; Ars., arsenite.


U2OS cells stably expressing WT and kinase - dead ( KD ) versions of S - HA - ZAKβ were pre - treated with ZAK inhibitor ( ZAKi - 0 . 5 h ) and 500 mM sorbitol ( 1 h ) as indicated . Whole Cell Extracts ( WCE ) were analyzed by immunoblotting , and strep - pulldown material was used in a kinase assay , separated by SDS - PAGE and analyzed by autoradiography .

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

U2OS cells stably expressing WT and kinase-dead (KD) versions of S-HA-ZAKβ were pre-treated with ZAK inhibitor (ZAKi - 0.5 h) and 500 mM sorbitol (1 h) as indicated. Whole Cell Extracts (WCE) were analyzed by immunoblotting, and strep-pulldown material was used in a kinase assay, separated by SDS-PAGE and analyzed by autoradiography.


U2OS and ∆ ZAK cells rescued with S - HA - ZAKβ , were DOX - induced and treated with 500 mM sorbitol ( 1 h ) as indicated . Lysates were analyzed by immunoblotting with the indicated antibodies .

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

U2OS and ∆ZAK cells rescued with S-HA-ZAKβ, were DOX-induced and treated with 500 mM sorbitol (1 h) as indicated. Lysates were analyzed by immunoblotting with the indicated antibodies.


U2OS and ∆ ZAK cells were transfected with the indicated siRNAs and treated with sorbitol ( 500 mM , 1 h ) as indicated . Lysates were analyzed by immunoblotting with the indicated antibodies . T1 , TAK1 ; M2 , MEKK2 .

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

U2OS and ∆ZAK cells were transfected with the indicated siRNAs and treated with sorbitol (500 mM, 1 h) as indicated. Lysates were analyzed by immunoblotting with the indicated antibodies. T1, TAK1; M2, MEKK2.


U2OS cells stably expressing Strep - HA - ZAKβ or - ZAKα were treated with sorbitol ( 500 mM , 1 h ) as indicated . Cells were fixed , immunostained with HA antibody and counter - stained with DAPI . Data information : scale bars , 10 μm .

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

U2OS cells stably expressing Strep-HA-ZAKβ or -ZAKα were treated with sorbitol (500 mM, 1 h) as indicated. Cells were fixed, immunostained with HA antibody and counter-stained with DAPI. Data information: scale bars, 10 μm.


U2OS cells stably expressing ZAKβ - GFP were treated with sorbitol ( 500 mM , 1 h ) , pre - extracted , fixed and immunostained with Lamin A / C antibody . Lower right : Intensity distribution graph showing fluorescence intensities along the magenta line . As in ( B ) except that Hela cells were transfected with ZAKbeta - GFP and immunostained with 53BP1 antibody . Data information : 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

U2OS cells stably expressing ZAKβ-GFP were treated with sorbitol (500 mM, 1 h), pre-extracted, fixed and immunostained with Lamin A/C antibody. Lower right: Intensity distribution graph showing fluorescence intensities along the magenta line. As in (B) except that Hela cells were transfected with ZAKbeta-GFP and immunostained with 53BP1 antibody. Data information: scale bars, 10 μm.


Cells from ( B ) were imaged by live cell fluorescence microscopy . Sorbitol ( final concentration 500 mM ) was added after acquisition of the first frame . Data information : scale bars , 10 μm .

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

Cells from (B) were imaged by live cell fluorescence microscopy. Sorbitol (final concentration 500 mM) was added after acquisition of the first frame. Data information: scale bars, 10 μm.


As in ( D ) , except that cells were co - transfected with mCherry - FAK . Inserts show higher magnification of the yellow and red regions , white arrow indicates the direction of stress fibers . Data information : scale bars , 10 μm .

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As in (D), except that cells were co-transfected with mCherry-FAK. Inserts show higher magnification of the yellow and red regions, white arrow indicates the direction of stress fibers. Data information: scale bars, 10 μm.


U2OS cells were transfected with constructs from ( F ) and imaged by live cell fluorescence microscopy . Sorbitol ( final concentration 500 mM ) was added after acquisition of the first frame . Data information : scale bars , 10 μm .

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

U2OS cells were transfected with constructs from (F) and imaged by live cell fluorescence microscopy. Sorbitol (final concentration 500 mM) was added after acquisition of the first frame. Data information: scale bars, 10 μm.


U2OS and ∆ ZAK cells stably expressing the indicated Strep - HA - tagged ZAKβ constructs were treated with sorbitol ( 500 mM , 5 min ) . Lysates were analyzed by immunoblotting with the indicated antibodies .

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

U2OS and ∆ZAK cells stably expressing the indicated Strep-HA-tagged ZAKβ constructs were treated with sorbitol (500 mM, 5 min). Lysates were analyzed by immunoblotting with the indicated antibodies.


U2OS and ∆ ZAK cells were embedded in agarose and subjected to cyclic compression ( 0 . 5 h ) . Lysates were analyzed by immunoblotting with the indicated antibodies .

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

U2OS and ∆ZAK cells were embedded in agarose and subjected to cyclic compression (0.5 h). Lysates were analyzed by immunoblotting with the indicated antibodies.


U2OS and ∆ ZAK cells stably expressing the indicated Strep - HA - tagged ZAKβ constructs were embedded in agarose and subjected to cyclic compression for 5 or 30 min . Lysates were analyzed as in ( B ) .

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

U2OS and ∆ZAK cells stably expressing the indicated Strep-HA-tagged ZAKβ constructs were embedded in agarose and subjected to cyclic compression for 5 or 30 min. Lysates were analyzed as in (B).


U2OS and ∆ ZAK cells were pretreated with ZAK inhibitor ( ZAKi ) for 30 min and subjected to equibiaxial ( Equi ) or uniaxial ( Uni ) cyclic stretch ( 0 . 5 h ) as indicated . Lysates were analyzed as in ( B ) .

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

U2OS and ∆ZAK cells were pretreated with ZAK inhibitor (ZAKi) for 30 min and subjected to equibiaxial (Equi) or uniaxial (Uni) cyclic stretch (0.5 h) as indicated. Lysates were analyzed as in (B).


U2OS and ∆ ZAK cells were transfected with the indicated siRNAs and subjected to equibiaxial cyclic stretch ( 5 min ) as indicated . Lysates were analyzed as in ( B ) .

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

U2OS and ∆ZAK cells were transfected with the indicated siRNAs and subjected to equibiaxial cyclic stretch (5 min) as indicated. Lysates were analyzed as in (B).


Expression levels of MAP3 kinase genes in human skeletal muscle ( GTEx Portal ) . TPM , Transcripts Per Million .

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Expression levels of MAP3 kinase genes in human skeletal muscle (GTEx Portal). TPM, Transcripts Per Million.


C2C12 cells were incubated in diff . medium for 3 or 6 days . Lysates were analyzed for ZAK isoform expression by immunoblotting .

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C2C12 cells were incubated in diff. medium for 3 or 6 days. Lysates were analyzed for ZAK isoform expression by immunoblotting.


C2C12 myoblasts and myotubes ( 6 d differentiation ) were pre - treated with ZAK inhibitor ( ZAKi , 0 . 5 h ) and treated with 500 mM sorbitol ( 1 h ) as indicated . Lysates were analyzed by immunoblotting with the indicated antibodies .

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C2C12 myoblasts and myotubes (6 d differentiation) were pre-treated with ZAK inhibitor (ZAKi, 0.5 h) and treated with 500 mM sorbitol (1 h) as indicated. Lysates were analyzed by immunoblotting with the indicated antibodies.


Skeletal muscle ( tibialis anterior , TA ) and liver from WT and ZAK - / - mice were lysed and analyzed for ZAK isoform expression by immunoblotting . Ponceau staining of the membrane indicates equal loading .

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Skeletal muscle (tibialis anterior, TA) and liver from WT and ZAK-/- mice were lysed and analyzed for ZAK isoform expression by immunoblotting. Ponceau staining of the membrane indicates equal loading.


Mouse embryonic fibroblasts ( MEF ) isolated from WT and ZAK - / - mice were treated with 500 mM sorbitol ( 1 h ) . Lysates were analyzed by immunoblotting with the indicated antibodies . * , unspecific band .

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Mouse embryonic fibroblasts (MEF) isolated from WT and ZAK-/- mice were treated with 500 mM sorbitol (1 h). Lysates were analyzed by immunoblotting with the indicated antibodies. *, unspecific band.


16 - 18 - week - old WT and ZAK - / - male mice ( n = 3 biological replicates ) were subjected to the protocol in ( D ) . TA lysates were analyzed by immunoblotting with the indicated antibodies .

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16-18-week-old WT and ZAK-/- male mice (n=3 biological replicates) were subjected to the protocol in (D). TA lysates were analyzed by immunoblotting with the indicated antibodies.


ZAKβ S335 and S339 phosphorylation sites upregulated in WT TA muscles from ( D ) . Values indicate absolute phospho - peptide abundances and error bars represent the standard deviation ( n = 3 biological replicates ) .

ZAKβ S335 and S339 phosphorylation sites upregulated in WT TA muscles from (D). Values indicate absolute phospho-peptide abundances and error bars represent the standard deviation (n=3 biological replicates).


TA muscle in an 8 - week old WT male mouse was electroporated with a ZAKβ - tdTomato construct . After 7 days the muscle was harvested , sectioned longitudinally and immunostained with an antibody against the Z - disc marker α - actinin1 . The right panel shows a higher magnification of the region highlighted in yellow . Data information : scale bars , 20 μm .

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TA muscle in an 8-week old WT male mouse was electroporated with a ZAKβ-tdTomato construct. After 7 days the muscle was harvested, sectioned longitudinally and immunostained with an antibody against the Z-disc marker α-actinin1. The right panel shows a higher magnification of the region highlighted in yellow. Data information: scale bars, 20 μm.


C2C12 cells were differentiated into myotubes for 14 days and transfected with the indicated GFP - ZAKβ constructs . Cells were fixed and immunostained with an antibody against α - actinin1 . Data information : scale bars , 20 μm .

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C2C12 cells were differentiated into myotubes for 14 days and transfected with the indicated GFP-ZAKβ constructs. Cells were fixed and immunostained with an antibody against α-actinin1. Data information: scale bars, 20 μm.


16 - 18 - week - old WT and ZAK - / - male mice were subjected to the experimental protocol in ( I ) . TA muscle lysates were analyzed by immunoblotting with the indicated antibodies .

16-18-week-old WT and ZAK-/- male mice were subjected to the experimental protocol in (I). TA muscle lysates were analyzed by immunoblotting with the indicated antibodies.


H & E staining of soleus muscle cross sections from 8 - and 22 - week - old WT and ZAK - / - male mice . Arrows indicate the presence of centralized nuclei . Scale bars , 50 μm . Quantification of ( A ) . Values indicate the percentage of fibers displaying centralized nuclei and error bars represent the standard deviation ( n = 3 biological replicates ) . * * , p < 0 . 01 and * * * , p < 0 . 001 in t - test with Bonferroni - Dunn correction for multiple comparison .

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H&E staining of soleus muscle cross sections from 8- and 22-week-old WT and ZAK-/- male mice. Arrows indicate the presence of centralized nuclei. Scale bars, 50 μm. Quantification of (A). Values indicate the percentage of fibers displaying centralized nuclei and error bars represent the standard deviation (n=3 biological replicates). **, p<0.01 and ***, p<0.001 in t-test with Bonferroni-Dunn correction for multiple comparison.


Muscles from ( A ) were immunostained for type I ( red ) and type IIa ( green ) fibers using myosin isoform - specific antibodies . Scale bars , 500 μm . Quantification of ( C ) . Values indicate the percentage of fibers positive for type I myosin and error bars represent the standard deviation ( n = 3 biological replicates ) . * * , p < 0 . 01 and ns , not significant in t - test with Bonferroni - Dunn correction for multiple comparison .

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Muscles from (A) were immunostained for type I (red) and type IIa (green) fibers using myosin isoform-specific antibodies. Scale bars, 500 μm. Quantification of (C). Values indicate the percentage of fibers positive for type I myosin and error bars represent the standard deviation (n=3 biological replicates). **, p<0.01 and ns, not significant in t-test with Bonferroni-Dunn correction for multiple comparison.


q - RT - PCR reveals that miR - 181a / b silencing leads to upregulation of miR - 181a / b predicted targets in SH - SY5Y cells . N = 3 independent experiments .

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q-RT-PCR reveals that miR-181a/b silencing leads to upregulation of miR-181a/b predicted targets in SH-SY5Y cells. N=3 independent experiments.


miR - 181 - mimic transfection specifically inhibits Luciferase activity of constructs containing WT 3 ' - UTR predicted target sequences . Point mutations ( mut ) in miR - 181a / b binding sites abolish Luciferase repression in all cases apart from PPARGC1A . Data are normalized to negative mimic transfection ( dashed line ) . N = 6 independent experiments .

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miR-181-mimic transfection specifically inhibits Luciferase activity of constructs containing WT 3'-UTR predicted target sequences. Point mutations (mut) in miR-181a/b binding sites abolish Luciferase repression in all cases apart from PPARGC1A. Data are normalized to negative mimic transfection (dashed line). N=6 independent experiments.


q - RT - PCR reveals upregulation of miR - 181a / b targets in the eyes of miR - 181a / b - 1 - / - vs . miR - 181a / b - 1 + / + animals . n ≥ 5 animals / genotype .

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q-RT-PCR reveals upregulation of miR-181a/b targets in the eyes of miR-181a/b-1-/- vs. miR-181a/b-1+/+ animals. n≥5 animals/genotype.


miR - 181a / b - 1 - / - mice show increased mtDNA content vs . miR - 181a / b - 1 + / + mice as measured by q - PCR . N = 4 animals / genotype .

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miR-181a/b-1-/- mice show increased mtDNA content vs. miR-181a/b-1+/+ mice as measured by q-PCR. N=4 animals/genotype.


WB analysis ( left panel ) reveals increased levels of mitochondrial proteins in the eyes of miR - 181a / b - 1 - / - ( - / - ) vs . miR - 181a / b - 1 + / + ( + / + ) mice ( quantified in the right panel ) . Data are normalized to either p115 or Gapdh . N ≥ 3 animals / genotype . Please note that all compared bands from + / + and - / - samples are from the same blots which were cropped and shown split for the sake of data presentation clarity .

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WB analysis (left panel) reveals increased levels of mitochondrial proteins in the eyes of miR-181a/b-1-/- (-/-) vs. miR-181a/b-1+/+ (+/+) mice (quantified in the right panel). Data are normalized to either p115 or Gapdh. N≥3 animals/genotype. Please note that all compared bands from +/+ and -/- samples are from the same blots which were cropped and shown split for the sake of data presentation clarity.


WB analysis on mitochondrial and cytosolic fractions ( left panel ) show increased levels of p62 and Parkin in mitochondrial fraction from the eye of - / - vs . + / + mice ( quantified in the right panel ) . Data are normalized to Ndufb8 or Gapdh for mitochondrial and cytosolic fractions , respectively . N = 3 animals / genotype .

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WB analysis on mitochondrial and cytosolic fractions (left panel) show increased levels of p62 and Parkin in mitochondrial fraction from the eye of -/- vs. +/+ mice (quantified in the right panel). Data are normalized to Ndufb8 or Gapdh for mitochondrial and cytosolic fractions, respectively. N=3 animals/genotype.


Cell death analysis shows that miR - 181a / b silencing protects SH - SY5Y cells from FCCP treatment . N ≥ 7 independent experiments .

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Cell death analysis shows that miR-181a/b silencing protects SH-SY5Y cells from FCCP treatment. N≥7 independent experiments.


TUNEL assays on eye and brain sections of stage ( st ) 30 medakafish control and MO - injected embryos reveal a decrease in the number of apoptotic cells in both hccs - MO / miR - 181a / b - MO - and cox7b - MO / miR - 181a / b - MO - injected compared to hccs - MO - and cox7b - MO - injected embryos . Scale bars are 20μm .

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TUNEL assays on eye and brain sections of stage (st)30 medakafish control and MO-injected embryos reveal a decrease in the number of apoptotic cells in both hccs-MO/miR-181a/b-MO- and cox7b-MO/miR-181a/b-MO-injected compared to hccs-MO- and cox7b-MO-injected embryos. Scale bars are 20μm.


TUNEL - positive cells / eye . n ≥ 5 eyes for each model .

TUNEL-positive cells/eye. n≥5 eyes for each model.


Caspase assays show restored levels of caspase - 3 and caspase - 9 activities in hccs - MO / miR - 181a / b - MO - and cox7b - MO / miR - 181a / b - MO - injected embryos with respect to hccs - MO - and cox7b - MO - injected embryos . n ≥ 5 embryos for each model .

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Caspase assays show restored levels of caspase-3 and caspase-9 activities in hccs-MO/miR-181a/b-MO- and cox7b-MO/miR-181a/b-MO-injected embryos with respect to hccs-MO- and cox7b-MO-injected embryos. n≥5 embryos for each model.


Representative images of st30 medaka embryos injected with hccs - MO ( B ) alone or co - injected with miR - 181a / b - MOs Co - injection of miR - 181a / b - MOs rescues microphthalmia and microcephaly in both hccs - MO embryos . hccs - MO / miR - 181a / b - MO - b - MO - injected embryos were treated with Baf - A1 , PD98059 or HA14 - 1 . Baf - A1 treatment counteracts the protective effect of miR - 181a / b downregulation in hccs - MO / miR - 181a / b - MO embryos PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR - 181a / b downregulation HA14 - 1 treatment counteracts the effect of miR - 181a / b downregulation in the hccs - MO / miR - 181a / b - MO model Scale bars are 100μm .

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Representative images of st30 medaka embryos injected with hccs-MO (B) alone or co-injected with miR-181a/b-MOs Co-injection of miR-181a/b-MOs rescues microphthalmia and microcephaly in both hccs-MO embryos. hccs-MO/miR-181a/b-MO- b-MO-injected embryos were treated with Baf-A1, PD98059 or HA14-1. Baf-A1 treatment counteracts the protective effect of miR-181a/b downregulation in hccs-MO/miR-181a/b-MO embryos PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR-181a/b downregulation HA14-1 treatment counteracts the effect of miR-181a/b downregulation in the hccs-MO/miR-181a/b-MO model Scale bars are 100μm.


Representative images of st30 medaka embryos injected with cox7b - MO ( H ) alone or co - injected with miR - 181a / b - MOs Co - injection of miR - 181a / b - MOs rescues microphthalmia and microcephaly in cox7b - MO embryos . cox7b - MO / miR - 181a / b - MO - injected embryos were treated with Baf - A1 , PD98059 or HA14 - 1 . Baf - A1 treatment counteracts the protective effect of miR - 181a / b downregulation in cox7b - MO / miR - 181a / b - MO embryos PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR - 181a / b downregulation Scale bars are 100μm .

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Representative images of st30 medaka embryos injected with cox7b-MO (H) alone or co-injected with miR-181a/b-MOs Co-injection of miR-181a/b-MOs rescues microphthalmia and microcephaly in cox7b-MO embryos. cox7b-MO/miR-181a/b-MO-injected embryos were treated with Baf-A1, PD98059 or HA14-1. Baf-A1 treatment counteracts the protective effect of miR-181a/b downregulation in cox7b-MO/miR-181a/b-MO embryos PD98059 treatment does not interfere with the modulation of the MLS phenotype mediated by miR-181a/b downregulation Scale bars are 100μm.


Percentage of embryos with or without MLS phenotype in the different conditions in hccs - MO embryos . N ≥ 300 embryos / conditions .

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Percentage of embryos with or without MLS phenotype in the different conditions in hccs-MO embryos. N≥300 embryos/conditions.


Percentage of embryos with or without MLS phenotype in the different conditions in cox7b - MO ( N ) embryos . N ≥ 300 embryos / conditions .

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Percentage of embryos with or without MLS phenotype in the different conditions in cox7b-MO (N) embryos. N≥300 embryos/conditions.


Immunofluorescence analysis with anti - NeuN antibody in the retina of miR - 181a / b - 1 + / + and miR - 181a / b - 1 - / - mice intravitreally injected with Rotenone or DMSO . RGCs are preserved in miR - 181a / b - 1 - / - rotenone - injected mice with respect to controls at both one and two weeks after injection . Scale bars are 50μm .

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Immunofluorescence analysis with anti-NeuN antibody in the retina of miR-181a/b-1+/+ and miR-181a/b-1-/- mice intravitreally injected with Rotenone or DMSO. RGCs are preserved in miR-181a/b-1-/- rotenone-injected mice with respect to controls at both one and two weeks after injection. Scale bars are 50μm.


Number of RGCs / mm ( indicated as % ) at one - and two - weeks post - injection . N = 10 .

Number of RGCs/mm (indicated as %) at one- and two-weeks post-injection. N=10.


NADH dehydrogenase histochemical reaction on retinal sections of miR - 181a / b - 1 + / + and miR - 181a / b - 1 - / - mice intravitreally injected with Rotenone or DMSO . At one week post - injection , NADH dehydrogenase activity is lost in RGCs ( GCL , areas within dashed lines ) of miR - 181a / b - 1 + / + Rotenone - injected eyes , while it is preserved in those of miR - 181a / b - 1 - / - Rotenone - injected eyes . GCL , Ganglion Cells Layer ONL ; INL , Inner Nuclear Layer ; Outer Nuclear Layer . Scale bars are 50μm .

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NADH dehydrogenase histochemical reaction on retinal sections of miR-181a/b-1+/+ and miR-181a/b-1-/- mice intravitreally injected with Rotenone or DMSO. At one week post-injection, NADH dehydrogenase activity is lost in RGCs (GCL, areas within dashed lines) of miR-181a/b-1+/+ Rotenone-injected eyes, while it is preserved in those of miR-181a/b-1-/- Rotenone-injected eyes. GCL, Ganglion Cells Layer ONL; INL, Inner Nuclear Layer; Outer Nuclear Layer. Scale bars are 50μm.


Immunofluorescence analysis , with an anti - CoxIV antibody , in the retina of miR - 181a / b - 1 + / + and miR - 181a / b - 1 - / - mice , injected with either Rotenone or DMSO , showed preserved mitochondria in miR - 181a / b - 1 - / - rotenone - injected eyes at one - week post - injection . Dashed boxes indicate the area of magnifications shown in right panels . Scale bars are 10μm .

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Immunofluorescence analysis, with an anti-CoxIV antibody, in the retina of miR-181a/b-1+/+ and miR-181a/b-1-/- mice, injected with either Rotenone or DMSO, showed preserved mitochondria in miR-181a/b-1-/- rotenone-injected eyes at one-week post-injection. Dashed boxes indicate the area of magnifications shown in right panels. Scale bars are 10μm.


Graphical representation of the results of the optokinetic tracking assays reported as fold change of cycles / degree . Visual acuity is preserved in miR - 181a / b - 1 - / - rotenone - injected mice with respect to controls at both one and two weeks after injection . N = 10 .

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Graphical representation of the results of the optokinetic tracking assays reported as fold change of cycles/degree. Visual acuity is preserved in miR-181a/b-1-/- rotenone-injected mice with respect to controls at both one and two weeks after injection. N=10.


Immunofluorescence analysis with anti - NeuN antibody in the retina of WT , Ndufs4 - / - , Ndufs4 - / - / miR - 181a / b - 1 - / - and miR - 181a / b - 1 - / - mice . RGCs are preserved in Ndufs4 - / - / miR - 181a / b - 1 - / - with respect to Ndufs4 - / - mice . Scale bars are 50μm .

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Immunofluorescence analysis with anti-NeuN antibody in the retina of WT, Ndufs4-/-, Ndufs4-/-/miR-181a/b-1-/- and miR-181a/b-1-/- mice. RGCs are preserved in Ndufs4-/-/miR-181a/b-1-/- with respect to Ndufs4-/- mice. Scale bars are 50μm.


Graphical representation of the results of the optokinetic tracking assays reported as fold change of cycles / degree . Visual acuity is preserved in Ndufs4 - / - / miR - 181a / b - 1 - / - with respect to Ndufs4 - / - mice . N ≥ 8 .

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Graphical representation of the results of the optokinetic tracking assays reported as fold change of cycles/degree. Visual acuity is preserved in Ndufs4-/-/miR-181a/b-1-/- with respect to Ndufs4-/- mice. N≥8.


Electroretinographic analysis reveals amelioration of b - wave patterns in Ndufs4 - / - / miR - 181a / b - 1 - / - with respect to Ndufs4 - / - mice . N ≥ 5 .

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Electroretinographic analysis reveals amelioration of b-wave patterns in Ndufs4-/-/miR-181a/b-1-/- with respect to Ndufs4-/-mice. N≥5.


Electron microscopy analysis shows amelioration of mitochondria morphology and increase of mitochondrial number in Ndufs4 - / - / miR - 181a / b - 1 - / - vs . Ndufs4 - / - mice . Scale bars are 1μm . The quantitative increase of mitochondria is reported in C as number of mitochondria / RGC , at p30 and p55 . N ≥ 2 animals / genotype .

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Electron microscopy analysis shows amelioration of mitochondria morphology and increase of mitochondrial number in Ndufs4-/-/miR-181a/b-1-/- vs. Ndufs4-/- mice. Scale bars are 1μm. The quantitative increase of mitochondria is reported in C as number of mitochondria/RGC, at p30 and p55. N≥2 animals/genotype.


Ndufs4 - / - / miR - 181a / b - 1 - / - mice show increased mtDNA content vs . Ndufs4 - / - mice as measured by q - PCR . N ≥ 4 animals / genotype .

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Ndufs4-/-/miR-181a/b-1-/- mice show increased mtDNA content vs. Ndufs4-/- mice as measured by q-PCR. N≥4 animals/genotype.


Biochemical activity of MRC complexes I , II and IV normalized by the percentage of citrate synthase ( CS ) activity in Ndufs4 - / - / miR - 181a / b - 1 - / - vs . Ndufs4 - / - mice . N ≥ 3 animals / genotype . p ‐ values were calculated by two tailed Student ' s t - test ; Error bars are SEM .

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Biochemical activity of MRC complexes I, II and IV normalized by the percentage of citrate synthase (CS) activity in Ndufs4-/-/miR-181a/b-1-/- vs. Ndufs4-/- mice. N≥3 animals/genotype. p‐values were calculated by two tailed Student's t-test; Error bars are SEM.


B . Identification of highly variable genes ( HVGs ) for the time - point ZT2 . The trend for the global relation ( red line ) between CV2 ( variance / mean2 ) and mean expression is defined using all genes , minus small and lowly expressed genes ( see material and methods for more detail ) and used to identify HVG ( blue ) . For each gene , a corrected CV2 is calculated : log2 ( CV2 / trend ) .

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B. Identification of highly variable genes (HVGs) for the time-point ZT2. The trend for the global relation (red line) between CV2 (variance/mean2) and mean expression is defined using all genes, minus small and lowly expressed genes (see material and methods for more detail) and used to identify HVG (blue). For each gene, a corrected CV2 is calculated: log2(CV2 /trend).


C . Expression profiles ( top ) in the 14 seedlings over a 24 hour time course ( with 12 time - points ) for a non - variable gene ( left ) , a highly variable gene ( middle ) and a gene with the level of variability changing across the 24 hour ( right ) . Each dot is the mean normalised expression level for a single seedling . Variability profiles ( bottom ) of the log2 ( CV2 / trend ) for the same genes are also shown . Blue stars indicate time - points for which the gene is identified as being highly variable .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Expression profiles (top) in the 14 seedlings over a 24 hour time course (with 12 time-points) for a non-variable gene (left), a highly variable gene (middle) and a gene with the level of variability changing across the 24 hour (right). Each dot is the mean normalised expression level for a single seedling. Variability profiles (bottom) of the log2(CV2 /trend) for the same genes are also shown. Blue stars indicate time-points for which the gene is identified as being highly variable.


A . Number of genes identified as being highly variable for each time - point . These genes are separated between those that are also selected as highly variable in at least one other time - point ( dark blue ) and those highly variable in only one time - point ( light blue ) . The top bar indicates time - points harvested during the day ( orange ) , just before dusk ( red ) , during the night ( black ) and just before dawn ( blue ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Number of genes identified as being highly variable for each time-point. These genes are separated between those that are also selected as highly variable in at least one other time-point (dark blue) and those highly variable in only one time-point (light blue). The top bar indicates time-points harvested during the day (orange), just before dusk (red), during the night (black) and just before dawn (blue).


B . Distribution of the number of time - points at which genes are identified as highly variable ( blue ) or lowly variable ( green ) . The distribution of average ( black ) and 95 % confidence interval ( dotted grey ) for the thousand random sets is also represented .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Distribution of the number of time-points at which genes are identified as highly variable (blue) or lowly variable (green). The distribution of average (black) and 95% confidence interval (dotted grey) for the thousand random sets is also represented.


C . Heatmap of the percentage of HVGs shared between time - points . Red indicates a high percentage of HVGs in common between two time - points . The top and side bars indicates time - points harvested during the day ( orange ) , just before dusk ( red ) , during the night ( black ) and just before dawn ( blue ) . D . Hierarchical clustering of HVGs based on the log2 ( CV2 / trend ) at each time point . The result is represented as a heatmap where yellow indicates a high log2 ( CV2 / trend ) . The genes were separated into four clusters , indicated by the side colored bar . The top bar indicates time - points harvested during the day ( orange ) , just before dusk ( red ) , during the night ( black ) and just before dawn ( blue ) . See Appendix Fig S3G for heatmaps of the log2 ( CV2 / trend ) with the same colour cutoffs for HVGs , LVGs and random genes . E . Heatmap of the mean normalised expression level for the genes in Fig 2D , keeping the same clustering organisation . The top bar indicates time - points harvested during the day ( orange ) , just before dusk ( red ) , during the night ( black ) and just before dawn ( blue ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Heatmap of the percentage of HVGs shared between time-points. Red indicates a high percentage of HVGs in common between two time-points. The top and side bars indicates time-points harvested during the day (orange), just before dusk (red), during the night (black) and just before dawn (blue). D. Hierarchical clustering of HVGs based on the log2(CV2/trend) at each time point. The result is represented as a heatmap where yellow indicates a high log2(CV2/trend). The genes were separated into four clusters, indicated by the side colored bar. The top bar indicates time-points harvested during the day (orange), just before dusk (red), during the night (black) and just before dawn (blue). See Appendix Fig S3G for heatmaps of the log2(CV2/trend) with the same colour cutoffs for HVGs, LVGs and random genes. E. Heatmap of the mean normalised expression level for the genes in Fig 2D, keeping the same clustering organisation. The top bar indicates time-points harvested during the day (orange), just before dusk (red), during the night (black) and just before dawn (blue).


C . Distribution of the number of TFs targeting a gene , based on the DAP - seq available dataset , for LVG ( green ) and HVG ( blue ) . The distribution of average ( black ) and 95 % confidence interval ( dotted grey ) for the thousand random sets is also represented .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Distribution of the number of TFs targeting a gene, based on the DAP-seq available dataset, for LVG (green) and HVG (blue). The distribution of average (black) and 95% confidence interval (dotted grey) for the thousand random sets is also represented.


A . Proportion of genes marked with several chromatin marks among all genes passing size and expression level thresholds ( black ) , HVGs ( blue ) , LVGs ( green ) and the average for the thousand random sets ( grey ) . Error bars indicate the 95 % confidence interval for the thousand random sets . Blue and green stars indicate statistical differences in the number of marked genes compare to all genes , for variable and stable genes respectively ( * Indicates a p - value < 0 . 05 , * * * Indicates a p - value < 0 . 001 , Chi - square test ) .

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

A. Proportion of genes marked with several chromatin marks among all genes passing size and expression level thresholds (black), HVGs (blue), LVGs (green) and the average for the thousand random sets (grey). Error bars indicate the 95% confidence interval for the thousand random sets. Blue and green stars indicate statistical differences in the number of marked genes compare to all genes, for variable and stable genes respectively (* Indicates a p-value < 0.05, *** Indicates a p-value < 0.001, Chi-square test).


B . Average profile for H3K27me3 , H2A . Z , H3K4me3 and H3K23ac at HVGs ( blue ) , LVGs ( green ) and random genes ( grey ) . C . Heatmap of the enrichment for H3K27me3 , H2A . Z , H3K4me3 and H3K23ac for HVGs ( top ) , random genes ( middle ) and LVG ( bottom ) . Red means a high level and blue means a low level for the chromatin marks .

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

B. Average profile for H3K27me3, H2A.Z, H3K4me3 and H3K23ac at HVGs (blue), LVGs (green) and random genes (grey). C. Heatmap of the enrichment for H3K27me3, H2A.Z, H3K4me3 and H3K23ac for HVGs (top), random genes (middle) and LVG (bottom). Red means a high level and blue means a low level for the chromatin marks.


B . Screening results of compounds competing Dvl - CXXC5 interaction . Each 2 μM of the compounds was used for competing as described in Fig 1A . [ 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

B . Screening results of compounds competing Dvl-CXXC5 interaction. Each 2 μM of the compounds was used for competing as described in Fig 1A. [n=3]


C , D . Primary calvaria cells were treated with 2 μM of each compound for 2 days . The cells were subjected to immunofluorescence analyses to visualize β - catenin ( C ; green ) . The relative numbers of β - catenin - positive nuclei were counted ( D ) . [ 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

C, D. Primary calvaria cells were treated with 2 μM of each compound for 2 days. The cells were subjected to immunofluorescence analyses to visualize β-catenin (C; green). The relative numbers of β-catenin-positive nuclei were counted (D). [n=3]


E , F . The calvariae from 4 - day - old mice were cultured ex vivo for 7 days with 2 µM of each compound . Representative calvaria sections were visualized by H & amp ; amp ; E staining ( E ) . The calvaria thicknesses were measured from the images using Image Pro software ( F ) . [ 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

E, F . The calvariae from 4-day-old mice were cultured ex vivo for 7 days with 2 µM of each compound. Representative calvaria sections were visualized by H&amp;E staining (E). The calvaria thicknesses were measured from the images using Image Pro software (F). [n=3]


B . A competition curve for the Dvl - CXXC5 interaction by KY - 02061 .

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

B . A competition curve for the Dvl-CXXC5 interaction by KY-02061.


C - E . NMR titration analyses for Dvl PZD domain with KY - 02061 . 1H - 15N - HSQC analyses were performed to analyze the interaction of 15N - labelled Dvl - PDZ domain with KY - 02061 . The 1H - 15N - HSQC spectrum of different molar ratios ( Dvl PDZ domain : KY - 02061 ) are displayed as red ( 1 : 0 ) , orange ( 1 : 10 ) , purple ( 1 : 20 ) , cyan ( 1 : 40 ) , green ( 1 : 60 ) and blue ( 1 : 80 ) ( C , residues with meaningful chemical shift change are indicated by arrows ) . Plot of chemical shift changes ( Δδ ) as a function of residue number in molecular ratio 1 : 80 ( D , a red - colored line indicates the line for Δδ = 0 . 05 ) . The residues with Δδ greater than 0 . 05 are visualized as a stick model on the ribbon representation of the Dvl PDZ domain structure ( E ) .

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

C-E. NMR titration analyses for Dvl PZD domain with KY-02061. 1H-15N-HSQC analyses were performed to analyze the interaction of 15N-labelled Dvl-PDZ domain with KY-02061. The 1H-15N-HSQC spectrum of different molar ratios (Dvl PDZ domain:KY-02061) are displayed as red (1:0), orange (1:10), purple (1:20), cyan (1:40), green (1:60) and blue (1:80) (C, residues with meaningful chemical shift change are indicated by arrows). Plot of chemical shift changes (Δδ) as a function of residue number in molecular ratio 1:80 (D, a red-colored line indicates the line for Δδ=0.05). The residues with Δδ greater than 0.05 are visualized as a stick model on the ribbon representation of the Dvl PDZ domain structure (E).


F . Molecular docking of Dvl binding motif ( DBM ) or KY - 02061 to Dvl PDZ domain were analyzed by in silico experiments . The superimposed structure of DBM ( green ) and KY - 02061 ( yellow ) on the surface of Dvl PDZ domain ( gray ) was visualized .

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 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

F. Molecular docking of Dvl binding motif (DBM) or KY-02061 to Dvl PDZ domain were analyzed by in silico experiments. The superimposed structure of DBM (green) and KY-02061 (yellow) on the surface of Dvl PDZ domain (gray) was visualized.


A . MC3T3E1 cells were transfected with pTOPFLASH together with pCMV - β - gal . After 24 h , the cells were treated with DMSO , KY - 02061 in DMSO , or 5 µM PolyR - DBM for 2 days . The luciferase activities of whole cell lysates were measured and normalized with β - galactosidase activities . [ 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 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

A . MC3T3E1 cells were transfected with pTOPFLASH together with pCMV-β-gal. After 24 h, the cells were treated with DMSO, KY-02061 in DMSO, or 5 µM PolyR-DBM for 2 days. The luciferase activities of whole cell lysates were measured and normalized with β-galactosidase activities. [n=3]


B . Primary calvaria cells were isolated from the calvariae of 4 - day - old mice ( Manton et al , 2007 ) , and treated with DMSO or KY - 02061 in DMSO for 4 days . ALP activity levels were visualized by ALP staining .

O O O O O O 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 . Primary calvaria cells were isolated from the calvariae of 4-day-old mice (Manton et al, 2007), and treated with DMSO or KY-02061 in DMSO for 4 days. ALP activity levels were visualized by ALP staining.


C . Primary calvaria cells were treated with DMSO or 5 µM KY - 02061 in DMSO for 14 days . The cells were subjected to immunofluorescence analyses to visualize Runx2 ( green ) and β - catenin ( red ) . The cell nuclei were counterstained with DAPI ( blue ) .

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

C. Primary calvaria cells were treated with DMSO or 5 µM KY-02061 in DMSO for 14 days. The cells were subjected to immunofluorescence analyses to visualize Runx2 (green) and β-catenin (red). The cell nuclei were counterstained with DAPI (blue).


D , E . Calvariae from 4 - day - old mice were cultured ex vivo for 7 days with KY - 02061 in DMSO ( D ) . The calvaria thicknesses were measured from the stained sections using Image Pro software ( E ) . [ n = 3 ]

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

D, E. Calvariae from 4-day-old mice were cultured ex vivo for 7 days with KY-02061 in DMSO (D). The calvaria thicknesses were measured from the stained sections using Image Pro software (E). [n=3]


B . A competition curve for the Dvl - CXXC5 interaction by KY - 02327 .

B. A competition curve for the Dvl-CXXC5 interaction by KY-02327.


C . Fluorescence quenching plot of Dvl PDZ domain upon addition of different amounts of KY - 02327 . From top to bottom final moral ratio between Dvl PDZ and KY - 02327 was , 1 : 0 , 1 : 1 , 1 : 2 , 1 : 3 , 1 : 4 , 1 : 5 , 1 : 6 , 1 : 7 , 1 : 8 , 1 : 9 , 1 : 10 , 1 : 15 , 1 : 20 and 1 : 25 , respectively .

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 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

C. Fluorescence quenching plot of Dvl PDZ domain upon addition of different amounts of KY-02327. From top to bottom final moral ratio between Dvl PDZ and KY-02327 was, 1:0, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20 and 1:25, respectively.


D - F . 1H - 15N - HSQC analyses were performed to analyze the interaction of 15N - labelled Dvl - PDZ domain with KY - 02327 . The 1H - 15N - HSQC spectrum of different molar ratios ( Dvl PDZ domain : KY - 02327 ) are displayed as red ( 1 : 0 ) , yellow ( 1 : 5 ) , green ( 1 : 10 ) , and magenta ( 1 : 20 ) ( D , residues with meaningful chemical shift change are indicated by arrows ) . Plot of chemical shift changes ( Δδ ) as a function of residue number in molecular ratio 1 : 20 ( E , a red - colored line indicates the line for Δδ = 0 . 05 ) . The residues with Δδ greater than 0 . 05 are visualized as a stick model on the ribbon representation of the Dvl PDZ domain structure ( F ) .

O 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F. 1H-15N-HSQC analyses were performed to analyze the interaction of 15N-labelled Dvl-PDZ domain with KY-02327. The 1H-15N-HSQC spectrum of different molar ratios (Dvl PDZ domain:KY-02327) are displayed as red (1:0), yellow (1:5), green (1:10), and magenta (1:20) (D, residues with meaningful chemical shift change are indicated by arrows). Plot of chemical shift changes (Δδ) as a function of residue number in molecular ratio 1:20 (E, a red-colored line indicates the line for Δδ=0.05). The residues with Δδ greater than 0.05 are visualized as a stick model on the ribbon representation of the Dvl PDZ domain structure (F).


G . Molecular docking of KY - 02061 or KY - 02327 to Dvl PDZ domain were analyzed by in silico experiments . The superimposed structure of KY - 02061 ( yellow ) and KY - 02327 ( cyan ) on the surface of Dvl PDZ domain ( gray ) was visualized .

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

G. Molecular docking of KY-02061 or KY-02327 to Dvl PDZ domain were analyzed by in silico experiments. The superimposed structure of KY-02061 (yellow) and KY-02327 (cyan) on the surface of Dvl PDZ domain (gray) was visualized.


A . MC3T3E1 cells were transfected with pTOPFLASH together with pCMV - β - gal . After 24 h , the cells were treated with indicated dose of KY - 02327 for 2 days . The luciferase activities of whole cell lysates were measured and normalized with β - galactosidase activities . [ n = 3 ]

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 . MC3T3E1 cells were transfected with pTOPFLASH together with pCMV-β-gal. After 24 h, the cells were treated with indicated dose of KY-02327 for 2 days. The luciferase activities of whole cell lysates were measured and normalized with β-galactosidase activities. [n=3]


B - C . MC3T3E1 cells were treated with indicated concentrations of KY - 02327 for 2 days . β - Catenin and α - tubulin were detected by immunoblot .

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

B-C . MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 2 days. β-Catenin and α-tubulin were detected by immunoblot.


B - C . MC3T3E1 cells were treated with indicated concentrations of KY - 02327 for 2 days . β - Catenin and α - tubulin were detected by immunoblot ( B ) or immunofluorescence ( C ) analyses . Nuclei were counterstained with DAPI ( C , blue ) .

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

B-C . MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 2 days. β-Catenin and α-tubulin were detected by immunoblot (B) or immunofluorescence (C) analyses. Nuclei were counterstained with DAPI (C, blue).


D . MC3T3E1 cells were treated with indicated concentrations of KY - 02327 for 14 days . The mRNA level of collagen 1a ( Col1a ) was measured by quantitative real - time qRT - PCR . [ n = 3 ]

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

D. MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 14 days. The mRNA level of collagen 1a (Col1a) was measured by quantitative real-time qRT-PCR. [n=3]


E . MC3T3E1 cells were treated with indicated concentrations of KY - 02327 for 21 days . The mRNA level of osteocalcin ( OCN ) was measured by qRT - PCR . [ n = 3 ]

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

E. MC3T3E1 cells were treated with indicated concentrations of KY-02327 for 21 days. The mRNA level of osteocalcin (OCN) was measured by qRT-PCR. [n=3]


A - F . Vehicle or 20 mg of KY - 02327 per kg of animal body weight ( mpk ) was orally administered or 0 . 06 mpk of the N - terminal fragment of PTH ( amino acids 1 - 34 ) was subcutaneously injected into the Sham - operated ( Sham ) or ovariectomized ( OVX ) mice on 5 sequential days per week for 4 weeks [ n = 4 ] . After calcein injection , the prepared femoral sections of the OVX mice were examined under fluorescence microscope to visualize integrated calcein ( A , green , arrows ) . Nuclei were counterstained with DAPI ( A , blue ) . Osteoblast numbers ( B ) and calcein double labelled surfaces ( C ) were measured in the femoral sections .

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

A-F. Vehicle or 20 mg of KY-02327 per kg of animal body weight (mpk) was orally administered or 0.06 mpk of the N-terminal fragment of PTH (amino acids 1-34) was subcutaneously injected into the Sham-operated (Sham) or ovariectomized (OVX) mice on 5 sequential days per week for 4 weeks [n=4]. After calcein injection, the prepared femoral sections of the OVX mice were examined under fluorescence microscope to visualize integrated calcein (A, green, arrows). Nuclei were counterstained with DAPI (A, blue). Osteoblast numbers (B) and calcein double labelled surfaces (C) were measured in the femoral sections.


A - F . Vehicle or 20 mg of KY - 02327 per kg of animal body weight ( mpk ) was orally administered or 0 . 06 mpk of the N - terminal fragment of PTH ( amino acids 1 - 34 ) was subcutaneously injected into the Sham - operated ( Sham ) or ovariectomized ( OVX ) mice on 5 sequential days per week for 4 weeks [ n = 4 ] . The mouse femurs were analyzed using micro - CT and BMD ( D ) and BV / TV ( E ) were calculated from the micro - CT data .

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

A-F. Vehicle or 20 mg of KY-02327 per kg of animal body weight (mpk) was orally administered or 0.06 mpk of the N-terminal fragment of PTH (amino acids 1-34) was subcutaneously injected into the Sham-operated (Sham) or ovariectomized (OVX) mice on 5 sequential days per week for 4 weeks [n=4]. The mouse femurs were analyzed using micro-CT and BMD (D) and BV/TV (E) were calculated from the micro-CT data.


A - F . Vehicle or 20 mg of KY - 02327 per kg of animal body weight ( mpk ) was orally administered or 0 . 06 mpk of the N - terminal fragment of PTH ( amino acids 1 - 34 ) was subcutaneously injected into the Sham - operated ( Sham ) or ovariectomized ( OVX ) mice on 5 sequential days per week for 4 weeks [ n = 4 ] . The three - dimensional images of femoral trabecular bone were reconstructed ( F ) , and trabecular number ( G ) and trabecular separation ( H ) were calculated .

A-F. Vehicle or 20 mg of KY-02327 per kg of animal body weight (mpk) was orally administered or 0.06 mpk of the N-terminal fragment of PTH (amino acids 1-34) was subcutaneously injected into the Sham-operated (Sham) or ovariectomized (OVX) mice on 5 sequential days per week for 4 weeks [n=4]. The three-dimensional images of femoral trabecular bone were reconstructed (F), and trabecular number (G) and trabecular separation (H) were calculated.


HEK293 cells were treated for 3 hours with 150 nM of LY411575 , 1 µM of MLN4924 , 10 µM of Roscovitine , 10 µM DRB or 10 µM XAV939 . DMSO served as vehicle control . Transfection with pCS2 - NICD vector served as positive control . Western blot analysis reveals that NICD levels were increased upon treatment with Roscovitine , DRB , XAV939 , or MLN4924 . NICD is undetectable following LY411575 treatment . NICD antibody detects a doublet following inhibitor treatment and the top band disappears following λ phosphatase treatment indicating that this top band is a phosphorylated isoform . β - Actin served as loading control . Quantification of the density of western blot bands in ( A ) using ImageJ software . Data are expressed as fold changes compared to DMSO treatment . All data represent the mean ± SEM from three independent experiments . One - way ANOVA analysis , followed by Dunnett test , was performed with * p ≤ 0 . 05 , * * p ≤ 0 . 01 and ns = not significant .

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

HEK293 cells were treated for 3 hours with 150 nM of LY411575, 1 µM of MLN4924, 10 µM of Roscovitine, 10 µM DRB or 10 µM XAV939. DMSO served as vehicle control. Transfection with pCS2-NICD vector served as positive control. Western blot analysis reveals that NICD levels were increased upon treatment with Roscovitine, DRB, XAV939, or MLN4924. NICD is undetectable following LY411575 treatment. NICD antibody detects a doublet following inhibitor treatment and the top band disappears following λ phosphatase treatment indicating that this top band is a phosphorylated isoform. β-Actin served as loading control.   Quantification of the density of western blot bands in (A) using ImageJ software. Data are expressed as fold changes compared to DMSO treatment. All data represent the mean ± SEM from three independent experiments. One-way ANOVA analysis, followed by Dunnett test, was performed with *p≤0.05, **p≤0.01 and ns=not significant.


iPS cells were treated for 3 hours with 150 nM of LY411575 , 1 µM of MLN4924 , 10 µM of Roscovitine , 10 µM DRB or 10 µM XAV939 . DMSO has been used as vehicle control . Western blot analysis reveals that NICD levels were increased upon treatment with Roscovitine , DRB , XAV939 , or MLN4924 . NICD is undetectable following LY411575 treatment . NICD antibody detects a doublet following Inhibitor treatment and the top band disappears following λ phosphatase treatment indicative that this top band is a phosphorylated isoform . β - Actin served as loading control . Quantification of the density of western blot bands in ( C ) using ImageJ software . Data are expressed as fold changes compared to DMSO treatment . All data represent the mean ± SEM from three independent experiments . One - way ANOVA analysis , followed by Dunnett test , was performed with * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 and ns = not significant .

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

iPS cells were treated for 3 hours with 150 nM of LY411575, 1 µM of MLN4924, 10 µM of Roscovitine, 10 µM DRB or 10 µM XAV939. DMSO has been used as vehicle control. Western blot analysis reveals that NICD levels were increased upon treatment with Roscovitine, DRB, XAV939, or MLN4924. NICD is undetectable following LY411575 treatment. NICD antibody detects a doublet following Inhibitor treatment and the top band disappears following λ phosphatase treatment indicative that this top band is a phosphorylated isoform. β-Actin served as loading control.   Quantification of the density of western blot bands in (C) using ImageJ software. Data are expressed as fold changes compared to DMSO treatment. All data represent the mean ± SEM from three independent experiments. One-way ANOVA analysis, followed by Dunnett test, was performed with *p≤0.05, **p≤0.01, ***p≤0.001 and ns= not significant.


HEK293 cells were treated with the inhibitors NICD phosphorylation status was analysed by a Phos - tag assay . DMSO served as vehicle control . NICD phosphorylation profile varies following Roscovitine , DRB , XAV939 or MLN4924 treatment . Following λ phosphatase treatment none of the high molecular weight bands are visible indicating they are all phosphorylated isoforms .

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HEK293 cells were treated with the inhibitors NICD phosphorylation status was analysed by a Phos-tag assay. DMSO served as vehicle control. NICD phosphorylation profile varies following Roscovitine, DRB, XAV939 or MLN4924 treatment. Following λ phosphatase treatment none of the high molecular weight bands are visible indicating they are all phosphorylated isoforms.


NICD interaction with FBXW7 at endogenous levels in HEK293 cells . 500 µg of HEK293 cell lysates treated with DMSO or MLN4924 were subjected to immunoprecipitation using FBXW7 antibody , or IgG antibody as negative control , and precipitated material was analysed by western blot using NICD antibody . Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency . 10 % of cell lysate before immunoprecipitation was used as input control and β - Actin served as loading control . Quantification of the density of western blot bands in ( A ) performed by ImageJ software . Data are expressed as percentage changes compared to MLN4924 . All data represent the mean ± SEM from three independent experiments . Student t - test was used to determine p values , with * * p ≤ 0 . 01 .

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NICD interaction with FBXW7 at endogenous levels in HEK293 cells. 500 µg of HEK293 cell lysates treated with DMSO or MLN4924 were subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β-Actin served as loading control.   Quantification of the density of western blot bands in (A) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924. All data represent the mean ± SEM from three independent experiments. Student t-test was used to determine p values, with **p≤0.01.


Roscovitine treatment reduced the NICD - FBXW7 interaction . 500 µg of HEK293 cell lysates treated with MLN4924 or MLN4924 together with Roscovitine were subjected to immunoprecipitation using FBXW7 antibody , or IgG antibody as negative control , and precipitated material was analysed by western blot using NICD antibody . Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency . 10 % of cell lysate before immunoprecipitation was used as input control and β - Actin served as loading control . Quantification of the density of western blot bands in ( C ) performed by ImageJ software . Data are expressed as percentage changes compared to MLN4924 treated samples . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * * * p ≤ 0 . 001 .

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Roscovitine treatment reduced the NICD-FBXW7 interaction. 500 µg of HEK293 cell lysates treated with MLN4924 or MLN4924 together with Roscovitine were subjected to immunoprecipitation using FBXW7 antibody, or IgG antibody as negative control, and precipitated material was analysed by western blot using NICD antibody. Western blot with FBXW7 antibody served as loading control for immunoprecipitation efficiency. 10% of cell lysate before immunoprecipitation was used as input control and β-Actin served as loading control.   Quantification of the density of western blot bands in (C) performed by ImageJ software. Data are expressed as percentage changes compared to MLN4924 treated samples. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with ***p≤0.001.


LC - MS - MS analysis of in - gel - digested HEK293 cells transfected with hNICD - GFP , and subjected to immunoprecipitation using GFP antibody , identified multiple phosphorylation sites in NICD , highlighted in green .

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LC-MS-MS analysis of in-gel-digested HEK293 cells transfected with hNICD-GFP, and subjected to immunoprecipitation using GFP antibody, identified multiple phosphorylation sites in NICD, highlighted in green.


Phosphorylation of serine 2513 , but not serine 2516 , appears to be essential for the NICD - FBXW7 interaction . hNICD - GFP phospho - mutant peptides encoding non - phosphorylatable residues at S2513 and / or 2516 ( serine to alanine ) were expressed in HEK293 cells . The exogenously expressed protein was subsequently immunoprecipitated with anti - GFP antibody and precipitated material was analysed by western blot using FBXW7 antibody . Wild - type hNICD - GFP and GFP only vectors were included as positive and negative controls , respectively . Western blot using GFP antibody served as immunoprecipitation efficiency control . β - Actin has been used as loading control for the input lanes .

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Phosphorylation of serine 2513, but not serine 2516, appears to be essential for the NICD-FBXW7 interaction. hNICD-GFP phospho-mutant peptides encoding non-phosphorylatable residues at S2513 and/or 2516 (serine to alanine) were expressed in HEK293 cells. The exogenously expressed protein was subsequently immunoprecipitated with anti-GFP antibody and precipitated material was analysed by western blot using FBXW7 antibody. Wild-type hNICD-GFP and GFP only vectors were included as positive and negative controls, respectively. Western blot using GFP antibody served as immunoprecipitation efficiency control. β-Actin has been used as loading control for the input lanes.


HEK293 cells were cultured for 48h after transfection with plasmids encoding scrambled siRNA ( - ) or siRNA specific for CDK2 ( + ) followed by western blot for NICD , CDK2 , CDK1 , CDK4 and CDK8 . β - Actin served as loading control . Quantification of the density of western blot bands in ( A ) performed by ImageJ software . Data are expressed as fold changes compared to control ( scrambled siRNA transfected ) cell lysate . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * * p ≤ 0 . 01 .

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HEK293 cells were cultured for 48h after transfection with plasmids encoding scrambled siRNA (-) or siRNA specific for CDK2 (+) followed by western blot for NICD, CDK2, CDK1, CDK4 and CDK8. β-Actin served as loading control.   Quantification of the density of western blot bands in (A) performed by ImageJ software. Data are expressed as fold changes compared to control (scrambled siRNA transfected) cell lysate. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with **p≤0.01.


HEK293 cells were cultured for 48h after transfection with plasmids encoding scrambled siRNA ( - ) or siRNA specific for CDK1 ( + ) followed by western blot for NICD , CDK1 , CDK2 , CDK4 and CDK8 . β - Actin has been used as loading control . Quantification of the density of western blot bands in ( C ) performed by ImageJ software . Data are expressed as fold changes compared to control ( scrambled siRNA transfected ) cell lysate . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * p ≤ 0 . 05 .

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HEK293 cells were cultured for 48h after transfection with plasmids encoding scrambled siRNA (-) or siRNA specific for CDK1 (+) followed by western blot for NICD, CDK1, CDK2, CDK4 and CDK8. β-Actin has been used as loading control.   Quantification of the density of western blot bands in (C) performed by ImageJ software. Data are expressed as fold changes compared to control (scrambled siRNA transfected) cell lysate. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with *p≤0.05.


Analysis of the inhibitory activity of three highly selective CDK2 inhibitors against a panel of kinases - a selection of those tested is shown here . At both 1 and 10µM , Roscovitine is able to inhibit more than 95 % of CDK2 activity , but is far less effective against other kinases . Purvalanol B ( 0 . 1 and 1µM ) inhibits more than 94 % of CDK2 activity . At both 1 and 10 µM , GSK650394A is able to inhibit more than 98 % of CDK2 activity . Source : The Kinase Profiling Inhibitor

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

Analysis of the inhibitory activity of three highly selective CDK2 inhibitors against a panel of kinases - a selection of those tested is shown here. At both 1 and 10µM, Roscovitine is able to inhibit more than 95% of CDK2 activity, but is far less effective against other kinases. Purvalanol B (0.1 and 1µM) inhibits more than 94% of CDK2 activity. At both 1 and 10 µM, GSK650394A is able to inhibit more than 98% of CDK2 activity. Source: The Kinase Profiling Inhibitor Database


HEK293 cells were treated with three highly selective CDK2 inhibitors ( 10 µM of Roscovitine , 0 . 1µM of Purvalanol B and 10µM of GSK650394A ) for 3 hours . Endogenous levels of NICD were detected by western blot . β - Actin served as loading control . Quantification of the density of western blot bands in ( F ) using ImageJ software . Data are expressed as fold changes compared to DMSO . All data represent the mean ± SEM from three independent experiments . One - way ANOVA analysis , followed by Dunnett test , was performed , with * p ≤ 0 . 05 , * * p ≤ 0 . 01 , and ns = not significant .

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

HEK293 cells were treated with three highly selective CDK2 inhibitors (10 µM of Roscovitine, 0.1µM of Purvalanol B and 10µM of GSK650394A) for 3 hours. Endogenous levels of NICD were detected by western blot. β-Actin served as loading control.   Quantification of the density of western blot bands in (F) using ImageJ software. Data are expressed as fold changes compared to DMSO. All data represent the mean ± SEM from three independent experiments. One-way ANOVA analysis, followed by Dunnett test, was performed, with *p≤0.05, **p≤0.01, and ns= not significant.


HEK293 cells were treated with RO - 3306 , a specific CDK1 inhibitor , for 3 hours at 10 µM . Levels of NICD , FBXW7 , phospho - Histone H3 and Histone H3 were detected by western blot . β - Actin was used as loading control . Quantification of the density of western blot bands in ( H ) using ImageJ software . Data are expressed as fold changes compared to DMSO . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * p ≤ 0 . 05 .

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HEK293 cells were treated with RO-3306, a specific CDK1 inhibitor, for 3 hours at 10 µM. Levels of NICD, FBXW7, phospho-Histone H3 and Histone H3 were detected by western blot. β-Actin was used as loading control.   Quantification of the density of western blot bands in (H) using ImageJ software. Data are expressed as fold changes compared to DMSO. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with *p≤0.05.


Cell cycle profile for HEK293 cells released from synchronization after double thymidine block . Cells were released and harvested at the indicated time points ( AS = asynchronous ) . Analysis of cell cycle arrest and release was performed using propidium iodide ( PI ) staining and flow cytometry . A representative experiment of three performed is shown .

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Cell cycle profile for HEK293 cells released from synchronization after double thymidine block. Cells were released and harvested at the indicated time points (AS= asynchronous). Analysis of cell cycle arrest and release was performed using propidium iodide (PI) staining and flow cytometry. A representative experiment of three performed is shown.


Expression of the indicated proteins in HEK293 cells was examined by western blotting , and β - Actin was used as loading control . This summary is a representation of three independent experiments .

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Expression of the indicated proteins in HEK293 cells was examined by western blotting, and β-Actin was used as loading control. This summary is a representation of three independent experiments.


Graph of flow cytometry data shows the percentage of cells in given cell ‐ cycle phases 48h after transfection with plasmids encoding scrambled siRNA or siRNA specific for CDK2 . Graph represents the mean of three independent experiments . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * * p ≤ 0 . 01 ( ns = not significant ) .

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Graph of flow cytometry data shows the percentage of cells in given cell‐cycle phases 48h after transfection with plasmids encoding scrambled siRNA or siRNA specific for CDK2. Graph represents the mean of three independent experiments. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with **p≤0.01 (ns=not significant).


Graph of flow cytometry data shows the percentage of cells in given cell ‐ cycle phases 48h after transfection with plasmids encoding scrambled siRNA or siRNA specific for CDK1 . Graph represents the mean of three independent experiments . All data represent the mean ± SEM from three independent experiments . Student ' s t - test analysis was performed , with * p ≤ 0 . 05 and * * p ≤ 0 . 01 .

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Graph of flow cytometry data shows the percentage of cells in given cell‐cycle phases 48h after transfection with plasmids encoding scrambled siRNA or siRNA specific for CDK1. Graph represents the mean of three independent experiments. All data represent the mean ± SEM from three independent experiments. Student's t-test analysis was performed, with *p≤0.05 and **p≤0.01.


iPS cells in G2 express high levels of Cyclin A and do not incorporate EdU ( marked in red within the NICD panel ) . EdU incorporation occurs during S phase ( marked by yellow in the NICD panel ) . Cells with low Cyclin A intensity and no EdU incorporation are either in G1 or M phase . M phase cells are distinguishable by their chromatin condensation , observable in the DAPI panel ( dividing cells are indicated with white in the NICD panel ) . G1 cells do not have any of the features described above ( blue in NICD panel ) . Scale bars are 20 µm .

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iPS cells in G2 express high levels of Cyclin A and do not incorporate EdU (marked in red within the NICD panel). EdU incorporation occurs during S phase (marked by yellow in the NICD panel). Cells with low Cyclin A intensity and no EdU incorporation are either in G1 or M phase. M phase cells are distinguishable by their chromatin condensation, observable in the DAPI panel (dividing cells are indicated with white in the NICD panel). G1 cells do not have any of the features described above (blue in NICD panel). Scale bars are 20 µm.