U2OS ATG2A / B double knock - out ( DKO ) CRISPR / Cas9 stably expressing Tandem tagged LC3B ( mCherry - GFP - LC3B ) were retrovirally transduced to express vector , or HA - tagged ATG2A - WT , - mLIR ( FCIL / AAAA ) or - mYFS ( YFS / AAA ) . were grown in complete medium ( CM ) or starved for 2 h ( EBSS ) or treated with CM plus BafilomycinA1 ( 200nM , 4 h ) , fixed analysed by confocal microscopy . Merged of GFP ( green ) mCherry ( red ) channels show presence of autophagosomes / phagophores ( GFP mCherry positive , yellow ) or autolysosomes ( mCherry only , red ) Scale bar 10µm . are representative of n = 3 independent experiments .

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

U2OS ATG2A/B double knock-out (DKO) CRISPR/Cas9 cells stably expressing Tandem tagged LC3B (mCherry-GFP-LC3B) were retrovirally transduced to express vector, or HA-tagged ATG2A-WT, -mLIR (FCIL/AAAA) or -mYFS (YFS/AAA). Cells were grown in complete medium (CM) or starved for 2 h (EBSS) or treated with CM plus BafilomycinA1 (200nM, 4 h), fixed and analysed by confocal microscopy. Merged images of GFP (green) and mCherry (red) channels show the presence of autophagosomes/phagophores (GFP and mCherry positive, yellow puncta) or autolysosomes (mCherry only, red puncta) Scale bar 10µm. Images are representative of n=3 independent experiments.


using flow cytometry of measuring GFP mCherry . were gated on GFP mCherry % mCherry positive gated used as an indication of autolysosome formation due to GFP quenching . Each symbol represents 1 independent experiment with 10 , 000 analysed per condition . A total of n = 3 independent experiments were performed , horizontal bar indicates mean ± SD .

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

Quantification using flow cytometry of measuring GFP and mCherry fluorescence. Cells were gated based on GFP and mCherry fluorescence and % mCherry positive cells gated used as an indication of autolysosome formation due to GFP quenching. Each symbol represents 1 independent experiment with 10,000 cells analysed per condition. A total of n=3 independent experiments were performed, and horizontal bar indicates mean ± SD.


U2OS ATG2A / B DKO reconstituted with vector only , HA - ATG2A - WT , - mLIR - mYFS were stimulated with complete medium , ( CM ) , 2 h starvation ( EBSS ) or 4 h BafilomycinA1 ( BafA1 , 200nM ) , lysed in total lysis buffer subjected to western blot . Blots were probed for presence of HA - tag ( ATG2A ) , p62 / SQSTM1 , LC3B , pan - GABARAP ( GABARAP , GABARAP - L1 GABARAP - L2 ) vinculin ( loading control ) .

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

U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS were stimulated with complete medium, (CM), 2 h starvation (EBSS) or 4 h BafilomycinA1 (BafA1, 200nM), lysed in total cell lysis buffer and subjected to western blot analysis. Blots were probed for the presence of HA-tag (ATG2A), p62/SQSTM1, LC3B, pan-GABARAP (GABARAP, GABARAP-L1 and GABARAP-L2) and vinculin (loading control).


D , E p62 / SQSTM1 ( D ) LC3B - II ( E ) were normalized to loading control quantified as fold change of DKO . Each symbol represents an independent experiment . of at least n = 3 independent experiments is shown . Horizontal bar represents mean ± SD .

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D, E p62/SQSTM1 (D) and LC3B-II (E) levels were normalized to loading control and quantified as fold change of DKO proteins levels. Each symbol represents an independent experiment. Quantification of at least n=3 independent experiments is shown. Horizontal bar represents mean ± SD.


U2OS ATG2A / B DKO reconstituted with vector only , HA - ATG2A - WT , - mLIR - mYFS were stimulated with complete medium , ( CM ) , 4 h starvation ( EBSS ) plus BafilomycinA1 ( BafA1 , 200nM ) treatment . were centrifuged resuspended in PBS digitonin , spun , washed fractions incubated with proteinase K with without 0 . 1 % TritonX - 100 . Sampels were then subjected to western blotting using anti - p62 / SQSTM1 anti - HA ( ATG2A ) antibodies . p62 / SQSTM1 remaining was calculated using densitometry . Blots are representative of n = 3 independent experiments .

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

U2OS ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS were stimulated with complete medium, (CM), 4 h starvation (EBSS) plus BafilomycinA1 (BafA1, 200nM) treatment. Cells were centrifuged and resuspended in PBS digitonin, spun, washed and the membrane fractions incubated with proteinase K with and without 0.1% TritonX-100. Sampels were then subjected to western blotting using anti-p62/SQSTM1 and anti-HA (ATG2A) antibodies. Percentage p62/SQSTM1 remaining was calculated using densitometry analysis. Blots are representative of n=3 independent experiments.


U2OS ATG2A / B DKO reconstituted with vector only , HA - ATG2A - WT , - mLIR - mYFS stably expressing GFP - Syntaxin17 ( STX17 ) were stimulated starvation ( EBSS ) plus BafilomycinA1 ( BafA1 , 200nM ) for 4 hours to stimulate autophagosome generation prevent their degradation in lysosome . were fixed immune - stained for LC3B ( magenta ) . DAPI was included ( blue ) to mark / nucleus . Confocal of LC3B GFP - STX17 ( green ) localization was performed . Closed arrows ( ATG2A / B DKO DKO + ATG2A mLIR ) highlight aggregate structures . Open arrows ( DKO + ATG2A - WT ATG2A - mYFS ) highlight STX17 / LC3B positive vesicles . Scale bar 10µm . of ( C ) expressed as a of with STX17 / LC3B positive vesicles . Each symbol represents a single field of with 5 - 10 per field . A total of 300 - 600 were analysed over n = 3 independent experiments . Data is shown as mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ATG2A/B DKO cells reconstituted with vector only, HA-ATG2A-WT, -mLIR and -mYFS and stably expressing GFP-Syntaxin17 (STX17) were stimulated starvation (EBSS) plus BafilomycinA1 (BafA1, 200nM) for 4 hours to stimulate autophagosome generation and prevent their degradation in the lysosome. Cells were fixed and immune-stained for LC3B (magenta). DAPI was included (blue) to mark the DNA/nucleus. Confocal analysis of LC3B and GFP-STX17 (green) localization was performed. Closed arrows (ATG2A/B DKO and DKO + ATG2A mLIR) highlight aggregate structures. Open arrows (DKO + ATG2A-WT and ATG2A-mYFS) highlight STX17/LC3B positive vesicles. Scale bar 10µm.   Quantification of (C) expressed as a percentage of cells with STX17/LC3B positive vesicles. Each symbol represents a single field of cells with 5-10 cells per field. A total of 300-600 cells were analysed over n=3 independent experiments. Data is shown as mean ± SD.


( A - C ) U2OS ATG2A / B double knock out ( DKO ) or DKO reconstituted with ATG2A - WT , - mLIR ( FCIL / AAAA ) or - mYFS ( YFS / AAA ) were starved for 2 h , fixed stained for LC3B ( green ) or WIPI2 ( magenta ) , ( B ) ATG9A ( magenta ) or ( C ) GABARAP ( Magenta ) imaged using a Zeiss 880 Airyscan super - resolution confocal microscope . are representatives of n = 3 independent experiments . Scale bars 10µm .

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(A-C) U2OS ATG2A/B double knock out (DKO) cells or DKO reconstituted with ATG2A-WT, -mLIR (FCIL/AAAA) or -mYFS (YFS/AAA) were starved for 2 h, fixed and stained for LC3B (green) or WIPI2 (magenta), (B) ATG9A (magenta) or (C) GABARAP (Magenta) and imaged using a Zeiss 880 Airyscan super-resolution confocal microscope. Images are representatives of n=3 independent experiments. Scale bars 10µm.


Transmission electron of ATG2A / B DKO ( upper left ) , DKO + ATG2A - WT ( upper right ) , DKO + ATG2A - mLIR ( lower left ) DKO + ATG2A - mYFS ( lower right ) starved ( EBSS ) for 2 h . Clustered small vesicles ( open arrowheads ; DKO DKO - mLIR ) autophagosomes ( closed arrow heads , DKO + ATG2A - WT - mYFS ) indicated . are representative of n = 3 independent experiments . Scale bars 500nm . ER = endoplasmic reticulum ; N = Nucleus ; Mt = Mitochondria ; G = Golgi ; AP = Autophagosome .

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Transmission electron micrographs of ATG2A/B DKO cells (upper left), DKO + ATG2A-WT (upper right), DKO+ ATG2A-mLIR (lower left) and DKO+ATG2A-mYFS( lower right) starved (EBSS) for 2 h. Clustered small vesicles (open arrowheads; DKO and DKO-mLIR) and autophagosomes (closed arrow heads, DKO+ATG2A-WT and -mYFS) indicated. Images are representative of n=3 independent experiments. Scale bars 500nm. ER= endoplasmic reticulum; N = Nucleus; Mt = Mitochondria; G = Golgi; AP = Autophagosome.


D Zygotic grk2i283 mutant at 4dpf compared to wild type ( N = 80 ) . Arrows indicate position of swim bladder . Scale bar ,

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D Zygotic grk2i283 mutant at 4dpf compared to wild type (N=80). Arrows indicate the position of swim bladder. Scale bar, 1mm


A Western blot of different forms of Gli2a in MZgrk2 compared to wild type , shh mRNA dnPKA mRNA injected wild type cyclopamine ( Cyc ( A ) ) treated wild type 20hpf . Gli2a - FL are low relative to Gli2aR in wild type , but are elevated in response to pathway activation ( shh dnPKA mRNA injected ) . Gli2aR are increased while Gli2a - FL is undetectable in CycA treated MZgrk2 mutant . Probing same blot with rabbit anti - GRK3 ( which recognizes zebrafish Grk2 ) reveals a complete loss of full Grk2 in MZgrk2 . Probing with anti - γ - tubulin was performed as a loading control . Three biological replicates of this were performed .

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 I-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 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

A Western blot analysis of the different forms of Gli2a in MZgrk2 embryos compared to wild type, shh mRNA and dnPKA mRNA injected wild type and cyclopamine (Cyc(A)) treated wild type 20hpf embryos. Gli2a-FL levels are low relative to Gli2aR levels in wild type embryos, but are elevated in response to pathway activation (shh and dnPKA mRNA injected). Gli2aR levels are increased while Gli2a-FL is undetectable in CycA treated and MZgrk2 mutant embryos. Probing the same blot with rabbit anti-GRK3 (which recognizes the zebrafish Grk2 protein) reveals a complete loss of full length Grk2 in MZgrk2 embryos. Probing with anti-γ-tubulin was performed as a loading control. Three biological replicates of this analysis were performed.


B Phenotype of wild type , MZgrk2 , grk2 - GFP mRNA injected MZgrk2 at 24hpf ( n = 20 for each sample ) . lines indicate shape of somites ( middle panels ) separation of eyes ( right panels ) . Scale bar ,

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B Phenotype of wild type, MZgrk2, and grk2-GFP mRNA injected MZgrk2 embryos at 24hpf (n=20 for each sample). The white lines indicate the shape of the somites (middle panels) and the separation of the eyes (right hand panels). Scale bar, 200μm


C In situ hybridisation of ptch2 , nkx2 . 2a olig2 transcripts in wild type , MZgrk2 grk2 - GFP injected MZgrk2 24hpf . Each panel shows a full view of on left a cross sectional view of a somite on right . ( n = 30 for each sample ) . Scale bars , 200μm ( whole mounts ) , 50μm ( sections ) .

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C In situ hybridisation of ptch2, nkx2.2a and olig2 transcripts in wild type, MZgrk2 and grk2-GFP injected MZgrk2 24hpf embryos. Each panel shows a full view of the embryo on the left and a cross sectional view of a somite on the right. (n=30 for each sample). Scale bars, 200μm (whole mounts), 50μm (sections).


D Expression of Prox1a Engrailed ( Eng ) in somites of wild - type , MZgrk2 grk2 - GFP mRNA injected MZgrk2 at 30hpf . Each panel shows Prox1a in green , Eng in red merge with DAPI in blue . ( n = 10 for each sample ) . Scale bar , 50μm .

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D Expression of Prox1a and Engrailed proteins (Eng) in somites of wild-type, MZgrk2 and grk2-GFP mRNA injected MZgrk2 embryos at 30hpf. Each panel shows Prox1a in green, Eng in red and the merge images with DAPI staining in blue. (n=10 for each sample). Scale bar, 50μm.


A Prox1a Eng expression in myotome of 30hpf wild type MZgrk2 injected with mRNAs encoding Shh , dnPKA or mSmoA1 - GFP . Each panel shows Prox1a in green , Eng in red , merged with DAPI in blue . co - labelling for Prox1a Eng ( orange ) is indicative of MP differentiation ( n = 30 for each sample ) Scale bar , 50μm .

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A Prox1a and Eng expression in the myotome of 30hpf wild type and MZgrk2 embryos injected with mRNAs encoding Shh, dnPKA or mSmoA1-GFP. Each panel shows Prox1a in green, Eng in red, and the merged images with DAPI staining in blue. The co-labelling for Prox1a and Eng (orange) is indicative of MP differentiation (n=30 for each sample) Scale bar, 50μm.


B Western blot showing of Gli2a - FL Gli2a - R forms in 20hpf wild type MZgrk2 injected with mRNA encoding Shh , dnPKA or mSmoA1 - GFP , respectively . γ - tubulin was used as loading control . Three biological replicates of this were performed .

O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot showing levels of Gli2a-FL and Gli2a-R forms in 20hpf wild type and MZgrk2 embryos injected with mRNA encoding Shh, dnPKA or mSmoA1-GFP, respectively. γ-tubulin was used as loading control. Three biological replicates of this analysis were performed.


A Western blot of GRK2 , GLI1 , PTCH1 , GLI3 in Flp - In - 3T3 with lentiCRISPR of 4 different guide RNAs in presence or absence of SAG . α - tubulin was used as loading control . Guide 2 was found to remove Grk2 efficiently used for subsequent experiments . GLI3 panel shows both full - ( GLI3FL ) repressor form of GLI3 ( GLI3R ) . ( 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 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

A Western blot analysis of GRK2, GLI1, PTCH1, and GLI3 protein levels in Flp-In-3T3 cells with lentiCRISPR of 4 different guide RNAs in presence or absence of SAG. α-tubulin was used as loading control. Guide RNA 2 was found to remove Grk2 efficiently and used for subsequent experiments. GLI3 panel shows both full-length (GLI3FL) and repressor form of GLI3 (GLI3R). (n=3)


B , C Hh reporter activity for wild type Grk2 - / - . were treated with PKA peptide inhibitor ( PKI ) mutant form of PKA peptide inhibitor ( PKI - M ) respectively , in presence or absence of SHH ( B ) were transfected with mSmoA1 - GFP expressing constructs in presence of SHH or SAG ( C ) . Data represent mean ± SD ( n = 3 ) . Unpaired Students t - was used for . * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 n . s . ( not significant ) .

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 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 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

B, C Hh reporter activity assay for wild type and Grk2-/- cells. Cells were treated with PKA peptide inhibitor (PKI) and mutant form of PKA peptide inhibitor (PKI-M) respectively, in presence or absence of SHH (B) Cells were transfected with mSmoA1-GFP expressing constructs in the presence of SHH or SAG (C). Data represent the mean and ± SD (n=3). Unpaired Students t-test was used for analysis. ***P<0.001; **P<0.01; *P<0.05 and n.s. (not significant).


A Expression of Prox1a ( green ) eng2a : GFP reporter ( red ) in otherwise wild - type 30hpf injected with mRNA encoding wild type mutant forms of mouse Smo . Ectopic MPs are indicated by fibres co - labelled with Prox1a GFP ; ectopic MFFs are labeled only with GFP . are representative of in following proportions of each sample : 28 / 28 ( mSmo ; mSmoA1 ; mSmoSD ; mSmoKRA ) ; 9 / 28 ( mSmoA1SA ) ; 12 / 28 ( mSmoA1SD ) . Scale bar , 50μm .

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A Expression of Prox1a (green) and the eng2a:GFP reporter (red) in otherwise wild-type 30hpf embryos injected with mRNA encoding wild type and mutant forms of mouse Smo. Ectopic MPs are indicated by fibres co-labelled with Prox1a and GFP; ectopic MFFs are labeled only with GFP. Images are representative of embryos in the following proportions of each sample: 28/28 (mSmo; mSmoA1; mSmoSD; mSmoKRA); 9/28 (mSmoA1SA); 12/28 (mSmoA1SD). Scale bar, 50μm.


B Average number of Prox1a + ve slow fibres in wild type injected with mRNA encoding different forms of mSmo . Prox1 positive were quantified in 4 somites in each of 4 for each sample . error bars indicate SD . Unpaired Student ' s t - was used to determine statistical significance between uninjected various Smo mutants ( black asterisk ) ; between mSmoA1 mSmoA1SA or mSmoA1SD ( red asterisk ) . * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 n . s . ( not significant ) .

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B Average number of Prox1a+ve slow fibres in wild type embryos injected with mRNA encoding different forms of mSmo. Prox1 positive cells were quantified in 4 somites in each of 4 embryos for each sample. The error bars indicate SD. Unpaired Student's t-test was used to determine the statistical significance between uninjected embryos and the various Smo mutants (black asterisk); and between mSmoA1 and mSmoA1SA or mSmoA1SD (red asterisk). ***P<0.001; **P<0.01; *P<0.05 and n.s. (not significant).


C Prox1a Eng expression in smohi1640 mutant injected with mRNA encoding mSmo ( n = 10 ) , mSmoSA ( n = 9 ) mSmo14SA ( n = 6 ) . Note full recovery of SSFs MPs compared to un - injected controls . Scale bar , 50μm .

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C Prox1a and Eng expression in smohi1640 mutant embryos injected with mRNA encoding mSmo (n=10), mSmoSA (n=9) and mSmo14SA (n=6). Note the full recovery of SSFs and MPs compared to the un-injected controls. Scale bar, 50μm.


D In situ hybridisation for transcripts of ptch2 , olig2 nkx2 . 2 in 24hpf smohi1640 mutant injected with mRNA encoding mSmo mSmoSA ( n = 6 for each sample ) . Scale bars , are 100μm ( lateral view ) , 50μm ( sections ) .

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D In situ hybridisation for transcripts of ptch2, olig2 and nkx2.2 in 24hpf smohi1640 mutant embryos injected with mRNA encoding mSmo and mSmoSA (n=6 for each sample). Scale bars, are 100μm (lateral view), 50μm (sections).


E Hh reporter of activity of wild type mutant forms of mSmo in Smo - / - MEFs in response to Shh or SAG stimulation . Note that mSmoA1 shows constitutive activity in absence of either Shh or SAG whereas mSmoKRA dose not ; mutants affecting phosphorylation failed to restore response to Shh or SAG . Data represent mean ± SD ( n = 3 ) . Unpaired Students t - was used for . * * * * P < 0 . 0001 ; * * P < 0 . 01 ; n . s . ( not significant ) .

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E Hh reporter assay of the activity of wild type and mutant forms of mSmo in Smo-/- MEFs in response to Shh or SAG stimulation. Note that mSmoA1 shows constitutive activity in the absence of either Shh or SAG whereas mSmoKRA dose not; all mutants affecting phosphorylation failed to restore the response to Shh or SAG. Data represent the mean and ± SD (n=3). Unpaired Students t-test was used for analysis. ****P<0.0001; **P<0.01; and n.s. (not significant).


A Wild type 18hpf injected with mRNA encoding GFP - tagged mSmo ( green ) showing localisation to PC of myotomal labeled with anti - Acetylated tubulin ( AcTub ; red ) , stimulated in response to Shh injection ( n = 4 ) . Differences in PC distribution are due to morphological changes in myotome induced by ectopic expression of Shh as revealed by distribution of nuclei ( DAPI stained ; blue ) . Scale bar , 10μm .

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A Wild type 18hpf embryos injected with mRNA encoding GFP-tagged mSmo (green) showing localisation to the PC of myotomal cells labeled with anti-Acetylated tubulin (AcTub; red), stimulated in response to Shh injection (n = 4). Differences in PC distribution are due to morphological changes in the myotome induced by ectopic expression of Shh as revealed by the distribution of nuclei (DAPI stained; blue). Scale bar, 10μm.


B Notochord of wild type expressing GFP tagged wild - type mutant forms of mSmo . Note localisation to PC ( labeled with anti - AcTub ; red ) in each case ( n = 4 for each sample ) . Scale bar , 10μm .

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B Notochord cells of wild type embryos expressing GFP tagged wild-type and mutant forms of mSmo. Note the localisation to the PC (labeled with anti-AcTub; red) in each case (n=4 for each sample). Scale bar, 10μm.


C MZgrk2 18hpf injected with mRNA encoding GFP - tagged wild type mSmo or mSmoSA ( green ) showing localisation to PC ( labeled with anti - AcTub ; red ) in myotomal . More Smo is localised to PC in MZgrk2 mutants compared to wild type ( panel A ) . Scale bar , 10μm .

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C MZgrk2 18hpf embryos injected with mRNA encoding GFP-tagged wild type mSmo or mSmoSA (green) showing localisation to the PC (labeled with anti- AcTub; red) in myotomal cells. More Smo is localised to the PC in MZgrk2 mutants compared to wild type (panel A). Scale bar, 10μm.


B Prox1a ( green ) Eng ( red ) expression in myotome of 30hpf MZgrk2 that were injected with mRNA encoding : Grk2K220R - GFP ; Grk2K220M - GFP ; Grk2D335N - GFP ; Grk2ΔN7aa - GFP mGrk2L4AV7AL8A - GFP . Merged panels show with DAPI in blue . ( n = 20 for each sample ) . Scale bar , 50μm .

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B Prox1a (green) and Eng (red) expression in the myotome of 30hpf MZgrk2 embryos that were injected with mRNA encoding: Grk2K220R-GFP; Grk2K220M-GFP; Grk2D335N-GFP; Grk2ΔN7aa-GFP and mGrk2L4AV7AL8A-GFP. Merged panels show nuclear staining with DAPI in blue. (n=20 for each sample). Scale bar, 50μm.


C Hh reporter activity in wild type Grk2 - / - 3T3 transfected with equivalent wild type mutant kinase domain forms of mouse Grk2 , Grk2K220R , Grk2K220M Grk2D335N after 24h Shh - N SAG treatment . Data represent mean ± SD ( n = 3 ) . Unpaired Students t - was used for . * * * * P < 0 . 0001 ; * * P < 0 . 01 ; * P < 0 . 05 ; n . s . ( not significant ) .

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

C Hh reporter activity assay in wild type and Grk2-/- 3T3 cells transfected with the equivalent wild type and mutant kinase domain forms of mouse Grk2, Grk2K220R, Grk2K220M and Grk2D335N after 24h Shh-N and SAG treatment. Data represent the mean and ± SD (n=3). Unpaired Students t-test was used for analysis. ****P<0.0001; **P<0.01; *P<0.05; and n.s. (not significant).


Transgenic ESZscan4 _ Emerald were sorted total RNAs were extracted subjected to RT - qPCR . graph shows mRNA expression level of Arg2 in ES subpopulations .

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Transgenic ESZscan4_Emerald cells were sorted and total RNAs were extracted and subjected to RT-qPCR. The graph shows the mRNA expression level of Arg2 in ES subpopulations.


( B ) Transgenic ESZscan4 _ Emerald were sorted total were extracted subjected to immunoblotting . image is a representative of three independent biological western blotting on arg2 expression in sorted ESZscan4 _ Emerald control .

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(B)Transgenic ESZscan4_Emerald cells were sorted and total proteins were extracted and subjected to immunoblotting. The image is a representative of three independent biological western blotting analysis on arg2 expression in sorted ESZscan4_Emerald and control cells.


Transcript ( left panel ) expression ( right panel ) of Arg2 in ESZscan4 _ LNGFR after transfection with a non - silencing siRNA ( siCtrl ) or siRNA against Arg2 ( siArg2 ) .

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Transcript (left panel) and protein expression (right panel) levels of Arg2 in ESZscan4_LNGFR cells after transfection with a non-silencing siRNA (siCtrl) or siRNA against Arg2 (siArg2).


Imaging flow cytometry on Zscan4 + siArg2 Zscan4 + siCtrl . Samples were stained by labelling histone H3 . of H3 signal was measured in region by creating a mask defined by DAPI . texture feature of signal was analysed by using modulation method ( for details see Materials Methods ) . Two populations ( R1 R2 ) correspond to two different patterns of signal : homogenous clustered distribution , respectively .

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

Imaging flow cytometry analysis on Zscan4+ siArg2 and Zscan4+ siCtrl cells. Samples were stained by labelling histone H3 protein. The intensity of H3 fluorescent signal was measured in the nuclear region by creating a mask defined by DAPI staining. The texture feature of fluorescent signal was analysed by using the modulation method (for details see Materials and Methods). Two cell populations (R1 and R2) correspond to two different patterns of fluorescent signal: homogenous and clustered distribution, respectively.


representative SeaHorse time courses of OCR ECAR respectively ; where are indicated following compounds added : oligomycin ( olig . ) , carbonyl cyanide 4 - ( trifluoromethoxy ) phenylhydrazone ( FCCP ) , rotenone plus antimycin A ( R / A ) , glucose ( Glu ) , 2 - deoxyglucose ( 2DG ) . Each point in OCR ECAR time - courses is average ± S . D . of four technical replicates .

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

representative SeaHorse time courses of OCR and ECAR respectively; where are indicated the following compounds added: oligomycin (olig.), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), rotenone plus antimycin A (R/A), glucose (Glu), 2-deoxyglucose (2DG). Each point in the OCR and ECAR time-courses is the average ± S.D. of four technical replicates.


OCR / ECAR ; ( 4 technical replicates each ) ; Basal , ( resting OCR ) / ( ECAR + Glu ) ; Max , ( OCR + FCCP ) / ( ECAR + Olig . )

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

OCR/ECAR ratio; (4 technical replicates each); Basal, (resting OCR)/(ECAR +Glu); Max, (OCR+FCCP)/(ECAR+Olig.)


Cellular NAD+ and NADH content and NAD+/NADH.


Confocal microscopy of mitochondrial potential DYm . Pictures on right , representative imaging of loaded with tetramethylrhodamine , ethyl ester ( TMRE ) ; histogram on left , averaged / ± SD of n = 3 independent biological replicates ; * , P < 0 . 05 .

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Confocal microscopy analysis of the mitochondrial membrane potential DYm. Pictures on the right, representative imaging of cells loaded with tetramethylrhodamine, ethyl ester (TMRE); histogram on the left, averaged fluorescence intensity/cell ± SD of n=3 independent biological replicates; *, P < 0.05.


Representative confocal microscopy of loaded with MitoTracker Green ( pictures on left ) ; digital enlargements false colors rendering of details shown as contoured squares ( pictures on right ) .

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Representative confocal microscopy analysis of cells loaded with MitoTracker Green (pictures on the left); digital enlargements and false colors rendering of details shown as white contoured squares (pictures on the right).


Electron microscopy of sorted ESZscan4 _ Emerald control . Scale bar

Electron microscopy images of sorted ESZscan4_Emerald and control cells. Scale bar 1μm


Representative confocal microscopy of loaded with dichlorofluorescein - diacetate converted in dichlorofluorescein ( DCF ) ( pictures on left ) ; digital enlargements false - color rendering of details shown as contoured squares ( pictures on right ) ; histogram on left , averaged / ± of n = 3 independent biological replicates .

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Representative confocal microscopy analysis of cells loaded with dichlorofluorescein-diacetate converted in dichlorofluorescein (DCF) (pictures on the left); digital enlargements and false-color rendering of details shown as white contoured squares (pictures on the right); histogram on the left, averaged fluorescence intensity/cell ± SEM of n=3 independent biological replicates.


Basal R / A - sensitive OCR measured as in Fig . 2A in absence in presence of a cocktail of inhibitors ( BPTES + etomoxir + UK5099 ) ; End . Sub . , OCR measured with endogenous substrates ; Exo . Sub . , OCR measured with glutamine ( Gln ) , fatty ( FA ) glucose ( Glc ) supplemented in medium . superimposed bars are means ± of n = 3 independent biological replicates ( 3 technical replicates each ) ;

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

Basal R/A-sensitive OCR measured as in Fig. 2A in the absence and in the presence of a cocktail of inhibitors (BPTES + etomoxir + UK5099); End. Sub., OCR measured with endogenous substrates; Exo. Sub., OCR measured with glutamine (Gln), fatty acids (FA) and glucose (Glc) supplemented in the assay medium. The superimposed bars are means ± SEM of n=3 independent biological replicates (3 technical replicates each);


Measurement of dependency flexibility of OCR to either of Gln , FA , Glc ; was carried out according to MitoFuel protocol ( see Material Methods ) ; superimposed bars ( whose sum corresponds to capacity ) are means ± of n = 3 independent biological replicates ( 3 technical replicates each ) ;

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

Measurement of dependency and flexibility of the OCR to either of Gln, FA, Glc; the assay was carried out according to the MitoFuel protocol (see Material and Methods); the superimposed bars (whose sum corresponds to the capacity) are means ± SEM of n=3 independent biological replicates (3 technical replicates each);


LC - MSMS of content of free aminoacids ; symbols of aminoacids are indicated in x - axis ; amount expressed as μmol / μg is indicated in y - axis . inset shows content of ornithine , citrulline arginine .

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LC-MSMS analysis of the cellular content of free aminoacids; the symbols of aminoacids are indicated in x-axis; the amount expressed as μmol/μg protein is indicated in y-axis. The inset shows the content of ornithine, citrulline and arginine.


( D ) LC - MSMS of content of acylcarnitines ; of acyl moiety is indicated by number of carbon atoms in abscissa ; bars values are means ± of n = 3 independent biological replicates ; inset shows content of carnitine ( C0 ) acetylcarnitine ( C2 ) .

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

(D)LC-MSMS analysis of the cellular content of acylcarnitines; the length of the acyl moiety is indicated by the number of carbon atoms in abscissa; bars values are means ± SEM of n=3 independent biological replicates; The inset shows the content of carnitine (C0) and acetylcarnitine (C2).


B , Multiplexed activation of Ascl1 , Lmx1a , NeuroD1 of Ascl1 , Lmx1a , Nr4a2 in astrocytic cultures . n = 3 , representative experiment from 2 - 3 independent experiments , additional data in supplement . Activation are depicted as fold change between transfected with without sgRNAs . were normalized to β - Actin . Error bars represent mean ± SD between technical replicates .

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B, Multiplexed activation of Ascl1, Lmx1a, NeuroD1 and of Ascl1, Lmx1a, Nr4a2 in primary astrocytic cultures. n=3, representative experiment from 2-3 independent experiments, additional data in supplement. Activation levels are depicted as fold change between cells transfected with and without sgRNAs. All levels were normalized to β-Actin. Error bars represent mean ± SD between technical replicates.


A , Representative photomicrographs taken from dorsal striatum 13 after AAV - injection . In mice injected with GFP control , virtually GFP positive depict an astrocytic , many GFP positive in ALNe - 218 ALN - treated show a neuron - like . Data information : Scale bars indicate 20 µm .

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A, Representative photomicrographs taken from the dorsal striatum 13 weeks after AAV-injection. In mice injected with GFP control virus, virtually all GFP positive cells depict an astrocytic morphology, many GFP positive cells in ALNe-218 and ALN-treated animals show a neuron-like morphology. Data information: Scale bars indicate 20 µm.


B , Immunohistochemical showing GFP + / GFAP + double positive 13 wpi . Arrows indicate double positive GFP + / GFAP + , arrowheads indicate GFP + / GFAP - . of GFAP + / GFP + shows a significant decrease upon ALNe - 218 ALN - treatment ( GFP vs . ALNe - 218 P = 0 . 0064 , GFP vs . ALN P = 0 . 0002 ALN vs . ALNe - 218 P = 0 . 0127 ) . Multiple comparison ANOVA F ( 2 , 7 ) = 32 . 06 . C , Double immunostaining for GFP neuronal marker NeuN . Arrowheads indicate double positive GFP + / NeuN + 13 wpi . demonstrate a significant increase in NeuN + / GFP + upon ALNe - 218 ALN - induction ( GFP vs . ALNe - 218 P < 0 . 0001 , GFP vs . ALN P < 0 . 0001 ALN vs . ALNe - 218 P = 0 . 0012 ) . Multiple comparison ANOVA F ( 2 , 6 ) = 170 . 3 . Data information : Scale bars indicate 20 µm . Tukey ' s multiple comparisons * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . n = 3 - 4 mice per condition . Error bars represent mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Immunohistochemical analysis showing GFP+/GFAP+ double positive cells 13 wpi. Arrows indicate double positive GFP+/GFAP+ cells, arrowheads indicate GFP+/GFAP- cells. Quantification of GFAP+/GFP+ cells shows a significant decrease upon ALNe-218 and ALN-treatment (GFP vs. ALNe-218 P=0.0064, GFP vs. ALN P=0.0002 and ALN vs. ALNe-218 P=0.0127). Multiple comparison ANOVA F(2,7)=32.06. C, Double immunostaining for GFP and the neuronal marker NeuN. Arrowheads indicate double positive GFP+/NeuN+ cell 13 wpi. Quantification demonstrate a significant increase in NeuN+/GFP+ cells upon ALNe-218 and ALN-induction (GFP vs. ALNe-218 P<0.0001, GFP vs. ALN P<0.0001 and ALN vs. ALNe-218 P=0.0012). Multiple comparison ANOVA F(2,6)=170.3. Data information: Scale bars indicate 20 µm. Tukey's multiple comparisons test * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. n = 3-4 mice per condition. Error bars represent mean ± SD.


D , Double immunostaining for GFP TH as a marker for dopaminergic neurons in dorsal striatum of GFP - control , ALNe - 218 ALN - treated . Arrowheads indicate GFP + / TH - with neuronal . No GFP + / TH + could be detected in any of experimental groups . Scale bars indicate 20 µm . Data information : Scale bars indicate 20 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

D, Double immunostaining for GFP and TH as a marker for dopaminergic neurons in the dorsal striatum of GFP-control, ALNe-218 and ALN-treated animals. Arrowheads indicate GFP+/TH- cells with neuronal morphology. No GFP+/TH+ cell could be detected in any of the experimental groups. Scale bars indicate 20 µm. Data information: Scale bars indicate 20 µm.


B , RT - qPCR of Ascl1 induction comparing activation capacity of full - versus split - dCas9 in Neuro2A ( data in fold change normalized to non - activated control ; dCas9 6116 ± 847 . 3 , split - dCas9 4415 ± 748 . 8 , n = 3 biological replicates , box - - whiskers plots indicate median , 25th - 75th percentile min / max whiskers ) .

B, RT-qPCR analysis of Ascl1 induction comparing the activation capacity of full-length versus split-dCas9 in Neuro2A cells (data in fold change normalized to non-activated control; dCas9 6116 ± 847.3, split-dCas9 4415 ± 748.8, n=3 biological replicates, box-and-whiskers plots indicate median, 25th-75th percentile and min/max whiskers).


C , Immunocytochemistry of reprogrammed astrocytes 16 days after lentiviral transduction revealed successful in vitro reprogramming into neurons using CRISPRa . Astrocytes are infected with two lentiviruses expressing dCas9 - VPR in a intein - split version similar to AAV - dCAS system but driven by a Tet - O promoter . Tet - O driven dsRed Ascl1 cDNA expressing construct were used as negative positive controls respectively . Arrows indicate single MAP2 positive background neurons , arrowheads indicate double positive induced neurons . Scale bar indicates 20 µm . Data information : Abbreviations : OE -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Immunocytochemistry analysis of reprogrammed primary astrocytes cells 16 days after lentiviral transduction revealed successful in vitro reprogramming into neurons using CRISPRa. Astrocytes are infected with two lentiviruses expressing dCas9-VPR in a intein-split version similar to the AAV-dCAS system but driven by a Tet-O promoter. Tet-O driven dsRed and Ascl1 cDNA expressing construct were used as negative and positive controls respectively. Arrows indicate single MAP2 positive background neurons, arrowheads indicate double positive induced neurons. Scale bar indicates 20 µm. Data information: Abbreviations: OE - overexpression


E , Multiplexed activation of Ascl1 / Lmx1a / NeuroD1 , Ascl1 / Lmx1a / Nr4a2 Ascl1 / Lmx1a / Nr4a2 / Pitx3 / FoxA2 in astrocytic . n = 2 - 3 biological replicates , one representative run is shown , additional data in supplement . Activation are depicted as fold change between transfected with without sgRNAs . were normalized to β - Actin . Error bars represent mean ± SD between technical replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Multiplexed activation of Ascl1/Lmx1a/NeuroD1, Ascl1/Lmx1a/Nr4a2 and Ascl1/ Lmx1a/ Nr4a2/Pitx3/FoxA2 in primary astrocytic cells. n=2-3 biological replicates, one representative run is shown, additional data in supplement. Activation levels are depicted as fold change between cells transfected with and without sgRNAs. All levels were normalized to β-Actin. Error bars represent mean ± SD between technical replicates.


A , Representative photomicrographs taken from dorsal striatum 13 after AAV - injection . In GFP control condition , virtually GFP positive depict an astrocytic , many GFP positive in ALNe - 218 ALN - treated show a neuron - like . Data information : Scale bars indicate 20 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

A, Representative photomicrographs taken from the dorsal striatum 13 weeks after AAV-injection. In the GFP control condition, virtually all GFP positive cells depict an astrocytic morphology, many GFP positive cells in ALNe-218 and ALN-treated animals show a neuron-like morphology. Data information: Scale bars indicate 20 µm.


B , Immunohistochemical showing GFP + / GFAP + double positive 13 wpi . Arrows indicate double positive GFP + / GFAP + , arrowheads indicate GFP + / GFAP - . of GFAP + / GFP + shows a significant decrease upon ALNe - 218 ALN - treatment ( GFP vs . ALNe - 218 P = 0 . 0083 , GFP vs . ALN P < 0 . 0001 ALN vs . ALNe - 218 P = 0 . 0006 , multiple comparison ANOVA F ( 2 , 6 ) = 79 . 76 ) . C , Photomicrographs showing GFP + / NeuN + neurons 13 wpi . Arrowheads indicating GFP + / NeuN + induced neurons . demonstrate a significant increase in NeuN + / GFP + upon ALNe - 218 ALN - induction ( GFP vs . ALN P = 0 . 0008 , ALN vs . ALNe - 218 P = 0 . 0092 , multiple comparison ANOVA F ( 2 , 7 ) = 21 . 74 ) . Data information : Scale bars indicate 20 µm . Error bars represent mean ± SD . n = 3 - 4 mice ( b , c ) per condition . Tukey ' s multiple comparisons * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Immunohistochemical analysis showing GFP+/GFAP+ double positive cells 13 wpi. Arrows indicate double positive GFP+/GFAP+ cells, arrowheads indicate GFP+/GFAP- cells. Quantification of GFAP+/GFP+ cells shows a significant decrease upon ALNe-218 and ALN-treatment (GFP vs. ALNe-218 P=0.0083, GFP vs. ALN P<0.0001 and ALN vs. ALNe-218 P=0.0006, multiple comparison ANOVA F(2,6)=79.76). C, Photomicrographs showing GFP+/NeuN+ neurons 13 wpi. Arrowheads indicating GFP+/NeuN+ induced neurons. Quantification demonstrate a significant increase in NeuN+/GFP+ cells upon ALNe-218 and ALN-induction (GFP vs. ALN P=0.0008, ALN vs. ALNe-218 P=0.0092, multiple comparison ANOVA F(2,7)=21.74). Data information: Scale bars indicate 20 µm. Error bars represent mean ± SD. n = 3-4 mice (b,c) per condition. Tukey's multiple comparisons test * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


D , In ALN - induced neurons , confocal is demonstrating a co - localization of GFP glutamic acid decarboxylase ( Gad65 / 67 ) , a marker specific for GABAergic neurons . E , of GAD6567 + / GFP + with neuronal shows that majority of ALN - induced neurons are colocalizing with this GABAergic marker both in AAV - dCAS as well as in dCAM setting . Data information : Scale bars indicate 20 µm . Error bars represent mean ± SD . n = 6 - 8 mice ( e ) per condition . Tukey ' s multiple comparisons * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, In ALN-induced neurons, confocal analysis is demonstrating a co-localization of GFP and glutamic acid decarboxylase (Gad65/67), a marker specific for GABAergic neurons. E, Quantification of GAD6567+/GFP+ cells with neuronal morphology shows that the majority of ALN-induced neurons are colocalizing with this GABAergic marker both in the AAV-dCAS as well as in the dCAM setting. Data information: Scale bars indicate 20 µm. Error bars represent mean ± SD. n= 6-8 mice (e) per condition. Tukey's multiple comparisons test * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


A , Gait using CatWalk XT system reveals motor defects in 6 - OHDA lesion model . Both dCAM AAV - dCAS transduce with AAV containing specific gRNAs show a significant improvement in different aspects of voluntary movement like average speed of tread . Naive vs . 6 - OHDA P = 0 . 0063 . AAV - dCAS : GFP vs . ALN P = 0 . 015 multiple comparison ANOVA F ( 2 , 17 ) = 12 . 81 ) Data information : Statistics : naive vs . 6 - OHDA unpaired t - ( two - ) * P < 0 . 05 , * * P < 0 . 01 . GFP vs ALN , GFP vs ALNe - 218 ALN vs ALNe - 218 Tukey ' s multiple comparisons * P < 0 . 05 , * * P < 0 . 01 . n = 4 - 8 mice per condition .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Gait analysis using the CatWalk XT system reveals motor defects in 6-OHDA lesion model. Both dCAM and AAV-dCAS animals transduce with AAV containing specific gRNAs show a significant improvement in different aspects of voluntary movement like the average speed of tread. Naive vs. 6-OHDA P=0.0063. AAV-dCAS: GFP vs. ALN P=0.015 multiple comparison ANOVA F(2,17)=12.81) Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP vs ALN, GFP vs ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice per condition.


stride of front paws : B , Naive vs . 6 - OHDA P = 0 . 0164 . AAV - dCAS : GFP vs . ALN P = 0 . 005 , ALN vs . ALNe - 218 P = 0 . 0042 , multiple comparison ANOVA F ( 4 , 22 ) = 9 . 9 ) Data information : Statistics : naive vs . 6 - OHDA unpaired t - ( two - ) * P < 0 . 05 , * * P < 0 . 01 . GFP vs ALN , GFP vs ALNe - 218 ALN vs ALNe - 218 Tukey ' s multiple comparisons * P < 0 . 05 , * * P < 0 . 01 . n = 4 - 8 mice per condition .

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 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

the stride length of front paws: B, Naive vs. 6-OHDA P=0.0164. AAV-dCAS: GFP vs. ALN P=0.005, ALN vs. ALNe-218 P=0.0042, multiple comparison ANOVA F(4,22)=9.9) Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP vs ALN, GFP vs ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice per condition.


duty cycle of left front paws . C , Naive vs . 6 - OHDA P = 0 . 0096 . dCAM : GFP vs . ALN P = 0 . 036 ALN vs . ALNe - 218 P = 0 . 0252 , multiple comparison ANOVA F ( 2 , 20 ) = 5 . 199 ) . Data information : Statistics : naive vs . 6 - OHDA unpaired t - ( two - ) * P < 0 . 05 , * * P < 0 . 01 . GFP vs ALN , GFP vs ALNe - 218 ALN vs ALNe - 218 Tukey ' s multiple comparisons * P < 0 . 05 , * * P < 0 . 01 . n = 4 - 8 mice per condition .

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

the duty cycle of left front paws. C, Naive vs. 6-OHDA P=0.0096. dCAM: GFP vs. ALN P=0.036 and ALN vs. ALNe-218 P=0.0252, multiple comparison ANOVA F(2,20)=5.199). Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP vs ALN, GFP vs ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice per condition.


D , In contrast to this , dopamine dependent drug - induced behavior does not show rescue effects : amphetamine - induced rotation : change in rotational behavior in lesioned upon treatment with dopamine releaser substance . Net rotation = rotation - rotation ( naïve vs . 6 - OHDA P = 0 . 09 ) . Data information : Statistics : naive vs . 6 - OHDA unpaired t - ( two - ) * P < 0 . 05 , * * P < 0 . 01 . GFP vs ALN , GFP vs ALNe - 218 ALN vs ALNe - 218 Tukey ' s multiple comparisons * P < 0 . 05 , * * P < 0 . 01 . n = 4 - 8 mice per condition . CatWalk error bars represent mean ± SD . Rotation error bars represent mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, In contrast to this, dopamine dependent drug-induced behavior does not show rescue effects: amphetamine-induced rotation analysis: change in rotational behavior in lesioned animals upon treatment with dopamine releaser substance. Net rotation = ipsilateral rotation-contralateral rotation (naïve vs. 6-OHDA P=0.09). Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP vs ALN, GFP vs ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice per condition. CatWalk error bars represent mean ± SD. Rotation analysis error bars represent mean ± SEM.


( A ) Fractionation of compartments using a standard NP - 40 lysis protocol immunoblotting with chaperone specific antibodies

(A) Fractionation of nuclear and cytosolic compartments using a standard NP-40 lysis protocol and immunoblotting with chaperone specific antibodies


( B ) Immunofluorescence of histone chaperones in fixed using same antibodies used in ( A ) . are segregated into those undergoing replication those that are not . Scale bar represents 5 μm

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(B) Immunofluorescence of histone chaperones in fixed cells using the same antibodies used in (A). Cells are segregated into those undergoing replication and those that are not. Scale bar represents 5 μm


( D ) Real - time imaging of undergoing biochemical fractionation . sNASP is tagged with mCherry , whilst LamnA / C is tagged with EGFP . left column of panels represents a maximum projection . time course on right represents a plane in z dimension reconstructed from 20 z - stacks penetrating 20 μm into medium . Scale bars represent 5

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Real-time imaging of cells undergoing biochemical fractionation. sNASP is tagged with mCherry, whilst LamnA/C is tagged with EGFP. The left column of panels represents a maximum intensity projection. The time course on the right represents a plane in the z dimension reconstructed from 20 z-stacks penetrating 20 μm into the medium. Scale bars represent 5 μm


( A ) HeLa lines stably expressing eGFP - sNASP , eGFP - ASF1 eGFP - HAT1 . Scale bars represent 10 μm

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(A) HeLa cell lines stably expressing eGFP-sNASP, eGFP-ASF1 and eGFP-HAT1. Scale bars represent 10 μm


( B ) Histone chaperone immunoprecipitations separated by 15 % SDS - PAGE stained with Coomassie . Major bands that could be confidently identified by mass spectrometry are indicated . Due to high of acrylamide used , many of higher molecular bands are not resolved . Asterisks represent partial degradation . Background - corrected densitometry profiles are shown for portions of gels covering H3 H4 . Gel filtration purified recombinant H3 - H4 dimer is shown on

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Histone chaperone immunoprecipitations separated by 15% SDS-PAGE and stained with Coomassie. Major bands that could be confidently identified by mass spectrometry are indicated. Due to the high percentage of acrylamide used, many of the higher molecular weight bands are not resolved. Asterisks represent partial degradation. Background-corrected densitometry profiles are shown for the portions of the gels covering H3 and H4. Gel filtration purified recombinant H3-H4 dimer is shown on the right


Western blot of Fxr1 during ( A ) , TTX ( 48h treatment ) inducedupscaling ( n = 6 in each condition ) of postnatal cortical cultures . Student ' s T - * p < 0 . 05 , * * * p < 0 . 001 .

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Western blot analysis of Fxr1 during (A), TTX (48h treatment) inducedupscaling (n=6 in each condition) of primary postnatal cortical cultures. Student's T-test *p<0.05, ***p<0.001.


Western blot of Fxr1 during ( B ) , BIC ( 48h treatment ) induced downscaling ( n = 8 in each condition ) of postnatal cortical cultures . Student ' s T - * p < 0 . 05 , * * * p < 0 . 001 .

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Western blot analysis of Fxr1 during (B), BIC (48h treatment) induced downscaling (n=8 in each condition) of primary postnatal cortical cultures. Student's T-test *p<0.05, ***p<0.001.


C , D , Western blot of ( C ) , Fxr2 ( Veh n = 8 , TTX n = 8 ) ( D ) , Fmrp ( Veh n = 10 , TTX n = 12 ) during upscaling , Student ' s T - * p < 0 . 05 .

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C, D, Western blot analysis of (C), Fxr2 (Veh n=8, TTX n=8) and (D), Fmrp (Veh n=10, TTX n=12) during upscaling, Student's T-test *p<0.05.


E - G , RT - qPCR measurement of mRNA for ( E ) , Fxr1 , ( F ) , Fxr2 , ( G ) , Fmr1 during upscaling . n = 4 in each condition , Student ' s T - * p < 0 . 05 .

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E-G, RT-qPCR measurement of mRNA for (E), Fxr1, (F), Fxr2, (G), Fmr1 during upscaling. n=4 in each condition, Student's T-test *p<0.05.


H , Western blot for Fxr1 , pGsk3α / β , Gsk3α / β GAPDH in neuronal cultures treated with 1µM TTX ( TTX ) or 1mM LiCI ( Li ) or 1mM NaCI ( Ctrl ) for 48h .

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H, Western blot analysis for Fxr1, pGsk3α/β, Gsk3α/β and GAPDH in neuronal cultures treated with 1µM TTX (TTX) or 1mM LiCI (Li) or 1mM NaCI (Ctrl) for 48h.


I , Expression of Fxr1 in TTX ( n = 5 ) Li ( n = 5 ) conditions relative to Ctrl ( n = 6 ) condition . One way ANOVA with Dunnett ' s Multiple Comparison * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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I, Expression of Fxr1 protein in TTX (n=5) and Li (n=5) conditions relative to Ctrl (n=6) condition. One way ANOVA with Dunnett's Multiple Comparison Test *p<0.05, **p<0.01, ***p<0.001.


J , Expression of pGsk3β / Gsk3β or pGsk3α / Gsk3α in TTX ( n = 5 ) Li ( n = 5 ) conditions relative to Ctrl ( n = 6 ) condition . One way ANOVA with Dunnett ' s Multiple Comparison * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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J, Expression of pGsk3β/Gsk3β or pGsk3α/Gsk3α in TTX (n=5) and Li (n=5) conditions relative to Ctrl (n=6) condition. One way ANOVA with Dunnett's Multiple Comparison Test *p<0.05, **p<0.01, ***p<0.001.


B , Western blot of total GluA1 expression in Ctrl or Fxr1 overexpression ( Fxr1 ) condition during upscaling ( Ctrl / Veh n = 5 , Ctrl / TTX n = 5 , Fxr1 / Veh n = 6 , Fxr1 / Veh n = 6 ) . One way Anova with Dunnett ' s Multiple Comparison * * * p < 0 . 001 .

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B, Western blot analysis of total GluA1 expression in Ctrl or Fxr1 overexpression (Fxr1) condition during upscaling (Ctrl/Veh n=5, Ctrl/TTX n=5, Fxr1/Veh n=6, Fxr1/Veh n=6). One way Anova with Dunnett's Multiple Comparison Test ***p<0.001.


C , Western blot of total GluA1 expression in Ctrl or Fxr1over condition during downscaling ( Ctrl / Veh n = 4 , Ctrl / TTX n = 4 , Fxr1 / Veh n = 4 , Fxr1 / Veh n = 4 ) .

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C, Western blot analysis of total GluA1 expression in Ctrl or Fxr1over condition during downscaling (Ctrl/Veh n=4, Ctrl/TTX n=4, Fxr1/Veh n=4, Fxr1/Veh n=4).


D , RT - qPCR measurement of Gria1 mRNA in Ctrl or Fxr1over condition during upscaling ( Ctrl / Veh n = 3 , Ctrl / TTX n = 3 , Fxr1 / Veh n = 3 , Fxr1 / Veh n = 3 ) . One way Anova with Bonferroni ' s Multiple Comparison * * * p < 0 . 001 .

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D, RT-qPCR measurement of Gria1 mRNA in Ctrl or Fxr1over condition during upscaling (Ctrl/Veh n=3, Ctrl/TTX n=3, Fxr1/Veh n=3, Fxr1/Veh n=3). One way Anova with Bonferroni's Multiple Comparison Test ***p<0.001.


F , Measurement of relative luciferase signal after co - transfection of different tagged luciferase constructs along with GFP ( Ctrl ) or GFP - Fxr1 ( Fxr1 ) plasmids . n = 5 in each condition , Student ' s T - * p < 0 . 05 , * * * p < 0 . 001 .

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F, Measurement of relative luciferase signal after co-transfection of different tagged luciferase constructs along with GFP (Ctrl) or GFP-Fxr1 (Fxr1) plasmids. n=5 in each condition, Student's T-test *p<0.05, ***p<0.001.


G , Immunostaining for surface GluA1 in GFP ( Ctrl ) or GFP - Fxr1 ( Fxr1 over ) infected cultures after treatment with Veh , TTX or BIC for 48h .

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G, Immunostaining for surface GluA1 in GFP (Ctrl) or GFP-Fxr1 (Fxr1 over) infected cultures after treatment with Veh, TTX or BIC for 48h.


H , of surface GluA1 relative to mean of Ctrl / Veh condition ( Ctrl condition : Veh n = 63 , TTX n = 34 , BIC n = 29 , Fxr1 condition : Veh n = 56 , TTX n = 30 , BIC n = 29 ) . One way Anova with Bonferroni ' s Multiple Comparison * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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H, Percentage of surface GluA1 relative to the mean of Ctrl/Veh condition (Ctrl condition: Veh n=63, TTX n=34, BIC n=29, Fxr1 condition: Veh n=56, TTX n=30, BIC n=29 ). One way Anova with Bonferroni's Multiple Comparison Test *p<0.05, **p<0.01, ***p<0.001.


A , Cumulative probability plots of mEPSCs amplitude ( 500 events per ) representative examples of mEPSCs ( left panel ) recorded from cultured cortical control neurons after 48 hours of 1 µM TTX or Veh exposure ( Ctrl / Veh n = 16 Ctrl / TTX n = 17 ) . B , A linear fit of Ctrl / TTX Ctrl / Veh amplitudes . C , degrees of overlap between Ctrl / TTX Ctrl / Veh data were assessed using various scaling factors . largest nonsignificant p - value was obtained with 1 . 47 scaling factor . D , Cumulative probability plots of mEPSCs amplitude of Ctrl / Veh , Ctrl / TTX Ctrl / TTX divided by scaling factor 1 . 47 , which yielded maximum overlap with Ctrl / Veh data .

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A, Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical control neurons after 48 hours of 1 µM TTX or Veh exposure (Ctrl/Veh n=16 and Ctrl/TTX n=17). B, A linear fit of Ctrl/TTX and Ctrl/Veh amplitudes. C, The degrees of overlap between Ctrl/TTX and Ctrl/Veh data were assessed using various scaling factors. The largest nonsignificant p-value was obtained with 1.47 scaling factor. D, Cumulative probability plots of the mEPSCs amplitude of Ctrl/Veh, Ctrl/TTX and Ctrl/TTX divided by scaling factor 1.47, which yielded the maximum overlap with Ctrl/Veh data.


Cumulative probability plots of mEPSCs amplitude ( 500 events per ) representative examples of mEPSCs ( left panel ) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure ( E ) Fxr1overexpressing neurons ( Fxr1 / Veh n = 10 Fxr1 / TTX n = 8 )

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Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure (E) Fxr1overexpressing neurons (Fxr1/Veh n=10 and Fxr1/TTX n=8)


Cumulative probability plots of mEPSCs amplitude ( 500 events per ) representative examples of mEPSCs ( left panel ) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure ( F ) Gsk3 KO neurons ( Gsk3KO / Veh n = 10 Gsk3KO / TTX n = 11 )

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Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure (F) Gsk3 KO neurons (Gsk3KO/Veh n=10 and Gsk3KO/TTX n=11)


Cumulative probability plots of mEPSCs amplitude ( 500 events per ) representative examples of mEPSCs ( left panel ) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure ( G ) Fxr1 KO neurons ( Fxr1KO / Veh n = 16 Fxr1KO / TTX n = 8 ) . H , A linear fit of Fxr1 KO / Veh Ctrl / Veh amplitudes . I , degrees of overlap between Fxr1 KO / Veh Ctrl / Veh data were assessed using various scaling factors . largest non - significant p - value was obtained with 1 . 27 scaling factor . J , Cumulative probability plots of mEPSCs amplitude of Ctrl / Veh , Fxr1 KO / Veh Fxr1 KO / Veh divided by scaling factor 1 . 27 , which yielded maximum overlap with Ctrl / Veh data .

O O O O O O 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 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

Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure (G) Fxr1 KO neurons (Fxr1KO/Veh n=16 and Fxr1KO/TTX n=8). H, A linear fit of Fxr1 KO/Veh and Ctrl/Veh amplitudes. I, The degrees of overlap between Fxr1 KO/Veh and Ctrl/Veh data were assessed using various scaling factors. The largest non-significant p-value was obtained with 1.27 scaling factor. J, Cumulative probability plots of the mEPSCs amplitude of Ctrl/Veh, Fxr1 KO/Veh and Fxr1 KO/Veh divided by scaling factor 1.27, which yielded the maximum overlap with Ctrl/Veh data.


K , Cumulative probability plots of mEPSCs amplitude ( 500 events per ) representative examples of mEPSCs ( left panel ) recorded from cultured cortical Gsk3 Fxr1 KO neurons after 48 hours of 1 µM TTX or Veh exposure ( Gsk3 / Fxr1KO / Veh n = 8 Gsk3 / Fxr1KO / TTX n = 11 ) . L , A linear fit of Gsk3 / Fxr1 KO / Veh Ctrl / Veh amplitudes . M , degrees of overlap between Gsk3 / Fxr1 KO / Veh Ctrl / Veh data were assessed using various scaling factors . largest non - significant p - value was obtained with 1 . 68 scaling factor . N , Cumulative probability plots of mEPSCs amplitude of Ctrl / Veh , Gsk3 / Fxr1 KO / Veh Gsk3 / Fxr1 KO / Veh divided by scaling factor 1 . 68 , which yielded maximum overlap with Ctrl / Veh data .

O O O O O O O O O O O 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 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

K, Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from cultured cortical Gsk3 and Fxr1 KO neurons after 48 hours of 1 µM TTX or Veh exposure (Gsk3/Fxr1KO/Veh n=8 and Gsk3/Fxr1KO/TTX n=11). L, A linear fit of Gsk3/Fxr1 KO/Veh and Ctrl/Veh amplitudes. M, The degrees of overlap between Gsk3/Fxr1 KO/Veh and Ctrl/Veh data were assessed using various scaling factors. The largest non-significant p-value was obtained with 1.68 scaling factor. N, Cumulative probability plots of the mEPSCs amplitude of Ctrl/Veh, Gsk3/Fxr1 KO/Veh and Gsk3/Fxr1 KO/Veh divided by scaling factor 1.68, which yielded the maximum overlap with Ctrl/Veh data.


O , mEPSC mean amplitude of cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure . One way Anova with Bonferroni ' s Multiple Comparison * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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, mEPSC mean amplitude of cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure. One way Anova with Bonferroni's Multiple Comparison Test *p<0.05, **p<0.01, ***p<0.001.


P , mEPSC frequency of cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure .

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P, mEPSC frequency of cultured cortical neurons after 48 hours of 1 µM TTX or Veh exposure.


A , Hourly distribution of wakefulness ( WAKE ) , slow - wave sleep ( SWS ) paradoxical sleep ( PS ) during a 24 - h baseline ( BL ) recording a second 24 - h starting with 6 - h sleep deprivation ( SD ) ( recovery : REC ) in Ctrl Fxr1 overexpressing ( Fxr1 ) mice . Significant Group - by - Hour interactions were found for wakefulness during BL ( F23 , 230 = 1 . 67 , p = 0 . 046 ) REC ( F23 , 230 = 2 . 83 , p = 0 . 0029 ) , for SWS during BL ( F23 , 230 = 1 . 66 , p = 0 . 045 ) REC ( F23 , 230 = 2 . 89 , p = 0 . 0028 ) , for PS during REC ( F16 , 160 = 2 . 54 , p = 0 . 0018 , Two - way ANOVA Huynh - Feldt corrected , * p < 0 . 05 ) .

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A, Hourly distribution of wakefulness (WAKE), slow-wave sleep (SWS) and paradoxical sleep (PS) during a 24-h baseline (BL) recording and a second 24-h starting with 6-h sleep deprivation (SD) (recovery: REC) in Ctrl and Fxr1 overexpressing (Fxr1) mice. Significant Group-by-Hour interactions were found for wakefulness during BL (F23,230 = 1.67, p = 0.046) and REC (F23,230 = 2.83, p = 0.0029), for SWS during BL (F23,230 = 1.66, p = 0.045) and REC (F23,230 = 2.89, p=0.0028), and for PS during REC (F16,160 = 2.54, p=0.0018, Two-way ANOVA Huynh-Feldt corrected, *p<0.05).


B , Power spectra for WAKE , SWS , PS in Ctrl Fxr1 mice computed between 0 . 75 50 Hz per 0 . 25 - Hz for full 24 - h of BL REC .

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B, Power spectra for WAKE, SWS, and PS in Ctrl and Fxr1 mice computed between 0.75 and 50 Hz per 0.25-Hz for the full 24-h of BL and REC.


C , D , spectral activity of Fxr1 mice expressed relative to that of Ctrl mice for WAKE , SWS PS during ( C ) 24 - h BL ( D ) 24 - h REC . A significant difference between groups was found for frequency band 8 to 11 . 75 Hz during wakefulness ( t = 2 . 30 , p = 0 . 044 Student ' s T - * p < 0 . 05 ) .

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C, D, The spectral activity of Fxr1 mice expressed relative to that of Ctrl mice for WAKE, SWS and PS during (C) the 24-h BL and (D) the 24-h REC. A significant difference between groups was found for the frequency band 8 to 11.75 Hz during wakefulness (t = 2.30, p=0.044 Student's T-test *p<0.05).


E , Time course of wakefulness spectral activity between low alpha ( 8 . 5 - 10 . 5 Hz ) low theta ( 4 - 6 Hz ) during BL REC in Ctrl Fxr1 mice . A significant Group - by - Interval interaction was found for REC ( F22 , 220 = 1 . 96 , Two - way ANOVA Huynh - Feldt corrected ; * p < 0 . 05 ) , also when using BL REC intervals of periods ( F16 , 160 = 3 . 11 , p < 0 . 01 ) , .

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E, Time course of wakefulness spectral activity ratio between low alpha (8.5-10.5 Hz) and low theta (4-6 Hz) during BL and REC in Ctrl and Fxr1 mice. A significant Group-by-Interval interaction was found for REC (F22,220 = 1.96, Two-way ANOVA Huynh-Feldt corrected; *p < 0.05), and also when using BL and REC intervals of the light periods (F16,160 = 3.11, p < 0.01),.


F , Time course of SWS relative delta activity during BL REC in Ctrl Fxr1 mice . A significant Group - by - Interval interaction was found during REC ( F13 , 130 = 1 . 89 , Two - way ANOVA Huynh - Feldt corrected ; * p < 0 . 05 ) .

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F, Time course of SWS relative delta activity during BL and REC in Ctrl and Fxr1 mice. A significant Group-by-Interval interaction was found during REC (F13,130 = 1.89, Two-way ANOVA Huynh-Feldt corrected; *p < 0.05).


Western blot of ( B ) p845GluA1 GluA1 expression ( S n = 9 , SD n = 10 ) in prefrontal cortex of sleeping sleep deprived mice . Student ' s t - * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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Western blot analysis of (B) p845GluA1 and GluA1 expression (S n=9, SD n=10) in the prefrontal cortex of sleeping and sleep deprived mice. Student's t-test *p < 0.05, **p < 0.01, ***p < 0.001.


Western blot of , ( C ) p880GluA2 GluA2 expression ( S n = 9 , SD n = 8 ) in prefrontal cortex of sleeping sleep deprived mice . Student ' s t - * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

Western blot analysis of , (C) p880GluA2 and GluA2 expression (S n=9, SD n=8) in the prefrontal cortex of sleeping and sleep deprived mice. Student's t-test *p < 0.05, **p < 0.01, ***p < 0.001.


Western blot of ( D ) Fxr1 expression ( S n = 11 , SD n = 10 ) in prefrontal cortex of sleeping sleep deprived mice . Student ' s t - * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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Western blot analysis of (D) Fxr1 expression (S n=11, SD n=10) in the prefrontal cortex of sleeping and sleep deprived mice. Student's t-test *p < 0.05, **p < 0.01, ***p < 0.001.


Western blot of ( E ) Fmrp expression ( S n = 11 , SD n = 10 ) in prefrontal cortex of sleeping sleep deprived mice . Student ' s t - * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

Western blot analysis of (E) Fmrp expression (S n=11, SD n=10) in the prefrontal cortex of sleeping and sleep deprived mice. Student's t-test *p < 0.05, **p < 0.01, ***p < 0.001.


F - H , RNAseq measurement of mRNA for ( F ) Fxr1 , ( G ) Fxr2 , ( H ) Fmr1 during SD . n = 3 in each condition , Student ' s T - * p < 0 . 05 .

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F-H, RNAseq measurement of mRNA for (F) Fxr1, (G) Fxr2, (H) Fmr1 during SD. n=3 in each condition, Student's T-test *p<0.05.


Cumulative probability plots of mEPSCs amplitude ( 500 events per ) representative examples of mEPSCs ( left panel ) recorded from brain slices of S SD mice . ( B ) Control neurons ( Ctrl / S n = 8 / 4 mice , Ctrl / SD n = 8 / 4 mice )

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Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from brain slices of S and SD mice. (B) Control neurons (Ctrl/S n=8 cells/4 mice, Ctrl/SD n=8 cells/4 mice)


Cumulative probability plots of mEPSCs amplitude ( 500 events per ) representative examples of mEPSCs ( left panel ) recorded from brain slices of S SD mice . ( C ) Fxr1 overexpressing ( Fxr1 / S n = 7 / 5 mice , Fxr1 over / SD n = 9 / 5 mice )

Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from brain slices of S and SD mice. (C) Fxr1 overexpressing (Fxr1/S n=7 cells/5 mice, Fxr1 over/SD n=9 cells/5 mice)


Cumulative probability plots of mEPSCs amplitude ( 500 events per ) representative examples of mEPSCs ( left panel ) recorded from brain slices of S SD mice . ( D ) Gsk3 sKO ( Gsk3sKO / S n = 6 / 3 mice , Gsk3sKO / SD n = 7 / 4 mice ) .

Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from brain slices of S and SD mice. (D) Gsk3 sKO (Gsk3sKO/S n=6 cells/3 mice, Gsk3sKO/SD n=7 cells/4 mice).


mEPSC ( E ) mean amplitude of cortical neurons of S or SD mice . Control neurons ( Ctrl / S n = 8 / 4 mice , Ctrl / SD n = 8 / 4 mice ) , Fxr1 overexpressing ( Fxr1 / S n = 7 / 5 mice , Fxr1 over / SD n = 9 / 5 mice ) , Gsk3 sKO ( Gsk3sKO / S n = 6 / 3 mice , Gsk3sKO / SD n = 7 / 4 mice ) . Student ' s T - * p < 0 . 05 .

mEPSC (E) mean amplitude of cortical neurons of S or SD mice. Control neurons (Ctrl/S n=8 cells/4 mice, Ctrl/SD n=8 cells/4 mice), Fxr1 overexpressing (Fxr1/S n=7 cells/5 mice, Fxr1 over/SD n=9 cells/5 mice), and Gsk3 sKO (Gsk3sKO/S n=6 cells/3 mice, Gsk3sKO/SD n=7 cells/4 mice). Student's T-test *p<0.05.


F , mEPSC ( F ) frequency of cortical neurons of S or SD mice . Control neurons ( Ctrl / S n = 8 / 4 mice , Ctrl / SD n = 8 / 4 mice ) , Fxr1 overexpressing ( Fxr1 / S n = 7 / 5 mice , Fxr1 over / SD n = 9 / 5 mice ) , Gsk3 sKO ( Gsk3sKO / S n = 6 / 3 mice , Gsk3sKO / SD n = 7 / 4 mice ) . Student ' s T - * p < 0 . 05 .

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F, mEPSC (F) frequency of cortical neurons of S or SD mice. Control neurons (Ctrl/S n=8 cells/4 mice, Ctrl/SD n=8 cells/4 mice), Fxr1 overexpressing (Fxr1/S n=7 cells/5 mice, Fxr1 over/SD n=9 cells/5 mice), and Gsk3 sKO (Gsk3sKO/S n=6 cells/3 mice, Gsk3sKO/SD n=7 cells/4 mice). Student's T-test *p<0.05.


G , Representative examples of current - voltage relationship of evoked EPSC amplitude recorded from S ( top panel ) SD ( bottom panel ) mice .

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G, Representative examples of the current-voltage relationship of evoked EPSC amplitude recorded from S (top panel) and SD (bottom panel) mice.


H , Summary bar graphs showing rectification index of control ( S n = 12 / 5 mice , SD n = 10 / 5 mice ) , Fxr1P overexpressing ( S n = 9 / 4 mice , SD n = 14 / 4 mice ) Gsk3 sKO ( S n = 9 / 3 mice , SD n = 9 / 3 mice ) neurons . One way ANOVA with Bonferroni ' s Multiple Comparison * p < 0 . 05 .

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H, Summary bar graphs showing rectification index of control (S n=12 cells/5 mice, and SD n=10 cells/5 mice), Fxr1P overexpressing (S n=9 cells/4 mice, and SD n=14 cells/4 mice) and Gsk3 sKO (S n=9 cells/3 mice, and SD n=9 cells/3 mice) neurons. One way ANOVA with Bonferroni's Multiple Comparison *p < 0.05.


B , Immunostaining for GFP HA in Ctrl Fxr1 mouse brain slices . Arrows indicate presence arrowheads indicate an absence of GFP tagged Fxr1 granules .

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B, Immunostaining for GFP and HA in Ctrl and Fxr1 mouse brain slices. Arrows indicate presence and arrowheads indicate an absence of GFP tagged Fxr1 granules.


C , of colocalization of GFP HA labeled neurons ( GFP + HA + 71 . 3 % ± 3 . 7 , n = 3 , GFP - Fxr1 + HA + 79 . 7 % ± 2 . 8 , n = 3 ) .

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C, Quantification of colocalization of GFP and HA labeled neurons (GFP+HA+ 71.3%±3.7, n=3, GFP-Fxr1+HA+ 79.7%±2.8, n=3).


G , Heat map showing transcripts that have bidirectional expression changes between Ctrl / S , Ctrl / SD , Fxr1 / SD conditions .

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G, Heat map showing transcripts that have bidirectional expression changes between Ctrl/S, Ctrl/SD, and Fxr1/SD conditions.


( B ) Electropherograms of sequencing , family pedigrees , audiograms of two unrelated . Electropherograms show a heterozygous c . 3610C > T substitution resulting in a stop codon ( R1204X ) . Arrows in family pedigrees indicate identified in present study . In audiograms : red blue indicate right left ; respectively , solid dashed lines square brackets show air conduction hearing bone conduction hearing ; respectively . bold ( at of 48 years ) thin ( at of 50 years ) in audiogram of 1 indicate progressive hearing loss ( green arrow ) .

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(B) Electropherograms of DNA sequencing, family pedigrees, and audiograms of two unrelated patients. Electropherograms show a heterozygous c.3610C>T substitution resulting in a stop codon (R1204X). Arrows in family pedigrees indicate the patients identified in the present study. In audiograms: red and blue indicate right and left; respectively, solid and dashed lines and square brackets show air conduction hearing and bone conduction hearing; respectively. The bold line (at the age of 48 years) and thin line (at the age of 50 years) in the audiogram of patient 1 indicate progressive hearing loss (green arrow).


GFP - tagged DIA1 , DIA1 ( M1190D ) , DIA1 ( R1204X ) plasmids were transfected into MDCKmCherry - ESPIN1 cultured on inserts . Twenty - four hours after transfection , a reconstructed lateral view of fixed was generated using a confocal microscope . Arrows indicate elongated microvilli visualized by mCherry - ESPIN1 with GFP - hDIA1 ( R1204X ) expression . Scale bars : 5 μm . Representative of 3 experiments .

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GFP-tagged DIA1, DIA1(M1190D), and DIA1(R1204X) plasmids were transfected into MDCKmCherry-ESPIN1 cells cultured on membrane inserts. Twenty-four hours after transfection, a reconstructed lateral view of fixed cells was generated using a confocal laser fluorescence microscope. Arrows indicate the elongated microvilli visualized by mCherry-ESPIN1 with GFP-hDIA1(R1204X) expression. Scale bars: 5 μm. Representative of 3 experiments.


Various GFP - tagged DIA1 plasmids were transfected into HeLa , fixed 24 h after transfection . Fixed were stained with Alexa568 - conjugated phalloidin DAPI ( A , B : representative of 5 experiments ) . ( A ) Stress fiber formation was observed with a confocal microscope . Note induction of stress by GFP - DIA1 ( M1190D ) ( asterisks ) . Arrow arrowheads indicate a plasma ( PM ) localization of GFP - DIA1 ( M1190D ) a dot - like localization of GFP - DIA1 ( R1204X ) adjacent to PM , respectively . Inset in top panel of DIA1 ( R1204X ) shows magnified image of region indicated by rectangle . Scale bars : 10 μm . .

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Various GFP-tagged DIA1 plasmids were transfected into HeLa cells, and fixed 24 h after transfection. Fixed cells were stained with Alexa568-conjugated phalloidin and DAPI (A, B: representative of 5 experiments).(A) Stress fiber formation was observed with a confocal laser microscope. Note the induction of stress fibers by GFP-DIA1(M1190D) (asterisks). Arrow and arrowheads indicate a plasma membrane (PM) localization of GFP-DIA1(M1190D) and a dot-like localization of GFP-DIA1(R1204X) adjacent to the PM, respectively. Inset in the top panel of DIA1 (R1204X) shows the magnified image of the region indicated by rectangle. Scale bars: 10 μm..


Various GFP - tagged DIA1 plasmids were transfected into HeLa , fixed 24 h after transfection . Fixed were stained with Alexa568 - conjugated phalloidin DAPI ( A , B : representative of 5 experiments ) . ( B ) Microvilli with or without GFP - DIA1 ( R1204X ) expression were observed under a confocal microscope . Note elongated thick microvilli ( arrowheads ) in GFP - DIA1 ( R1204X ) - transfected , but not in non - transfected ( circles ) . 3D movies are available in Movie EV1 ( R1204X ) , Movie EV2 ( WT ) , Movie EV3 ( ttaa ) . Scale bar : 10 μm .

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Various GFP-tagged DIA1 plasmids were transfected into HeLa cells, and fixed 24 h after transfection. Fixed cells were stained with Alexa568-conjugated phalloidin and DAPI (A, B: representative of 5 experiments).(B) Microvilli with or without GFP-DIA1(R1204X) expression were observed under a confocal laser microscope. Note elongated and thick microvilli (arrowheads) in GFP-DIA1(R1204X)- transfected cells, but not in non-transfected cells (circles). 3D movies are available in Movie EV1 (R1204X), Movie EV2 (WT), and Movie EV3 (ttaa). Scale bar: 10 μm.


( B ) Purified GST GST - tagged DID ( 50 nM ) were mixed with biotin - labeled DADs ( WT ; R1204X ; M1190D , 50 nM ) in binding buffer . After rotation , streptavidin - coupled magnetic beads were added to solution , mixture was agitated . material absorbed to beads was eluted in Laemmli ' s sample buffer , were subjected to SDS - PAGE , followed by immunoblotting using an HRP - conjugated GST Ab . Comparable of input ( GSTproteins ) are confirmed in right panel . Representative of 5 experiments .

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(B) Purified GST and GST-tagged DID proteins (50 nM) were mixed with biotin-labeled DADs (WT; R1204X; and M1190D, 50 nM) in binding buffer. After rotation, streptavidin-coupled magnetic beads were added to the solution, and the mixture was agitated. The material absorbed to the beads was eluted in Laemmli's sample buffer, and were subjected to SDS-PAGE, followed by immunoblotting using an HRP-conjugated GST Ab. Comparable levels of input (GSTproteins) are confirmed in the right panel. Representative of 5 experiments.


GFP - tagged DIA1 ( WT ) , DIA1 ( R1204X ) , DIA1 ( M1190D ) were transfected into XTC . XTC were spread on PLL - coated coverslips time - lapse were acquired every 300 ms , illuminating restricted areas near edges , by using a single - molecule speckle microscope . Representative are shown for DIA1 ( WT ) ( A , Movie EV5 ) , DIA1 ( R1204X ) ( B , Movie EV6 ) , DIA1 ( M1190D ) ( C , Movie EV7 ) . Circles indicate speckles showing directional movement over a of several micrometers . Trajectories of these speckles are also illustrated , crosses were plotted at end of directional movement . Speckles showing directional movement were frequently observed in expressing GFP - DIA1 ( R1204X ) ( B ) , whereas such speckles were scarce in expressing GFP - DIA1 ( WT ) ( A ) . However , density of these speckles in GFP - DIA1 ( R1204X ) - expressing appeared lower than that in GFP - DIA1 ( M1190D ) - expressing , which is considered a fully active mutant ( C ) . To evaluate frequency of speckles indicating directional movement , we performed a quantitative ( D ) . number of speckles showing directional movement during 3 continuous planes was counted were normalized by of each , which corresponds to expression level of GFP - tagged . * P = 0 . 0288 * * P = 0 . 0002 Bonferroni ' s post hoc following one - way ANOVA ( WT ; n = 5 , R1204X ; n = 6 , M1190D ; n = 6 ) . Time is in seconds . Scale bars : 5 µm .

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GFP-tagged DIA1(WT), DIA1(R1204X), and DIA1(M1190D) were transfected into XTC cells. XTC cells were spread on the PLL-coated coverslips and time-lapse images were acquired every 300 ms, illuminating the restricted areas near the cell edges, by using a single-molecule speckle microscope. Representative images are shown for DIA1(WT) (A, Movie EV5), DIA1(R1204X) (B, Movie EV6), and DIA1(M1190D) (C, Movie EV7). Circles indicate speckles showing directional movement over a distance of several micrometers. Trajectories of these speckles are also illustrated, and crosses were plotted at the end of the directional movement. Speckles showing directional movement were frequently observed in cells expressing GFP-DIA1(R1204X) (B), whereas such speckles were scarce in cells expressing GFP-DIA1(WT) (A). However, the density of these speckles in GFP-DIA1(R1204X)-expressing cells appeared lower than that in GFP-DIA1(M1190D)-expressing cells, which is considered a fully active mutant (C). To evaluate the frequency of speckles indicating directional movement, we performed a quantitative analysis (D). The number of speckles showing directional movement during 3 continuous planes was counted and were normalized by the fluorescent intensity of each cell, which corresponds to the expression level of the GFP-tagged protein. *P= 0.0288 and **P= 0.0002 Bonferroni's post hoc test following one-way ANOVA (WT; n = 5, R1204X; n = 6, M1190D; n = 6). Time is in seconds. Scale bars: 5 µm.


Cochleae from control , heterozygous Dia1 - KO ( Dia1 - / + ) homozygous Dia1 - KO ( Dia1 - / - ) mice were fixed at 5 of ( A , B ; representative of n = 6 ) . ( A ) Paraffin - embedded 4 μm sections of cochlea were stained with hematoxylin eosin . No hair ( HC ) loss ( either or OHC ) is observed at basal turn of Dia1 - / + or Dia1 - / - cochleae . No obvious differences are observed in spiral ganglion ( SV ) , spiral ligament ( SL ) , stria vascularis ( SV ) , Reissner ' s ( RM ) in Dia1 - / + , or Dia1 - / - mice compared with control mice . Lower panels ( Scale bars : 20 µm ) are magnified of region indicated rectangle in upper panels ( Scale bars : 100 µm ) .

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Cochleae from control, heterozygous Dia1-KO (Dia1-/+) and homozygous Dia1-KO (Dia1-/-) mice were fixed at 5 weeks of age (A, B; representative of n=6).(A) Paraffin-embedded 4 μm sections of cochlea were stained with hematoxylin and eosin. No hair cell (HC) loss (either IHC or OHC) is observed at the basal turn of the Dia1-/+ or Dia1-/-cochleae. No obvious differences are observed in the spiral ganglion (SV), spiral ligament (SL), stria vascularis (SV), and Reissner's membrane (RM) in Dia1-/+, or Dia1-/-mice compared with control mice. Lower panels (Scale bars: 20 µm) are magnified images of the region indicated rectangle in upper panels (Scale bars: 100 µm).


Cochleae from control , heterozygous Dia1 - KO ( Dia1 - / + ) homozygous Dia1 - KO ( Dia1 - / - ) mice were fixed at 5 of ( A , B ; representative of n = 6 ) . ( B ) Basal turns of dissected cochleae were immunostained using a Myo7a Ab followed by an Alexa488 secondary Ab with DAPI as a counterstain . No HC loss ( either or OHC ) is observed in Dia1 - / - mice . Scale bars : 20 µm .

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Cochleae from control, heterozygous Dia1-KO (Dia1-/+) and homozygous Dia1-KO (Dia1-/-) mice were fixed at 5 weeks of age (A, B; representative of n=6).(B) Basal turns of the dissected cochleae were immunostained using a Myo7a Ab followed by an Alexa488 secondary Ab with DAPI as a nuclear counterstain. No HC loss (either IHC or OHC) is observed in Dia1-/-mice. Scale bars: 20 µm.


( C ) ABR thresholds ( 10 , 20 , 40 kHz ) in control Dia1 - / - mice at 5 of ( dB SPL , mean SE ) . No hearing loss was observed in Dia1 - / - mice ( n = 8 ) compared with control mice ( n = 4 ) .

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(C) ABR thresholds (10, 20, 40 kHz) in control and Dia1-/-mice at 5 weeks of age (dB SPL, mean SE). No hearing loss was observed in Dia1-/-mice (n = 8) compared with control mice (n = 4).


( B ) Expression of FLAG - tagged DIA1 ( R1204X ) in inner ear heart of TG , but not WT , mice is confirmed by immunoblotting using a FLAG Ab . Representative of 3 experiments .

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(B) Expression of FLAG-tagged DIA1(R1204X) in the inner ear and heart of TG, but not WT, mice is confirmed by immunoblotting using a FLAG Ab. Representative of 3 experiments.


( C ) Expression of FLAG - tagged DIA1 ( R1204X ) relative to endogenous Dia1 in inner ear , thymus , heart were determined by immunoblotting using a Dia1 / DIA1 Ab ( inner ear ; n = 4 , thymus heart ; n = 3 ) , quantified in inner ear thymus ( * P = 0 . 0226 * * P = 0 . 0016 by two - Student ' s t - ) . Lysates of HEK293 overexpressing equal amounts of GFP - Dia1 GFP - DIA1 ( determined using a GFP Ab : right end ) are detected with equal affinity by Dia1 / DIA1 antibody . Comparable loading of is confirmed using an α − tubulin or GAPDH Ab .

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(C) Expression levels of FLAG-tagged DIA1(R1204X) relative to endogenous Dia1 in the inner ear, thymus, and heart were determined by immunoblotting using a Dia1/DIA1 Ab (inner ear; n = 4, thymus and heart; n = 3), and quantified in the inner ear and thymus (*P= 0.0226 and **P= 0.0016 by two-tailed Student's t-test). Lysates of HEK293 cells overexpressing equal amounts of GFP-Dia1 and GFP-DIA1 (determined using a GFP Ab: right end) are detected with equal affinity by the Dia1/DIA1 antibody. Comparable loading of proteins is confirmed using an α−tubulin or GAPDH Ab.


( A ) , - related click pure tone - burst ( 8 , 16 , 24 , 32 kHz ) ABR thresholds ( dB SPL , mean SE ) in DIA1 ( R1204X ) - TG ( 5 , n = 6 ; 10 , n = 16 ; 25 , n = 42 ) control mice ( 5 , n = 6 ; 10 , n = 16 ; 25 , n = 33 ) . Note progressive hearing loss of DIA1 ( R1204X ) - TG mice starting at high frequency sounds from 10 . * * P = 0 . 0039 ( 24 kHz ) * * P < 0 . 0001 ( 32 kHz ) at 10 * * P < 0 . 0001 ( click ) , * * P < 0 . 0001 ( 8 kHz ) , * * P = 0 . 0001 ( 16 kHz ) , * * P < 0 . 0001 ( 24 kHz ) , * * P < 0 . 0001 ( 32 kHz ) at 25 by Bonferroni ' s post hoc following two - way ANOVA .

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(A), Age-related click and pure tone-burst (8, 16, 24, 32 kHz) ABR thresholds (dB SPL, mean SE) in DIA1(R1204X)-TG (5 weeks, n = 6; 10 weeks, n = 16; 25 weeks, n = 42) and control mice (5 weeks, n = 6; 10 weeks, n = 16; 25 weeks, n = 33). Note the progressive hearing loss of DIA1(R1204X)-TG mice starting at high frequency sounds from 10 weeks. **P= 0.0039 (24 kHz) and **P< 0.0001 (32 kHz) at 10 weeks and **P< 0.0001 (click), **P< 0.0001 (8 kHz), **P= 0.0001 (16 kHz), **P< 0.0001 (24 kHz), and **P< 0.0001 (32 kHz) at 25 weeks by Bonferroni's post hoc test following two-way ANOVA.


( B ) Representative of organ of Corti at basal turn obtained from DIA1 ( R1204X ) - TG control mice at 5 , 10 , 25 of . red circles show OHC losses , respectively . Note OHC - dominant HC loss . Scale bars : 5 µm .

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(B) Representative SEM images of the organ of Corti at the basal turn obtained from DIA1(R1204X)-TG and control mice at 5, 10, and 25 weeks of age. White and red circles show IHC and OHC losses, respectively. Note OHC-dominant HC loss. Scale bars: 5 µm.


( C ) percentages of remaining OHC in each turn in DIA1 ( R1204X ) - TG control mice at 25 of ( n = 5 ) . Schematic illustration shows regions of cochlea divided into three parts : Ap ; apical turn , Md ; middle turn , Bs ; basal turn . * * P = 0 . 0017 ( OHC at Md ) , * * P < 0 . 0001 ( OHC at Bs ) , * P = 0 . 0117 ( at Ap ) , * P = 0 . 0330 ( at Md ) , * * P = 0 . 0003 ( at Bs ) by Bonferroni ' s post hoc following two - way ANOVA .

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(C) The percentages of remaining OHC and IHC in each turn in DIA1(R1204X)-TG and control mice at 25 weeks of age (n = 5). Schematic illustration shows the regions of the cochlea divided into three parts: Ap; apical turn, Md; middle turn, Bs; basal turn. **P= 0.0017 (OHC at Md), **P< 0.0001 (OHC at Bs), *P= 0.0117 (IHC at Ap), *P= 0.0330 (IHC at Md), and **P= 0.0003 (IHC at Bs) by Bonferroni's post hoc test following two-way ANOVA.