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 ) .

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

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 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 .

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

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O B-EXP_ASSAY 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 ratio ; ( 4 technical replicates each ) ; Basal , ( resting OCR ) / ( ECAR + Glu ) ; Max , ( OCR + FCCP ) / ( ECAR + Olig . )

B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-SMALL_MOLECULE 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 .

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

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


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 .

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

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 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 ) .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 images of sorted ESZscan4 _ Emerald and control cells . Scale bar

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


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 .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

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 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 ) ;

O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O 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 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 ) ;

O O O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O

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 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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE 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 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 .

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

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 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 .

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

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 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 .

O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM 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 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 .

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

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 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 ) .

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

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 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 -

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

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 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 .

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

O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O O B-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 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 .

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


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 .

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


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 .

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

O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM 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 nuclear and cytosolic compartments using a standard NP - 40 lysis protocol and immunoblotting with chaperone specific antibodies

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

(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 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

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 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

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

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

O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O

(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 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

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

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

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 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 .

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

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 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 .

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

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 - test * p < 0 . 05 .

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

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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O

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 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 .

O O B-EXP_ASSAY O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) 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 .

O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

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

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 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 ) .

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

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 Test * * * p < 0 . 001 .

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

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 - test * p < 0 . 05 , * * * p < 0 . 001 .

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

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 .

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

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 , 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 .

O O O O B-SUBCELLULAR B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

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

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 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 )

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD B-CELL_TYPE O B-GENEPROD O O O O O O B-GENEPROD O B-SMALL_MOLECULE 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 (E) Fxr1overexpressing neurons (Fxr1/Veh n=10 and Fxr1/TTX n=8)


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 )

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O O O O O O B-GENEPROD O B-SMALL_MOLECULE 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 (F) Gsk3 KO neurons (Gsk3KO/Veh n=10 and Gsk3KO/TTX n=11)


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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

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 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 O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-TISSUE B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O 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 Test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O B-EXP_ASSAY O B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

O, 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 .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O

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 ) 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 ) .

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

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 , 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 .

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

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 , 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 ) .

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

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 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 ) , .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 ) .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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 - test * p < 0 . 05 .

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

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 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 )

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

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 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 )

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

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 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 ) .

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

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 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 .

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

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 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 .

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

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

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

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 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 .

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

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

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

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 , 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 ) .

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

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 , and Fxr1 / SD conditions .

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

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


( 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 ) .

O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) , 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 .

B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O

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 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 . .

O B-GENEPROD O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

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 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 .

O B-GENEPROD O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

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

(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 ) , 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 .

B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-CELL_LINE O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 - / + ) 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 ) .

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

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 - / + ) 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 .

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

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 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 ) .

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

(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 the inner ear and heart of TG , but not WT , mice is confirmed by immunoblotting using a FLAG Ab . Representative of 3 experiments .

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

(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 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 .

O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O B-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O

(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 ) , 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 .

O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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), 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 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 .

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

(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 ) 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 .

O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O

(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.


( D ) DPOAE ( f2 frequency at 8 , 12 , 16 , 20 kHz ) amplitude ( dB SPL , mean SE ) in DIA1 ( R1204X ) - TG ( n = 37 ) and control mice ( n = 30 ) at 25 weeks of age . * P = 0 . 0127 , * * P = 0 . 0029 ( at 12 kHz ) , and * * P = 0 . 0013 ( at 16 kHz ) by Bonferroni ' s post hoc test following two - way ANOVA .

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

(D) DPOAE (f2 frequency at 8, 12, 16, 20 kHz) amplitude (dB SPL, mean SE) in DIA1(R1204X)-TG (n = 37) and control mice (n = 30) at 25 weeks of age. *P= 0.0127, **P= 0.0029 (at 12 kHz), and **P= 0.0013 (at 16 kHz) by Bonferroni's post hoc test following two-way ANOVA.


( E ) High magnification SEM images of OHCs and IHCs at the basal turn of cochlea obtained from DIA1 ( R1204X ) - TG and control mice at 25 weeks of age . Note the sparse and short ( arrow ) stereocilia of OHC , and sparse and fused ( arrowheads ) stereocilia of IHC in DIA1 ( R1204X ) - TG mice . Scale bars : 1 µm in OHCs and 5 µm in IHCs .

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

(E) High magnification SEM images of OHCs and IHCs at the basal turn of cochlea obtained from DIA1(R1204X)-TG and control mice at 25 weeks of age. Note the sparse and short (arrow) stereocilia of OHC, and sparse and fused (arrowheads) stereocilia of IHC in DIA1(R1204X)-TG mice. Scale bars: 1 µm in OHCs and 5 µm in IHCs.


A MMS - induced checkpoint activation is not altered in irc5 - Δ1 cells . The level of Rad53 phosphorylation was analyzed in wild type ( W303 - 1a ) and irc5 - Δ1 ( IL012 ) cells by Western blot with an anti - Rad53 antibody

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

A MMS-induced checkpoint activation is not altered in irc5-Δ1 cells. The level of Rad53 phosphorylation was analyzed in wild type (W303-1a) and irc5-Δ1 (IL012) cells by Western blot with an anti-Rad53 antibody


B Prolonged checkpoint activation in irc5 - Δ1 cells during recovery from MMS treatment . Rad53 phosphorylation status was analyzed in wild type ( W303 - 1a ) and irc5 - Δ1 ( IL012 ) cells by Western blot with an anti - Rad53 antibody ; M , MMS

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

B Prolonged checkpoint activation in irc5-Δ1 cells during recovery from MMS treatment. Rad53 phosphorylation status was analyzed in wild type (W303-1a) and irc5-Δ1 (IL012) cells by Western blot with an anti-Rad53 antibody; M, MMS


C Lack of Irc5 results in an elevated number of cells with Rad52 - YFP foci . At indicated time points samples of wild type ( W3749 - 14C ) and irc5 - Δ1 ( TB039 ) cells were collected , washed and processed for microscopic analysis . Error bars represent mean value ± standard deviations of mean ( n = 5 ) . Unpaired t - test was used to calculate the P - value . Representative microscopic photos of cells are shown . Scale bars : 5 μm

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

C Lack of Irc5 results in an elevated number of cells with Rad52-YFP foci. At indicated time points samples of wild type (W3749-14C) and irc5-Δ1 (TB039) cells were collected, washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=5). Unpaired t-test was used to calculate the P-value. Representative microscopic photos of cells are shown. Scale bars: 5 μm


D Disruption of IRC5 leads to accumulation of ssDNA - containing lesions . ChEC analysis of wild type ( BYR52MN ) and irc5 - Δ1 ( TB040 ) cells cultured in the presence or absence of 0 . 03 % MMS for 2 hours ( n = 3 )

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

D Disruption of IRC5 leads to accumulation of ssDNA-containing lesions. ChEC analysis of wild type (BYR52MN) and irc5-Δ1 (TB040) cells cultured in the presence or absence of 0.03% MMS for 2 hours (n=3)


F Increased levels of bright Rfa1 - YFP foci in cells lacking Irc5 . At indicated time points culture samples of wild type ( W3775 - 12C ) and irc5 - Δ1 ( TB041 ) cells were collected , washed and processed for microscopic analysis . Error bars represent mean value ± standard deviations of mean ( n = 5 ) . Unpaired t - test was used to calculate the P - value . Representative microscopic photos of cells are shown . Scale bars : 5

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

F Increased levels of bright Rfa1-YFP foci in cells lacking Irc5. At indicated time points culture samples of wild type (W3775-12C) and irc5-Δ1 (TB041) cells were collected, washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=5). Unpaired t-test was used to calculate the P-value. Representative microscopic photos of cells are shown. Scale bars: 5 μm


A The irc5 - Δ1 mutant exhibits S - phase delay in the presence of MMS . DNA content of wild type ( W303 - 1a ) and irc5 - Δ1 ( IL012 ) cells was measured by FACS and plotted as histograms

O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O

A The irc5-Δ1 mutant exhibits S-phase delay in the presence of MMS. DNA content of wild type (W303-1a) and irc5-Δ1 (IL012) cells was measured by FACS and plotted as histograms


B Completion of DNA replication is delayed in the irc5 - Δ1 mutant after MMS treatment . Intact chromosomes were isolated from wild type ( W303 - 1a ) and irc5 - Δ1 ( IL012 ) cells and processed for PFGE . M , 0 . 03 % MMS

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

B Completion of DNA replication is delayed in the irc5-Δ1 mutant after MMS treatment. Intact chromosomes were isolated from wild type (W303-1a) and irc5-Δ1 (IL012) cells and processed for PFGE. M, 0.03% MMS


D Samples used for PFGE in B were processed for FACS analysis

O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O


A Lack of Irc5 translocase activity results in elevated number of cells with Rfa1 - YFP foci . At indicated time points samples of wild type ( TB042 ) and irc5DAEA ( TB043 ) cells were collected , washed and processed for microscopic analysis . Error bars represent mean value ± standard deviations of mean ( n = 5 ) . Unpaired t - test was used to calculate the P -

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

A Lack of Irc5 translocase activity results in elevated number of cells with Rfa1-YFP foci. At indicated time points samples of wild type (TB042) and irc5DAEA (TB043) cells were collected, washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=5). Unpaired t-test was used to calculate the P-value


B Increased levels of bright Rad52 - YFP foci in cells expressing Irc5DAEA lacking ATPase activity . At indicated time points samples of wild type ( TB044 ) and irc5DAEA ( TB045 ) cells were collected , washed and processed for microscopic analysis . Error bars represent mean value ± standard deviations of mean ( n = 5 ) . Unpaired t - test was used to calculate the P -

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

B Increased levels of bright Rad52-YFP foci in cells expressing Irc5DAEA lacking ATPase activity. At indicated time points samples of wild type (TB044) and irc5DAEA (TB045) cells were collected, washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=5). Unpaired t-test was used to calculate the P-value


C Translocase activity of Irc5 is important for completion of DNA replication after MMS treatment . PFGE was performed as in Fig 2B using wild type ( EMD09 ) and irc5DAEA ( EMD10 )

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

C Translocase activity of Irc5 is important for completion of DNA replication after MMS treatment. PFGE was performed as in Fig 2B using wild type (EMD09) and irc5DAEA (EMD10) strains


E Samples used for PFGE in C were processed for FACS analysis


A , B ChIP - qPCR analysis of Scc1 association with early replication origin ARS305 or ARS607 . PK - tagged Scc1 interaction with chromatin was monitored in wild type ( JC1513 ) and irc5Δ - 1 ( TB064 ) cells . Error bars represent mean value ± standard deviations of mean ( n = 3 ) . ARS305 + 7 kb locus was used as a control for cohesin - free chromosomal region . POA1 was used as a control locus for the chromosomal region that is bound by cohesin but is located ~ 30 . 000 bp from the nearest replication origin

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

A, B ChIP-qPCR analysis of Scc1 association with early replication origin ARS305 or ARS607. PK-tagged Scc1 interaction with chromatin was monitored in wild type (JC1513) and irc5Δ-1 (TB064) cells. Error bars represent mean value ± standard deviations of mean (n=3). ARS305 + 7 kb locus was used as a control for cohesin-free chromosomal region. POA1 was used as a control locus for the chromosomal region that is bound by cohesin but is located ~ 30.000 bp from the nearest replication origin


C , D ChIP - qPCR analysis of Pol2 association with early replication origin ARS305 or ARS607 as well as late replication origin ARS501 . HA - tagged Pol2 interaction with chromatin was monitored in wild type ( TB065 ) and irc5Δ - 1 ( TB066 ) cells . Error bars represent mean value ± standard deviations of mean ( n = 3 )

O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

C, D ChIP-qPCR analysis of Pol2 association with early replication origin ARS305 or ARS607 as well as late replication origin ARS501. HA-tagged Pol2 interaction with chromatin was monitored in wild type (TB065) and irc5Δ-1 (TB066) cells. Error bars represent mean value ± standard deviations of mean (n=3)


A , B ChIP - qPCR analysis of Scc1 association with early replication origin ARS305 or ARS607 . HA - tagged Scc1 interaction with chromatin was monitored in wild type ( Y39 ) and scc2 - 4 ( Y4366 ) cells . Error bars represent mean value ± standard deviations of mean ( n = 3 )

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

A, B ChIP-qPCR analysis of Scc1 association with early replication origin ARS305 or ARS607. HA-tagged Scc1 interaction with chromatin was monitored in wild type (Y39) and scc2-4 (Y4366) cells. Error bars represent mean value ± standard deviations of mean (n=3)


C , D ChIP - qPCR analysis of Pol2 association with early replication origin ARS305 or ARS607 . HA - tagged Pol2 interaction with chromatin was monitored in wild type ( TB067 ) and scc2 - 4 ( TB068 ) cells . Error bars represent mean value ± standard deviations of mean ( n = 3 )

O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

C, D ChIP-qPCR analysis of Pol2 association with early replication origin ARS305 or ARS607. HA-tagged Pol2 interaction with chromatin was monitored in wild type (TB067) and scc2-4 (TB068) cells. Error bars represent mean value ± standard deviations of mean (n=3)


E Cohesin loading complex is required for completion of chromosome replication after MMS treatment . Intact chromosomes were isolated from wild type ( Y39 ) and scc2 - 4 ( Y4366 ) cells cultivated at permissive temperature ( 25 ° C ) and processed for PFGE . M ,

O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O

E Cohesin loading complex is required for completion of chromosome replication after MMS treatment. Intact chromosomes were isolated from wild type (Y39) and scc2-4 (Y4366) cells cultivated at permissive temperature (25°C) and processed for PFGE. M, MMS