Automated microinjection was performed on organotypic slices of mouse E14 . 5 dorsal telencephalon using Dx - A488 along with mRNA for RFP Organotypic slices of mouse E14 . 5 dorsal telencephalon injected with Dextran - A488 ( green ) and RFP poly - A + - mRNA . After 24h in culture the progeny expresses RFP ( magenta ; blue : DAPI , n = 17 cells injected , 8 RFP positive ) . Percentage of RFP - positive and RFP - negative cells ( n = 17 cells injected ) .

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Automated microinjection was performed on organotypic slices of mouse E14.5 dorsal telencephalon using Dx-A488 along with mRNA for RFP Organotypic slices of mouse E14.5 dorsal telencephalon injected with Dextran-A488 (green) and RFP poly-A+-mRNA. After 24h in culture the progeny expresses RFP (magenta; blue: DAPI, n = 17 cells injected, 8 RFP positive). Percentage of RFP-positive and RFP-negative cells (n = 17 cells injected).


Automated microinjection was performed on organotypic slices of mouse E14 . 5 - E15 . 5 dorsal telencephalon using a solution containing Dextran - A555 ( not shown ) and Alexa488 ( green ) . Slices were fixed after microinjection ( n = 71 cells total ) and were stained for Tbr2 ( white , H - J ) . ( A - C ) Representative examples of a non - coupled cell ( A , cartoon on the right ) , a 2 - cell cluster ( B , cartoon on the right ) and a 5 - cell cluster ( C , cartoon on the right ) . The asterisks indicate the Dx - A555 - positive , microinjected cell . Percentage of microinjected cells found in a coupled cluster ( coupled , green ) . Cluster size , expressed as % of total . Distribution of coupled cells expressed as the distance from the ventricular surface ( 0 µm = ventricle surface . Distribution of coupled cells divided into 11 bins and expressed as % of total . the images on the left are maximum intensity projections ( MIP ) of 18 , 17 , 23 , 12 and 30 - focal planes ,

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Automated microinjection was performed on organotypic slices of mouse E14.5 - E15.5 dorsal telencephalon using a solution containing Dextran-A555 (not shown) and Alexa488 (green). Slices were fixed after microinjection (n = 71 cells total) and were stained for Tbr2 (white, H-J). (A-C) Representative examples of a non-coupled cell (A, cartoon on the right), a 2-cell cluster (B, cartoon on the right) and a 5-cell cluster (C, cartoon on the right). The asterisks indicate the Dx-A555-positive, microinjected cell. Percentage of microinjected cells found in a coupled cluster (coupled, green). Cluster size, expressed as % of total. Distribution of coupled cells expressed as the distance from the ventricular surface (0 µm = ventricle surface. Distribution of coupled cells divided into 11 bins and expressed as % of total. the images on the left are maximum intensity projections (MIP) of 18, 17, 23, 12 and 30-focal planes, respectively


A representative picture of a 2 - cell cluster ( cartoon in the middle , nuclei are numbered from 1 to 2 . Microinjected cells are stained for Tbr2 . A representative picture of ( H ) a 2 - cell cluster ( cartoon in the middle , nuclei are numbered from 1 to 2 ) and ( I ) a 7 - cell cluster ( cartoon in the middle , nuclei are numbered from 1 to 7 ) , the latter containing one Tbr2 positive cell . Microinjected cells are stained for Tbr2 . The asterisks indicate the Dx - A555 - positive , microinjected cell . comparison of Tbr2 - positive cells ( expressed as % of total ) among all the cells in the VZ ( VZ ; n = 1003 from 4 different confocal images from 3 independent experiments ; error bar represents standard deviation ) and in coupled clusters in the VZ ( coupled clusters , VZ ; n = 104 from two independent experiments ) . Coupled cells were scored based one presence / absence of apical and / or basal polarity cues , and results are expressed as % of total . the images on the right are single optical sections corresponding to the nucleus ( white dotted line ) ; scale bars are 20 µm for MIP and 10 µm for single focal planes . VZ , ventricular zone ; SVZ , subventricular zone ; CP , cortical plate .

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A representative picture of a 2-cell cluster (cartoon in the middle, nuclei are numbered from 1 to 2. Microinjected cells are stained for Tbr2. A representative picture of (H) a 2-cell cluster (cartoon in the middle, nuclei are numbered from 1 to 2) and (I) a 7-cell cluster (cartoon in the middle, nuclei are numbered from 1 to 7), the latter containing one Tbr2 positive cell. Microinjected cells are stained for Tbr2. The asterisks indicate the Dx-A555-positive, microinjected cell. comparison of Tbr2-positive cells (expressed as % of total) among all the cells in the VZ (VZ; n=1003 from 4 different confocal images from 3 independent experiments; error bar represents standard deviation) and in coupled clusters in the VZ (coupled clusters, VZ; n= 104 from two independent experiments). Coupled cells were scored based one presence/absence of apical and/or basal polarity cues, and results are expressed as % of total. the images on the right are single optical sections corresponding to the nucleus (white dotted line); scale bars are 20 µm for MIP and 10 µm for single focal planes. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.


Automated microinjection of mouse neurons . Automated microinjection was performed on organotypic slices of mouse E16 . 5 dorsal telencephalon using Dx - A555 Phase contrast image of automated microinjection into mouse neurons . Scale bar is 100 µm .

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Automated microinjection of mouse neurons. Automated microinjection was performed on organotypic slices of mouse E16.5 dorsal telencephalon using Dx-A555 Phase contrast image of automated microinjection into mouse neurons. Scale bar is 100 µm.


Automated microinjection of mouse neurons . Automated microinjection was performed on organotypic slices of mouse E16 . 5 12 wpc dorsal telencephalon using Dx - A555 ( not shown ) and LY ( green , Slices were fixed after microinjection and stained for Reelin ( white , C ) Left : overview image of a mouse microinjected area ( arrow indicates a pyramidal neuron , arrowhead indicates a Cajal - Retzius neuron ; image is MIP of 20 focal planes ) . Microinjected neurons show different morphology and positivity for the Cajal - Retzius marker Reelin ( white ) . Right , top row : the pyramidal neuron indicated with an arrow in the overview image is negative for Reelin . Right , bottom row : the Cajal - Retzius neuron indicated with an arrowhead in the overview image is positive for Reelin . Images on the top and bottom row left ( LY MIP ) are a MIP of 2 and 3 focal planes , respectively . The three images on the top and bottom right ( LY , Reelin and DAPI ) are single optical sections . Scale bar is 20 µm for MIP and 10 µm for single focal planes . VZ , ventricular zone ; SVZ , subventricular zone ; CP , cortical plate .

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Automated microinjection of mouse neurons. Automated microinjection was performed on organotypic slices of mouse E16.5 12 wpc dorsal telencephalon using Dx-A555 (not shown) and LY (green, Slices were fixed after microinjection and stained for Reelin (white, C) Left: overview image of a mouse microinjected area (arrow indicates a pyramidal neuron, arrowhead indicates a Cajal-Retzius neuron; image is MIP of 20 focal planes). Microinjected neurons show different morphology and positivity for the Cajal-Retzius marker Reelin (white). Right, top row: the pyramidal neuron indicated with an arrow in the overview image is negative for Reelin. Right, bottom row: the Cajal-Retzius neuron indicated with an arrowhead in the overview image is positive for Reelin. Images on the top and bottom row left (LY MIP) are a MIP of 2 and 3 focal planes, respectively. The three images on the top and bottom right (LY, Reelin and DAPI) are single optical sections. Scale bar is 20 µm for MIP and 10 µm for single focal planes. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.


Automated microinjection of mouse neurons . Automated microinjection was performed on organotypic slices of mouse E16 . 5 12 wpc dorsal telencephalon using Dx - A555 ( not shown ) and LY ( green Slices were fixed after microinjection and stained for SatB2 ( white , Overview image of a mouse microinjected area . The arrow indicates a pyramidal neuron ; the image is MIP of 30 focal planes , scale bar : 50 µm . Row on the right : the microinjected neuron with pyramidal morphology indicated with an arrow in the overview is positive for the upper - layers marker SatB2 ( white ) . Images are single focal planes . Scale bar is 10 µm . Proportion of neurons positive or negative for Reelin ( n = 12 cells total ) . Proportion of neurons positive or negative for SatB2 ( n = 39 cells in total ) . VZ , ventricular zone ; SVZ , subventricular zone ; CP , cortical plate .

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Automated microinjection of mouse neurons. Automated microinjection was performed on organotypic slices of mouse E16.5 12 wpc dorsal telencephalon using Dx-A555 (not shown) and LY (green Slices were fixed after microinjection and stained for SatB2 (white, Overview image of a mouse microinjected area. The arrow indicates a pyramidal neuron; the image is MIP of 30 focal planes, scale bar: 50 µm. Row on the right: the microinjected neuron with pyramidal morphology indicated with an arrow in the overview is positive for the upper-layers marker SatB2 (white). Images are single focal planes. Scale bar is 10 µm. Proportion of neurons positive or negative for Reelin (n = 12 cells total). Proportion of neurons positive or negative for SatB2 (n = 39 cells in total). VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.


Automated microinjection of human ( G , H ) neurons . Automated microinjection was performed on organotypic slices of human 12 wpc dorsal telencephalon using Dx - A555 ( not shown ) and LY ( green , Slices were fixed after microinjection Microinjected neurons in the developing human telencephalon . Top , MIP of 20 focal planes ( scale bar : 50µm ) . Bottom : high magnification ( scale bar : 10 µm ) . VZ , ventricular zone ; SVZ , subventricular zone ; CP , cortical plate .

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Automated microinjection of human (G,H) neurons. Automated microinjection was performed on organotypic slices of human 12 wpc dorsal telencephalon using Dx-A555 (not shown) and LY (green, Slices were fixed after microinjection Microinjected neurons in the developing human telencephalon. Top, MIP of 20 focal planes (scale bar: 50µm). Bottom: high magnification (scale bar: 10 µm). VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.


D Distributions of pairwise Ka / Ks ( nonsynonymous to synonymous nucleotide substitution rates ) ratios between PIWI duplicates in arthropods ( n = 17 in Drosophila and n = 37 in others , Mann - Whitney U test , * P < 0 . 05 ) . The center line of the boxplots represents median value , the bounds of the box represent 75th and 25th percentile , and the whiskers represent maximum and minimum value .

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D Distributions of pairwise Ka/Ks (nonsynonymous to synonymous nucleotide substitution rates) ratios between PIWI duplicates in arthropods (n = 17 in Drosophila and n = 37 in others, Mann-Whitney U test, *P<0.05). The center line of the boxplots represents median value, the bounds of the box represent 75th and 25th percentile, and the whiskers represent maximum and minimum value.


A PIWI genes are present in the locust brain . Rp49 served as a loading control ( n = 4 biological replicates ) . Data information : data are presented as mean ± SEM .

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A PIWI genes are present in the locust brain. Rp49 served as a loading control (n=4 biological replicates). Data information: data are presented as mean ± SEM.


C The fluorescence signals of PIWI proteins were determined in locust brains . Hochest33342 was used to label cell nuclei in locust brains . The scale bars represent 20 µm .

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C The fluorescence signals of PIWI proteins were determined in locust brains. Hochest33342 was used to label cell nuclei in locust brains. The scale bars represent 20 µm.


G Food intake by dsPiwi1 - treated locusts and dsGFP - treated control locusts within 1 h ( n = 5 biological replicates ) , * * * P < 0 . 001 ( Student ' s t - test ) . Data information : All assays were performed on fourth - instar locusts .

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G Food intake by dsPiwi1-treated locusts and dsGFP-treated control locusts within 1 h (n=5 biological replicates) , ***P<0.001 (Student's t-test). Data information: All assays were performed on fourth-instar locusts.


C Representative images of Nile red staining of lipid droplets in the fat bodies of dsPiwi1 - treated locusts and dsGFP - treated control locusts ( n = 5 biological replicates ) . The scale bars represent 50 µm . D Quantification of lipid droplet staining in dsPiwi1 - treated locusts and dsGFP - treated control locusts ( n = 5 biological replicates ) . E Data information : All data are presented as the mean ± SEM ( Student ' s t - test , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant ) . All assays were performed on four - day - old fourth - instar locusts .

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C Representative images of Nile red staining of lipid droplets in the fat bodies of dsPiwi1-treated locusts and dsGFP-treated control locusts (n=5 biological replicates). The scale bars represent 50 µm. D Quantification of lipid droplet staining in dsPiwi1-treated locusts and dsGFP-treated control locusts (n=5 biological replicates). E Data information: All data are presented as the mean ± SEM (Student's t-test, **P<0.01, ***P<0.001, ns: not significant). All assays were performed on four-day-old fourth-instar locusts.


E , F Measurements of glycogen ( n = 5 biological replicates ) and TAGs ( n = 10 biological replicates ) in fat bodies . Data information : All data are presented as the mean ± SEM ( Student ' s t - test , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant ) . All assays were performed on four - day - old fourth - instar locusts .

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E, F Measurements of glycogen (n=5 biological replicates) and TAGs (n=10 biological replicates) in fat bodies. Data information: All data are presented as the mean ± SEM (Student's t-test, **P<0.01, ***P<0.001, ns: not significant). All assays were performed on four-day-old fourth-instar locusts.


B Food intake within 1 h following treatment with candidate genes ( dsNPF1 - treated , n = 4 biological replicates ; dsCG10621 - treated , n = 5 biological replicates ; dsMFS3 - treated , n = 5 biological replicates ) , the data are shown as the mean ± SEM ( Student ' s t - test , * * P < 0 . 01 ) .

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B Food intake within 1 h following treatment with candidate genes (dsNPF1-treated, n=4 biological replicates; dsCG10621-treated, n=5 biological replicates; dsMFS3-treated, n=5 biological replicates), the data are shown as the mean ± SEM (Student's t-test, **P<0.01).


D , E Representative images ( D ) and quantification ( E ) of Nile red staining of fat body lipid droplets in dsNPF1 - treated locusts and dsGFP - treated controls ( n = 5 biological replicates ) . The scale bars represent 50 µm , the data are shown as the mean ± SEM ( Student ' s t - test , * * P < 0 . 01 ) .

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D, E Representative images (D) and quantification (E) of Nile red staining of fat body lipid droplets in dsNPF1-treated locusts and dsGFP-treated controls (n=5 biological replicates). The scale bars represent 50 µm, the data are shown as the mean ± SEM (Student's t-test, **P<0.01).


G Food intake within 1 h in the food instake assays ( n = 5 biological replicates ) , the data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * * P < 0 . 001 ) . H Food intake within 2 h in the food instake assays ( n = 5 biological replicates ) . Five locusts were included in each replicate , the data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 ) .

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G Food intake within 1 h in the food instake assays (n=5 biological replicates), the data are shown as the mean ± SEM (Student's t-test, *P<0.05, ***P<0.001) . H Food intake within 2 h in the food instake assays (n=5 biological replicates). Five locusts were included in each replicate, the data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01).


B mRNA levels of differentially expressed piRNAs located within the flanking region of the NPF1 gene in dsPiwi1 - treated locusts and dsGFP - treated control locusts ( n = 5 biological replicates ) . Data information : The qPCR data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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B mRNA levels of differentially expressed piRNAs located within the flanking region of the NPF1 gene in dsPiwi1-treated locusts and dsGFP-treated control locusts (n=5 biological replicates). Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).


C The mRNA expression of NPF1 was measured by qPCR after a mixture of inhibitors or mimics of piRs - 3 - I2 and piRs - 3 - I3 was injected ( n = 5 biological replicates ) . Data information : The qPCR data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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C The mRNA expression of NPF1 was measured by qPCR after a mixture of inhibitors or mimics of piRs-3-I2 and piRs-3-I3 was injected (n=5 biological replicates). Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).


E Body weight measurement of locusts administered inhibitors ( NC , n = 25 locusts ; Inhibitors - piRs - 3 - I2 + I3 , n = 23 locusts ) and mimics ( NC , n = 37 locusts ; Inhibitors - piRs - 3 - I2 + I3 , n = 39 locusts ) of piRs - 3 - I2 and piRs - 3 - I3 . The center line of the boxplots represents median value , the bounds of the box represent 75th and 25th percentile , and the whiskers represent maximum and minimum value .

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E Body weight measurement of locusts administered inhibitors (NC, n=25 locusts; Inhibitors- piRs-3-I2+I3, n=23 locusts) and mimics (NC, n=37 locusts; Inhibitors-piRs-3-I2+I3, n=39 locusts) of piRs-3-I2 and piRs-3-I3. The center line of the boxplots represents median value, the bounds of the box represent 75th and 25th percentile, and the whiskers represent maximum and minimum value.


F RNA - binding protein immunoprecipitation assay of piRs - 3 - I2 and piRs - 3 - I3 in Piwi1 immunoprecipitates from brain tissue extracts ( n = 6 biological replicates ) . Normal mouse IgG was used as a negative control .

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

F RNA-binding protein immunoprecipitation assay of piRs-3-I2 and piRs-3-I3 in Piwi1 immunoprecipitates from brain tissue extracts (n=6 biological replicates). Normal mouse IgG was used as a negative control.


A U6 RNA was used as a nuclear RNA control ( relative value set to 1 in the nuclear fraction ) , and glycine tRNA was used as the cytoplasmic RNA control ( relative value set to 1 in the cytoplasmic fraction ) ( n = 4 biological replicates ) , U6 and tRNA - Gly was used as an endogenous control .

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

A U6 RNA was used as a nuclear RNA control (relative value set to 1 in the nuclear fraction), and glycine tRNA was used as the cytoplasmic RNA control (relative value set to 1 in the cytoplasmic fraction) (n=4 biological replicates), U6 and tRNA-Gly was used as an endogenous control.


B Relative expressions of piRs - 3 - I2 and piRs - 3 - I3 determined by qPCR and high - throughput sequencing in the nucleus ( n = 3 biological replicates ) . The brain tissues were homogenized and then divided into two parts of equal volume ; one was prepared for high - throughput sequencing and the other was used for piRNA quantification in nuclear and cytoplasmic fractions after subcellular RNA fractionation . The relative piRNA expression in nucleus was determined by multiplying the relative nuclear piRNA fraction estimate by qPCR quantification by the normalized count of piRNA in high - throughput sequencing libraries . Data information : The qPCR data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

B Relative expressions of piRs-3-I2 and piRs-3-I3 determined by qPCR and high-throughput sequencing in the nucleus (n=3 biological replicates). The brain tissues were homogenized and then divided into two parts of equal volume; one was prepared for high-throughput sequencing and the other was used for piRNA quantification in nuclear and cytoplasmic fractions after subcellular RNA fractionation. The relative piRNA expression in nucleus was determined by multiplying the relative nuclear piRNA fraction estimate by qPCR quantification by the normalized count of piRNA in high-throughput sequencing libraries. Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).


C Expression level of pre - NPF1 in the nucleus after a mixture of inhibitors ( piRs - 3 - I2 and piRs - 3 - I3 ) was injected ( n = 4 biological replicates ) . D qPCR expression analyses of pre - NPF1 . The precursor mRNA expression level was measured by qPCR after the dsPiwi1 treatments ( n = 6 biological replicates ) . E Data information : The qPCR data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

C Expression level of pre-NPF1 in the nucleus after a mixture of inhibitors (piRs-3-I2 and piRs-3-I3) was injected (n=4 biological replicates). D qPCR expression analyses of pre-NPF1. The precursor mRNA expression level was measured by qPCR after the dsPiwi1 treatments (n=6 biological replicates). E Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).


F Analysis was performed to amplify pre - NPF1 and piRs - 3 - I3 in hnRNP F / H immunoprecipitates from extracts of brain tissue treated with piRs - 3 - I3 mimics compared to the negative controls ( pre - NPF1 n = 3 biological replicates ; piRs - 3 - I3 n = 4 biological replicates ) . G Analysis was performed to amplify pre - NPF1 in U2AF65 immunoprecipitates from extracts of brain tissue treated with piRs - 3 - I3 mimics compared to the negative controls ( pre - NPF1 n = 3 biological replicates ) .

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

F Analysis was performed to amplify pre-NPF1 and piRs-3-I3 in hnRNP F/H immunoprecipitates from extracts of brain tissue treated with piRs-3-I3 mimics compared to the negative controls (pre-NPF1 n=3 biological replicates; piRs-3-I3 n=4 biological replicates). G Analysis was performed to amplify pre-NPF1 in U2AF65 immunoprecipitates from extracts of brain tissue treated with piRs-3-I3 mimics compared to the negative controls (pre-NPF1 n=3 biological replicates). H


J Expression level of the mature NPF1 gene and pre - NPF1 in 293T cells transfected with pre - NPF1 and mutated pre - NPF1 ( n = 5 biological replicates ) . NC means negative control . The center line of the boxplots represents median value , the bounds of the box represent 75th and 25th percentile , and the whiskers represent maximum and minimum value . Data information : The qPCR data are shown as the mean ± SEM ( Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

J Expression level of the mature NPF1 gene and pre-NPF1 in 293T cells transfected with pre-NPF1 and mutated pre-NPF1 (n=5 biological replicates). NC means negative control. The center line of the boxplots represents median value, the bounds of the box represent 75th and 25th percentile, and the whiskers represent maximum and minimum value. Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).


( A ) Gating strategy used to identify ILCs and ILC subsets in humans ( hILCs ) .

O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O B-ORGANISM O B-CELL_TYPE O O

(A) Gating strategy used to identify ILCs and ILC subsets in humans (hILCs).


( B ) Representative histogram and summary of the percentage and of the gMFI of c - Maf expression in hILC subsets ( ANOVA analysis : F ( 2 , 18 ) = 305 . 4 , * * * * p < 0 . 0001 ) . Data information : Each dot represents the expression level in one donor ( n = 7 ) . Bars represent mean ± SEM .

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

(B) Representative histogram and summary of the percentage and of the gMFI of c-Maf expression in hILC subsets (ANOVA analysis: F(2,18)=305.4, ****p<0.0001). Data information: Each dot represents the expression level in one donor (n=7). Bars represent mean ± SEM.


( C Gating strategy for cKitlow and cKithigh ILC2s ( C )

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

(C Gating strategy for cKitlow and cKithigh ILC2s (C)


D ) expression level ( percentage and gMFI ) of c - Maf in cKitlow and cKithigh ILC2s ( D ) ( paired T tests , * * * * p < 0 . 0001 ) . Data information : Each dot represents the expression level in one donor ( n = 7 ) . Bars represent mean ± SEM .

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

D) expression level (percentage and gMFI) of c-Maf in cKitlow and cKithigh ILC2s (D) (paired T tests, ****p<0.0001). Data information: Each dot represents the expression level in one donor (n=7). Bars represent mean ± SEM.


( G ) Human ILC2 sorted from peripheral blood and in vitro expanded were transduced with Luc - sh or Maf - sh as in ( C ) . hILC2s were then stimulated for 48h with IL - 33 and the concentration of the indicated cytokines in the supernatants was measured ( Multiple Mann - Whitney tests , * p < 0 . 05 , * * p < 0 . 01 ) Bars represent mean ± SEM . Data information : Each dot represents data from one individual mouse or from one donor .

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

(G) Human ILC2 sorted from peripheral blood and in vitro expanded were transduced with Luc-sh or Maf-sh as in (C). hILC2s were then stimulated for 48h with IL-33 and the concentration of the indicated cytokines in the supernatants was measured (Multiple Mann-Whitney tests, *p<0.05, **p<0.01) Bars represent mean ± SEM. Data information: Each dot represents data from one individual mouse or from one donor.


( E ) ChIP - qPCR analysis of c - Maf occupancy on IL - 4 , IL - 5 and IL - 13 promoters in untreated mouse and human ILC2s ( IL - 2 stimulation ) and in mouse and human ILC2s treated with IL - 33 ( n = 3 biological replicates ) .

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

(E) ChIP-qPCR analysis of c-Maf occupancy on IL-4, IL-5 and IL-13 promoters in untreated mouse and human ILC2s (IL-2 stimulation) and in mouse and human ILC2s treated with IL-33 (n=3 biological replicates).


( C ) Representative histograms of c - Maf and GATA3 expression in ILC2s from adult PBMC or cord blood cells and percentages of c - Maf + and GATA3 + ILC2s from adult PBMC or cord blood ( n = 5 different donors ; T test , * * * * p < 0 . 0001 ) .

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

(C) Representative histograms of c-Maf and GATA3 expression in ILC2s from adult PBMC or cord blood cells and percentages of c-Maf+ and GATA3+ ILC2s from adult PBMC or cord blood (n=5 different donors; T test, ****p<0.0001).


( D ) ILC2 expression of the indicated markers . Each dot represent sample from one donor . Bars represent mean ± SEM .

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

(D) ILC2 expression of the indicated markers. Each dot represent sample from one donor. Bars represent mean ± SEM.


( E , F ) c - Maf quantification by qPCR ( E ) and cytokine concentration ( F ) in the supernatants of adult and cord blood ILC2s unstimulated , stimulated for 3h with PMA - Ionomycin ( 3h stim ) and stimulated for an overnight with IL - 33 and then 3h with PMA - Ionomycin ( o . n . stim ) . ILC2s were sorted from 3 different donors . ( E ) Multiple T tests , unstimulated : p = 0 . 000101 ; 3h stim : p = 0 . 0036 . ( F ) Multiple T tests , IL - 13 : p = 0 . 022 ; IL - 5 : p = 0 . 015 ( * p < 0 . 05 ) . Bars represent mean ± SEM .

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

(E, F) c-Maf quantification by qPCR (E) and cytokine concentration (F) in the supernatants of adult and cord blood ILC2s unstimulated, stimulated for 3h with PMA-Ionomycin (3h stim) and stimulated for an overnight with IL-33 and then 3h with PMA-Ionomycin (o.n. stim). ILC2s were sorted from 3 different donors. (E) Multiple T tests, unstimulated: p=0.000101; 3h stim: p=0.0036. (F) Multiple T tests, IL-13: p=0.022; IL-5: p=0.015 (*p<0.05). Bars represent mean ± SEM.


C - Left : Percentage of total live cells expressing ChAT - eGFP derived from IWAT ( including inguinal lymph node ; n = 7 ) , IWAT alone ( n = 8 ) and lymph node alone ( n = 8 ) . Right : Proportion of ChAT - eGFP + cells comprised by T cells , B cells and MΦ in each tissue type .

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

C - Left: Percentage of total live cells expressing ChAT-eGFP derived from IWAT (including inguinal lymph node; n = 7), IWAT alone (n = 8) and lymph node alone (n = 8). Right: Proportion of ChAT-eGFP+ cells comprised by T cells, B cells and MΦ in each tissue type.


F - Left : relative mRNA expression of Chat in ChATfl / fl and ChATfl / fl ; Vav - iCre IWAT ( n = 9 ) . Chat expression was analyzed by qPCR and normalized to levels of Tbp using the 2 - ΔΔCt method . Right : quantification by LC / MS - MS of Ach concentration in SVF isolated from ChATfl / fl and ChATfl / fl ; Vav - iCre IWAT ( n = 6 ) .

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

F - Left: relative mRNA expression of Chat in ChATfl/fl and ChATfl/fl;Vav-iCre IWAT (n = 9). Chat expression was analyzed by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method. Right: quantification by LC/MS-MS of Ach concentration in SVF isolated from ChATfl/fl and ChATfl/fl;Vav-iCre IWAT (n = 6).


Body weight ( C ) of ChATfl / fl ; LysM - Cre ( n = 7 for ChATfl / fl , n = 8 for Cre ChATfl / fl ; Cd4 - Cre ( n = 6 for ChATfl / fl and n = 7 for Cre ChATfl / fl ; Mb1 - Cre ( n = 12 in C , n = 9 for ChATfl / fl , n = 8 for Cre and littermate ChATfl / fl mice housed at RT .

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

Body weight (C) of ChATfl/fl;LysM-Cre (n = 7 for ChATfl/fl, n = 8 for Cre ChATfl/fl;Cd4-Cre (n = 6 for ChATfl/fl and n = 7 for Cre ChATfl/fl;Mb1-Cre (n = 12 in C, n = 9 for ChATfl/fl, n = 8 for Cre and littermate ChATfl/fl mice housed at RT.


E - G - mRNA expression of Chrna2 and thermogenic genes in IWAT of ChATfl / fl ; LysM - Cre ( n = 24 for ChATfl / fl _ RT , n = 25 for Cre _ RT , n = 20 for ChATfl / fl _ CE , n = 20 for Cre _ CE ) ( E ) , ChATfl / fl ; Cd4 - Cre ( n = 17 for ChATfl / fl _ RT , n = 22 - 23 for Cre _ RT , n = 19 for ChATfl / fl _ CE , n = 22 for Cre _ CE ) ( F ) , ChATfl / fl ; Mb1 - Cre ( n = 10 for ChATfl / fl _ RT , n = 11 for Cre _ RT , n = 9 - 12 for ChATfl / fl _ CE , n = 13 for Cre _ CE ) ( G ) and littermate ChATfl / fl mice housed at RT or 4oC ( CE ) for 6 h . An insert graph in ( E ) highlights mRNA expression of Chrna2 and thermogenic genes in IWAT of ChATfl / fl and ChATfl / fl ; LysM - Cre mice after 6 h CE .

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

E-G - mRNA expression of Chrna2 and thermogenic genes in IWAT of ChATfl/fl;LysM-Cre (n = 24 for ChATfl/fl_RT, n = 25 for Cre_RT, n = 20 for ChATfl/fl_CE, n = 20 for Cre_CE) (E), ChATfl/fl;Cd4-Cre (n = 17 for ChATfl/fl_RT, n = 22-23 for Cre_RT, n = 19 for ChATfl/fl_CE, n = 22 for Cre_CE) (F), ChATfl/fl;Mb1-Cre (n = 10 for ChATfl/fl_RT, n = 11 for Cre_RT, n = 9-12 for ChATfl/fl_CE, n = 13 for Cre_CE) (G) and littermate ChATfl/fl mice housed at RT or 4oC (CE) for 6 h. An insert graph in (E) highlights mRNA expression of Chrna2 and thermogenic genes in IWAT of ChATfl/fl and ChATfl/fl;LysM-Cre mice after 6 h CE.


K - Average whole - body oxygen consumption rate ( OCR ) of ChATfl / fl ( n = 10 ) and ChATfl / fl ; LysM - Cre ( n = 14 ) mice housed in metabolic chambers at RT or CE for 6 h ( from 9 a . m . to 3 p . m . ) .

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

K - Average whole-body oxygen consumption rate (OCR) of ChATfl/fl (n = 10) and ChATfl/fl;LysM-Cre (n = 14) mice housed in metabolic chambers at RT or CE for 6 h (from 9 a.m. to 3 p.m.).


A - ChAT - eGFP + cells as a percentage of total IWAT SVF cells from ChAT - eGFP mice housed at RT ( n = 7 ) or 10 ° C for 4 h ( n = 3 ) . B - Percentages of total T cells , B cells and MΦ that are ChAT - eGFP + in IWAT from ChAT - eGFP mice housed at RT ( n = 7 ) or 10 ° C for 4 h ( n = 3 ) . C - ChAT - eGFP + cells as a percentage of total IWAT SVF cells from β - less ChAT - eGFP mice housed at RT or 10 ° C for 4 h ( n = 4 ) . D - Percentages of total T cells , B cells and MΦ that are ChAT - eGFP + in IWAT from β - less ChAT - eGFP mice housed at RT or 10 ° C for 4 h ( n = 4 ) . E - ChAT - eGFP + cells as a percentage of total IWAT SVF cells from ChAT - eGFP mice treated with vehicle ( veh ) or 1 mg / kg NE for 2 h ( n = 6 ) . F - Percentages of total T cells , B cells and MΦ that are ChAT - eGFP + in IWAT from ChAT - eGFP mice treated with veh or 1 mg / kg NE for 2 h ( n = 6 ) .

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

A - ChAT-eGFP+ cells as a percentage of total IWAT SVF cells from ChAT-eGFP mice housed at RT (n = 7) or 10°C for 4 h (n = 3). B - Percentages of total T cells, B cells and MΦ that are ChAT-eGFP+ in IWAT from ChAT-eGFP mice housed at RT (n = 7) or 10°C for 4 h (n = 3). C - ChAT-eGFP+ cells as a percentage of total IWAT SVF cells from β-less ChAT-eGFP mice housed at RT or 10°C for 4 h (n = 4). D - Percentages of total T cells, B cells and MΦ that are ChAT-eGFP+ in IWAT from β-less ChAT-eGFP mice housed at RT or 10°C for 4 h (n = 4). E - ChAT-eGFP+ cells as a percentage of total IWAT SVF cells from ChAT-eGFP mice treated with vehicle (veh) or 1 mg/kg NE for 2 h (n = 6). F - Percentages of total T cells, B cells and MΦ that are ChAT-eGFP+ in IWAT from ChAT-eGFP mice treated with veh or 1 mg/kg NE for 2 h (n = 6).


J - Relative mRNA expression of Adrb1 , Adrb2 and Adrb3 in IWAT MΦ sorted from WT mice ( n = 4 ) . Expression was measured by qPCR and normalized to levels of Tbp using the 2 - ΔΔCt method .

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

J - Relative mRNA expression of Adrb1, Adrb2 and Adrb3 in IWAT MΦ sorted from WT mice (n = 4). Expression was measured by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method.


D - E - Percentage of ( D ) total IWAT SVF cells and ( E ) IWAT MΦ expressing ChAT - eGFP in reporter mice with genetic deletion combinations of β - ARs 1 , 2 and 3 following treatment with veh ( white bar : n = 7 ) or 1 mg / kg NE ( black bars : from left to right , n = 7 , 5 , 4 , 5 , 4 , 7 ) for 2 h .

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

D-E - Percentage of (D) total IWAT SVF cells and (E) IWAT MΦ expressing ChAT-eGFP in reporter mice with genetic deletion combinations of β-ARs 1, 2 and 3 following treatment with veh (white bar: n = 7) or 1 mg/kg NE (black bars: from left to right, n = 7, 5, 4, 5, 4, 7) for 2 h.


F - LC / MS - MS quantification of acetylcholine levels secreted by SVF cells derived from IWAT of β2WT and β2KO mice treated with veh or 1 mg / kg Form for 2 h ( n = 3 ) .

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

F - LC/MS-MS quantification of acetylcholine levels secreted by SVF cells derived from IWAT of β2WT and β2KO mice treated with veh or 1 mg/kg Form for 2 h (n = 3).


M - Left : BMDMs were treated for 2 h with veh or pan β - AR agonist ( 100 μM NE ) , β2 - AR antagonist ( 5 μM butoxamine , Buto ) or a combination of NE and β2 antagonist ( Buto ) ( n = 6 ) . Chat mRNA expression was measured by qPCR and normalized to levels of Tbp using the 2 - ΔΔCt method . Right : Total number of ChAT - eGFP + BMDMs . ChAT - eGFP BMDMs were treated for 2 h with veh or pan β - AR agonist ( 100 μM NE ) , β2 - AR antagonist ( 5 μM butoxamine , Buto ) or a combination of NE and β2 - AR antagonist ( Buto ) ( n = 4 ) . An equal number of events ( 50 , 000 ) were analyzed by flow cytometry .

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

M - Left: BMDMs were treated for 2 h with veh or pan β-AR agonist (100 μM NE), β2-AR antagonist (5 μM butoxamine, Buto) or a combination of NE and β2 antagonist (Buto) (n = 6). Chat mRNA expression was measured by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method. Right: Total number of ChAT-eGFP+ BMDMs. ChAT-eGFP BMDMs were treated for 2 h with veh or pan β-AR agonist (100 μM NE), β2-AR antagonist (5 μM butoxamine, Buto) or a combination of NE and β2-AR antagonist (Buto) (n = 4). An equal number of events (50,000) were analyzed by flow cytometry.


N - Left : Bicompartmental co - culture system with media alone ( Ctrl ) or WT SVF cells isolated from IWAT in the upper compartment ( transwell insert ) and freshly isolated IWAT explants from β - less mice in the lower compartment . Cells were co - cultured for 4 h in the presence or absence of β2 - AR agonist ( 2 . 5 μM Form ) . 150 μM rivastigmine was added to the media to prevent degradation of Ach . Right : qPCR analyses of Chat and Ucp1 mRNA levels in β ‑ less explants following co - culture with media ( n = 4 ) , vehicle ( n = 10 ) or β2 - AR agonist ( n = 10 ) treated SVF cells . mRNA expression was measured by qPCR and normalized to levels of Tbp using the 2 - ΔΔCt method .

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

N - Left: Bicompartmental co-culture system with media alone (Ctrl) or WT SVF cells isolated from IWAT in the upper compartment (transwell insert) and freshly isolated IWAT explants from β-less mice in the lower compartment. Cells were co-cultured for 4 h in the presence or absence of β2-AR agonist (2.5 μM Form). 150 μM rivastigmine was added to the media to prevent degradation of Ach. Right: qPCR analyses of Chat and Ucp1 mRNA levels in β‑less explants following co-culture with media (n = 4), vehicle (n = 10) or β2-AR agonist (n = 10) treated SVF cells. mRNA expression was measured by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method.


( D , E ) Change in pO2 of osteoclasts in the mice upon exposure to various concentration of oxygen from 21 % to 14 % pO2 . Magnified PLIM images of osteoclasts under different peripheral oxygen saturation ( SpO2 , D ) . The pO2 of each osteoclast in these mice was plotted ( E , n = 47 from three mice for each SpO2 ) . Scale bar , 20 μm .

O O O O O O O O O B-CELL_TYPE O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-CELL_TYPE O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O O

(D, E) Change in pO2 of osteoclasts in the mice upon exposure to various concentration of oxygen from 21% to 14% pO2. Magnified PLIM images of osteoclasts under different peripheral oxygen saturation (SpO2, D). The pO2 of each osteoclast in these mice was plotted (E, n = 47 from three mice for each SpO2). Scale bar, 20 μm.


( F ) Effect of physioxia ( 5 % pO2 ) or physiological hypoxia ( 2 % pO2 ) on osteoclastogenesis . TRAP - stained cells ( left panel ) and the number of TRAP - positive cells with more than three nuclei ( right ) . Scale bar , 100 μm .

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

(F) Effect of physioxia (5% pO2) or physiological hypoxia (2% pO2) on osteoclastogenesis. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right). Scale bar, 100 μm.


( A ) μCT analysis of the femurs of 10 - week - old male mice upon exposure to low oxygen concentration ( 14 % pO2 , n = 5 ) or ambient air ( n = 7 ) , and of 10 - week - old Hif1aRank - / - ; Hif2aRank - / - male mice upon exposure to low oxygen concentration ( 14 % pO2 , n = 6 ) or ambient air ( n = 5 ) for 10 days ( axial view of the metaphyseal region ) . Scale , 0 . 5 mm . ( B ) Histological analysis of the proximal tibias of 10 - week - old male mice upon exposure to low oxygen concentration ( 14 % pO2 ) or ambient air for 10 days , and of 10 - week - old Hif1aRank - / - ; Hif2aRank - / - male mice upon exposure to low oxygen concentration ( 14 % pO2 ) or ambient air ( toluidine blue staining [ arrow , osteoclasts ] ) . Scale , 100 μm . ( C ) Parameters for osteoclasts and osteoblasts during bone morphometric analysis of 10 - week - old male mice under low oxygen concentration ( n = 3 ) or ambient air ( n = 5 ) , and 10 - week - old Hif1aRank - / - ; Hif2aRank - / - male under low oxygen concentration ( n = 3 ) or ambient air ( n = 5 ) for 10 days .

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

(A) μCT analysis of the femurs of 10-week-old male mice upon exposure to low oxygen concentration (14% pO2, n = 5) or ambient air (n = 7), and of 10-week-old Hif1aRank-/-; Hif2aRank-/- male mice upon exposure to low oxygen concentration (14% pO2, n = 6) or ambient air (n = 5) for 10 days (axial view of the metaphyseal region). Scale, 0.5 mm. (B) Histological analysis of the proximal tibias of 10-week-old male mice upon exposure to low oxygen concentration (14% pO2) or ambient air for 10 days, and of 10-week-old Hif1aRank-/-; Hif2aRank-/- male mice upon exposure to low oxygen concentration (14% pO2 ) or ambient air (toluidine blue staining [arrow, osteoclasts]). Scale, 100 μm. (C) Parameters for osteoclasts and osteoblasts during bone morphometric analysis of 10-week-old male mice under low oxygen concentration (n = 3) or ambient air (n = 5), and 10-week-old Hif1aRank-/-; Hif2aRank-/- male under low oxygen concentration (n = 3) or ambient air (n = 5) for 10 days.


( A ) Protein expression of HIF - 1α , HIF - 2α , Blimp1 , NFATc1 , Ctsk and TRAP in control and Hif1aRank - / - ; Hif2aRank - / - BMMs stimulated with RANKL and cultured under different conditions of oxygen concentration for 2 days .

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

(A) Protein expression of HIF-1α, HIF-2α, Blimp1, NFATc1, Ctsk and TRAP in control and Hif1aRank-/-; Hif2aRank-/- BMMs stimulated with RANKL and cultured under different conditions of oxygen concentration for 2 days.


( B ) Effect of physioxia ( 5 % pO2 ) or physiological hypoxia ( 2 % pO2 ) on the formation of osteoclast from control and Hif1aRank - / - ; Hif2aRank - / - BMMs . TRAP - stained cells ( left panel ) and the number of TRAP - positive cells with more than three nuclei ( right ) . Scale bar , 100 μm .

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

(B) Effect of physioxia (5% pO2) or physiological hypoxia (2% pO2) on the formation of osteoclast from control and Hif1aRank-/-; Hif2aRank-/- BMMs. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right). Scale bar, 100 μm.


( C ) Expression of osteoclastogenic and canonical HIF - target genes in wild - type control and Hif1aRank - / - ; Hif2aRank - / - BMMs under conditions of 5 % or 2 % oxygen .

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

(C) Expression of osteoclastogenic and canonical HIF-target genes in wild-type control and Hif1aRank-/-; Hif2aRank-/- BMMs under conditions of 5% or 2% oxygen.


( A ) Representative intravital image of calvarial bone marrow of Rosa26GO - ATeam / + ; Tg ( Acp5 - tdTomato ) male mice showing osteoclasts ( left , tdTomato fluorescence ) and FLIM image for ATP changes ( middle and right , fluorescence lifetime of EGFP ) . The FRET efficiencies of each osteoclast were plotted from the mice upon exposure to low oxygen atmosphere ( 14 % pO2 ) or ambient air ( right , n = 11 from three mice for each SpO2 ) .

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

(A) Representative intravital image of calvarial bone marrow of Rosa26GO-ATeam/+; Tg(Acp5-tdTomato) male mice showing osteoclasts (left, tdTomato fluorescence) and FLIM image for ATP changes (middle and right, fluorescence lifetime of EGFP). The FRET efficiencies of each osteoclast were plotted from the mice upon exposure to low oxygen atmosphere (14% pO2) or ambient air (right, n = 11 from three mice for each SpO2).


( B ) Representative intravital image of calvarial bone marrow of Tg ( Acp5 - tdTomato ) male mice showing osteoclasts ( left , tdTomato fluorescence ) and NAD ( P ) H autofluorescence lifetime images ( middle and right ) . Average of autofluorescence lifetime of each osteoclast was plotted from the mice upon exposure to low oxygen concentration ( 14 % pO2 ) or ambient air ( right , n = 64 from three mice for each SpO2 ) .

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

(B) Representative intravital image of calvarial bone marrow of Tg(Acp5-tdTomato) male mice showing osteoclasts (left, tdTomato fluorescence) and NAD(P)H autofluorescence lifetime images (middle and right). Average of autofluorescence lifetime of each osteoclast was plotted from the mice upon exposure to low oxygen concentration (14% pO2) or ambient air (right, n = 64 from three mice for each SpO2).


( B ) μCT analysis of the femurs of 10 - week - old control ( n = 6 ) , Tet2Rank - / - ( n = 5 ) , Tet3Rank - / - ( n = 5 ) , Tet2Rank - / - ; Tet3aRank + / - ( n = 6 ) , Tet2Rank + / - ; Tet3aRank - / - ( n = 6 ) and Tet2Rank - / - ; Tet3aRank - / - ( n = 4 ) male mice ( axial view of the metaphyseal region ) . ( C ) Histological analysis of the proximal tibias of 10 - week - old control , Tet2Rank - / - , Tet3Rank - / - , Tet2Rank - / - ; Tet3aRank + / - , Tet2Rank + / - ; Tet3aRank - / - and Tet2Rank - / - ; Tet3aRank - / - male mice ( TRAP staining [ red , osteoclasts ] ) .

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

(B) μCT analysis of the femurs of 10-week-old control (n = 6), Tet2Rank-/- (n = 5), Tet3Rank-/- (n = 5), Tet2Rank-/-; Tet3aRank+/- (n = 6), Tet2Rank+/-; Tet3aRank-/- (n = 6) and Tet2Rank-/-; Tet3aRank-/- (n = 4) male mice (axial view of the metaphyseal region). (C) Histological analysis of the proximal tibias of 10-week-old control, Tet2Rank-/-, Tet3Rank-/-, Tet2Rank-/-; Tet3aRank+/-, Tet2Rank+/-; Tet3aRank-/- and Tet2Rank-/-; Tet3aRank-/- male mice (TRAP staining [red, osteoclasts]).


( E ) Effect of Tet2 and Tet3 double deficiency on osteoclastogenesis . TRAP - stained cells ( left panel ) and the number of TRAP - positive cells with more than three nuclei ( right ) . ( n = 6 ( control ) , n = 10 ( Tet2Rank - / - ; Tet3aRank + / - ) , n = 3 ( Tet2Rank + / - ; Tet3aRank - / - ) and n = 4 ( Tet2Rank - / - ; Tet3aRank - / - ) biological

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

(E) Effect of Tet2 and Tet3 double deficiency on osteoclastogenesis. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right). (n = 6 (control), n = 10 (Tet2Rank-/-; Tet3aRank+/-), n = 3 (Tet2Rank+/-; Tet3aRank-/-) and n = 4 (Tet2Rank-/-; Tet3aRank-/-) biological replicates


( B ) MeDIP - qPCR analysis to validate regions that are hypermethylated in Tet2Rank - / - ; Tet3aRank + / - BMMs stimulated with RANKL for 2 days . ( C ) Gene expression of TET target candidates in BMMs stimulated with RANKL for 2 days .

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

(B) MeDIP-qPCR analysis to validate regions that are hypermethylated in Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 2 days. (C) Gene expression of TET target candidates in BMMs stimulated with RANKL for 2 days.


( E ) Bisulfate sequencing analysis of selected region of the second intron of the Prdm1 gene in control and Tet2Rank - / - ; Tet3aRank + / - BMMs stimulated with RANKL for 2 days . ( F ) Effect of retroviral NFATc1 expression on osteoclastogenesis of control and Tet2Rank - / - ; Tet3aRank + / - BMMs stimulated with RANKL for 3 days . TRAP - stained cells ( left panel ) and the number of TRAP - positive cells with more than three nuclei ( right ) . ( G ) Bisulfate sequencing analysis of the selected region of the second intron of the Prdm1 gene in BMMs stimulated with RANKL and culture under 5 % and 2 % oxygen for 3 days . ( H ) Effect of retroviral NFATc1 expression on osteoclastogenesis of BMMs stimulated with RANKL and culture under 5 % and 2 % oxygen for 5 days . Control and Tet2Rank - / - ; Tet3aRank + / - BMMs stimulated with RANKL for 3 days . TRAP - stained cells ( left panel ) and the number of TRAP - positive cells with more than three nuclei ( right ) .

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

(E) Bisulfate sequencing analysis of selected region of the second intron of the Prdm1 gene in control and Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 2 days. (F) Effect of retroviral NFATc1 expression on osteoclastogenesis of control and Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 3 days. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right). (G) Bisulfate sequencing analysis of the selected region of the second intron of the Prdm1 gene in BMMs stimulated with RANKL and culture under 5% and 2% oxygen for 3 days. (H) Effect of retroviral NFATc1 expression on osteoclastogenesis of BMMs stimulated with RANKL and culture under 5% and 2% oxygen for 5 days. Control and Tet2Rank-/-; Tet3aRank+/- BMMs stimulated with RANKL for 3 days. TRAP-stained cells (left panel) and the number of TRAP-positive cells with more than three nuclei (right).


( A ) Representative micrographs of human skin sections stained with hematoxylin - eosin ( H & E ) ( left ) and anti - IL - 38 antibody ( right ) from normal patients ( n = 11 ) and tumors of cSCC patients ( n = 13 ) . Scale bars represent 100 μm . The graph shows the quantification of mean IL - 38 expression per high - powered field in tissues . ( B ) Relative expression of IL - 38 in human normal tissues ( n = 9 ) and cSCC ( n = 18 ) were analyzed using Geo Datasets ( GSE98767 ) . Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test .

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

(A) Representative micrographs of human skin sections stained with hematoxylin-eosin (H&E) (left) and anti-IL-38 antibody (right) from normal patients (n = 11) and tumors of cSCC patients (n = 13). Scale bars represent 100 μm. The graph shows the quantification of mean IL-38 expression per high-powered field in tissues. (B) Relative expression of IL-38 in human normal tissues (n = 9) and cSCC (n = 18) were analyzed using Geo Datasets (GSE98767). Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.


( C and D ) The dorsal hair of normal C57 / BL6 mice were shaved and treated with DMBA / TPA twice a week for 32 weeks to induce skin tumors . ( C ) Representative micrographs of mouse normal skin ( n = 6 ) and tumor ( n = 6 ) sections stained with anti - IL - 38 antibody . The graph shows the quantification of mean IL - 38 expression per high - powered field in tissues . Scale bars represent 100 μm . ( D ) Relative expression levels of Il - 38 in normal skin ( n = 5 ) and tumors ( n = 5 ) of mice were quantified using qPCR . Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test .

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

(C and D) The dorsal hair of normal C57/BL6 mice were shaved and treated with DMBA/TPA twice a week for 32 weeks to induce skin tumors. (C) Representative micrographs of mouse normal skin (n = 6) and tumor (n = 6) sections stained with anti-IL-38 antibody. The graph shows the quantification of mean IL-38 expression per high-powered field in tissues. Scale bars represent 100 μm. (D) Relative expression levels of Il-38 in normal skin (n = 5) and tumors (n = 5) of mice were quantified using qPCR. Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.


( F ) Representative western blot bands indicating IL - 38 in mouse normal skin ( n = 3 ) and DMBA / TPA - induced tumors ( n = 3 ) . The graph shows the quantification of mean IL - 38 expression in tissues . Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test .

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

(F) Representative western blot bands indicating IL-38 in mouse normal skin (n = 3) and DMBA/TPA-induced tumors (n = 3). The graph shows the quantification of mean IL-38 expression in tissues. Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.


( B ) Representative photographs of Il - 38f / f ( n = 12 ) and K14Cre / + - Il - 38f / f mice ( n = 8 ) treated with DMBA / TPA for 32 weeks . White triangles indicate the tumors on the back of mice . ( C Average tumor number ( C ) per mouse from Il - 38f / f ( n = 12 ) and K14Cre / + - Il - 38f / f mice ( n = 8 ) treated with DMBA / TPA . . Error bars represent the mean ± SD ( C ) All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test (

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

(B) Representative photographs of Il-38f/f (n = 12) and K14Cre/+-Il-38f/f mice (n = 8) treated with DMBA/TPA for 32 weeks. White triangles indicate the tumors on the back of mice. (C Average tumor number (C) per mouse from Il-38f/f (n = 12) and K14Cre/+-Il-38f/f mice (n = 8) treated with DMBA/TPA.. Error bars represent the mean ± SD (C) All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test (C


( A ) Representative immunohistochemical staining micrographs of γH2AX in the skin of Il - 38f / f ( n = 5 ) and K14Cre / + - Il - 38f / f ( n = 5 ) mice after treated with DMBA for 24h . Red triangles indicate the γH2AX + positive cells . Scale bars represent 100 μm . The graph shows the number of γH2AX + cells per high - powered field . Data information : Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test .

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

(A) Representative immunohistochemical staining micrographs of γH2AX in the skin of Il-38f/f (n = 5) and K14Cre/+-Il-38f/f (n = 5) mice after treated with DMBA for 24h. Red triangles indicate the γH2AX+ positive cells. Scale bars represent 100 μm. The graph shows the number of γH2AX+ cells per high-powered field. Data information: Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.


( A The dorsal hair of normal C57 / BL6 mice were shaved and treated with DMBA / TPA twice a week for 3 weeks to induce the skin inflammation . ( A ) Representative micrograph sections stained with hematoxylin - eosin ( H & E ) from the skin of Il - 38f / f ( n = 5 ) and K14Cre / + - Il - 38f / f mice ( n = 5 ) Scale bars represent 100 μm . The graph shows average epidermal thickness .

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

(A The dorsal hair of normal C57/BL6 mice were shaved and treated with DMBA/TPA twice a week for 3 weeks to induce the skin inflammation. (A) Representative micrograph sections stained with hematoxylin-eosin (H&E) from the skin of Il-38f/f (n = 5) and K14Cre/+-Il-38f/f mice (n = 5) Scale bars represent 100 μm. The graph shows average epidermal thickness.


B ) The dorsal hair of normal C57 / BL6 mice were shaved and treated with DMBA / TPA twice a week for 3 weeks to induce the skin inflammation . ( B ) Representative micrograph sections stained with anti - Ki67 antibody from the skin of Il - 38f / f ( n = 6 ) and K14Cre / + - Il - 38f / f mice ( n = 6 ) . Scale bars represent 100 μm . The graph shows average numbers of Ki67 + cells per high - powered field .

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

B) The dorsal hair of normal C57/BL6 mice were shaved and treated with DMBA/TPA twice a week for 3 weeks to induce the skin inflammation. (B) Representative micrograph sections stained with anti-Ki67 antibody from the skin of Il-38f/f (n = 6) and K14Cre/+-Il-38f/f mice (n = 6). Scale bars represent 100 μm. The graph shows average numbers of Ki67+ cells per high-powered field.


( G and H ) Representative images of the scratch assay ( left ) and wound closure rate ( right ) of A431 cells ( n = 5 ) after overexpression ( G ) or knockdown ( H ) of IL - 38 . Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test . NC , Negative control ; LV , Lentivirus ; Oe , Overexpression .

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

(G and H) Representative images of the scratch assay (left) and wound closure rate (right) of A431 cells (n = 5) after overexpression (G) or knockdown (H) of IL-38. Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test. NC, Negative control; LV, Lentivirus; Oe, Overexpression.


( I and J ) Representative western blot bands indicating cyclin E and cyclin A in A431 cells after IL - 38 overexpression ( I ) or knockdown ( J ) . NC , Negative control ; LV , Lentivirus ; Oe , Overexpression .

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

(I and J) Representative western blot bands indicating cyclin E and cyclin A in A431 cells after IL-38 overexpression (I) or knockdown (J). NC, Negative control; LV, Lentivirus; Oe, Overexpression.


( A and B ) Pull - down assay of IL - 38 ( 1 - 152 aa ) ( A ) or IL - 38 ( 20 - 152 aa ) ( B ) with IL - 1Rrp2 - Fc , IL - 1RAPL1 - Fc , IL1RAcp - Fc , and IgG - Fc fusion proteins .

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

(A and B) Pull-down assay of IL-38 (1-152 aa) (A) or IL-38 (20-152 aa) (B) with IL-1Rrp2-Fc, IL-1RAPL1-Fc, IL1RAcp-Fc, and IgG-Fc fusion proteins.


( C ) Cell extracts prepared from 293T cells transfected with a mock mammalian expression vector ( left ) or an expression vector encoding IL - 1Rrp2 ( right ) were probed using anti - phospho - JNK , anti - JNK , or anti - β - actin antibodies after stimulation with IL - 38 ( 200 ng / ml ) or IL - 36β ( 200 ng / ml ) at different times .

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

(C) Cell extracts prepared from 293T cells transfected with a mock mammalian expression vector (left) or an expression vector encoding IL-1Rrp2 (right) were probed using anti-phospho-JNK, anti-JNK, or anti-β-actin antibodies after stimulation with IL-38 (200 ng/ml) or IL-36β (200 ng/ml) at different times.


( D ) Cell extracts prepared from 293T cells transfected with an expression vector encoding IL - 1Rrp2 were probed using anti - phospho - JNK , anti - JNK , or anti - β - actin antibodies after stimulation with IL - 38 at different concentrations .

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

(D) Cell extracts prepared from 293T cells transfected with an expression vector encoding IL-1Rrp2 were probed using anti-phospho-JNK, anti-JNK, or anti-β-actin antibodies after stimulation with IL-38 at different concentrations.


( F ) Cell extracts prepared from 293T cells transfected with HA - tagged full - length or deletion mutants of IL - 1Rrp2 , were probed using anti - HA or anti - β - actin antibodies . Oe , overexpression .

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

(F) Cell extracts prepared from 293T cells transfected with HA-tagged full-length or deletion mutants of IL-1Rrp2, were probed using anti-HA or anti-β-actin antibodies. Oe, overexpression.


( G ) Cell extracts prepared from 293T cells transfected with full - length or deletion mutants of IL - 1Rrp2 were probed using anti - phospho - JNK , anti - JNK , or anti - β - actin antibodies after stimulation with IL - 38 ( 200 ng / ml ) at different times .

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

(G) Cell extracts prepared from 293T cells transfected with full-length or deletion mutants of IL-1Rrp2 were probed using anti-phospho-JNK, anti-JNK, or anti-β-actin antibodies after stimulation with IL-38 (200 ng/ml) at different times.


Representative images of the scratch assay ( left ) and wound closure rate ( right ) of A431 cells ( n = 5 ) treated with recombinant IL - 38 ( 200 ng / mL ) ( F ) after IL - 1Rrp2 interference . Error bars represent the mean ± SD . All data are biological replicates . ns , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using one - way ANOVA ,

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

Representative images of the scratch assay (left) and wound closure rate (right) of A431 cells (n = 5) treated with recombinant IL-38 (200 ng/mL) (F) after IL-1Rrp2 interference. Error bars represent the mean ± SD. All data are biological replicates. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using one-way ANOVA , F


Representative images of the scratch assay ( left ) and wound closure rate ( right ) of A431 cells ( n = 5 ) treated with recombinant IL - 38 ( 200 ng / mL ) ( F ) after JNK interference . Error bars represent the mean ± SD . All data are biological replicates . ns , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using one - way ANOVA ,

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of the scratch assay (left) and wound closure rate (right) of A431 cells (n = 5) treated with recombinant IL-38 (200 ng/mL) (F) after JNK interference. Error bars represent the mean ± SD. All data are biological replicates. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using one-way ANOVA , F


B - J : Representative immunofluorescences of the cell types composing the WT and Mecp2 null cultures differentiated from NPCs . Antibodies against Tuj1 , Gfap and Olig2 were used to recognize neurons , astrocytes and oligodendrocytes . In D , G and J the percentage of cells positive ( mean ± SEM ) for each marker was counted over the total of DAPI positive cells ( 100 % ) . No difference between genotypes was highlighted ( Two - way ANOVA ) ; the variation through time resulted significant for the levels of Tuj1 and Gfap ( Two - way ANOVA : F ( 1 , 16 ) = 9 . 542 , p - value < 0 . 05 and F ( 1 , 10 ) = 68 . 95 , p - value < 0 . 0001 respectively ) . Sample size : n ≥ 3 wells obtained from two independent preparations ; for each well 10 random fields were acquired . Scale bars : 30 µm .

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 B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O B-CELL_TYPE 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 B-EXP_ASSAY 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 B-EXP_ASSAY 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 O O 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-J: Representative immunofluorescences of the cell types composing the WT and Mecp2 null cultures differentiated from NPCs. Antibodies against Tuj1, Gfap and Olig2 were used to recognize neurons, astrocytes and oligodendrocytes. In D, G and J the percentage of cells positive (mean ± SEM) for each marker was counted over the total of DAPI positive cells (100%). No difference between genotypes was highlighted (Two-way ANOVA); the variation through time resulted significant for the levels of Tuj1 and Gfap (Two-way ANOVA: F(1,16)=9.542, p-value<0.05 and F(1,10)=68.95, p-value<0.0001 respectively). Sample size: n ≥3 wells obtained from two independent preparations; for each well 10 random fields were acquired. Scale bars: 30 µm.


P : Scatter plots describing the transcriptional levels of selected differentially expressed genes in WT and Mecp2 null cultures differentiated from NPCs and analysed at DIV3 and DIV14 . For each gene Two - way ANOVA and Šidák multiple comparison test were used to assess difference between genotypes at the two selected time points ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ) . Values are represented as mean ± SEM . Sample size : ≥ 5 samples obtained from two independent preparations .

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

P: Scatter plots describing the transcriptional levels of selected differentially expressed genes in WT and Mecp2 null cultures differentiated from NPCs and analysed at DIV3 and DIV14. For each gene Two-way ANOVA and Šidák multiple comparison test were used to assess difference between genotypes at the two selected time points (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Values are represented as mean ± SEM. Sample size: ≥5 samples obtained from two independent preparations.


H - K : Raster plots ( H and I ) and their quantitation under basal conditions and upon 100µM 4 - AP exposure . The magnitude of the response after stimulation is expressed either as fold change ( J ) or as the averaged maximal values reached after 4 - AP exposure ( K ) . Dotted line in J represents untreated controls ( set to 1 ) . Values are represented as mean ± SEM . Student ' s t - test , * : p - value < 0 . 05 . Sample size n = 8 wells deriving from three independent preparations .

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 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 O O O O O O O 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-K: Raster plots (H and I) and their quantitation under basal conditions and upon 100µM 4-AP exposure. The magnitude of the response after stimulation is expressed either as fold change (J) or as the averaged maximal values reached after 4-AP exposure (K). Dotted line in J represents untreated controls (set to 1). Values are represented as mean ± SEM. Student's t-test, *: p-value<0.05. Sample size n=8 wells deriving from three independent preparations.


L : Wt and null neurons counterstained with an antibody against Tuj1 and DAPI at different time points . The binary mask is representative of a neuron used to study morphology . Scale bars : 30 µm . M - Q : Each graph represents a different morphological parameter analysed at selected time point . Two - way ANOVA indicated both a significant time effect ( ( F 4 , 20 ) = 282 , 5 and p < 0 , 0001 for the number of intersections ; ( F 4 , 26 ) = 104 , 5 and p < 0 , 0001 for the total dendritic length ; ( F 4 , 20 ) = 58 , 51 and p < 0 , 0001 for the maximal dendritic length ; ( F 3 , 22 ) = 38 , 32 and p < 0 , 0001 for polarization ) and genotype effect ( ( F 1 , 20 ) = 25 , 1 and p < 0 , 0001 for the number of intersections ; ( F 4 , 26 ) = 16 , 0 and p = 0 . 005 for the total dendritic length ; ( F 1 , 20 ) = 17 , 24 and p = 0 , 005 for the maximal dendritic length ; ( F 1 , 22 ) = 18 , 76 and p < 0 . 003 for polarization ) . Bonferroni multiple comparison test was used to assess difference between wt and null neurons at each analysed time point . The dotted line in panel P represents the cut - off above which a culture is defined polarized ( Horton et al , 2006 ) . Values are represented as mean ± SEM ; * : p - value < 0 . 05 ; * * : p - value < 0 . 01 ; * * * * : p - value < 0 . 0001 . Sample size : n ≥ 3 wells deriving from three independent preparations ; for each well we measured at least 15 cells .

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

L: Wt and null neurons counterstained with an antibody against Tuj1 and DAPI at different time points. The binary mask is representative of a neuron used to study morphology. Scale bars: 30 µm. M-Q: Each graph represents a different morphological parameter analysed at selected time point. Two-way ANOVA indicated both a significant time effect ((F 4,20)=282,5 and p<0,0001 for the number of intersections; (F 4,26)=104,5 and p<0,0001 for the total dendritic length; (F 4,20)=58,51 and p<0,0001 for the maximal dendritic length; (F 3,22)=38,32 and p<0,0001 for polarization) and genotype effect ((F 1,20)=25,1 and p<0,0001 for the number of intersections; (F 4,26)=16,0 and p=0.005 for the total dendritic length; (F 1,20)=17,24 and p=0,005 for the maximal dendritic length; (F 1,22)=18,76 and p<0.003 for polarization). Bonferroni multiple comparison test was used to assess difference between wt and null neurons at each analysed time point. The dotted line in panel P represents the cut-off above which a culture is defined polarized (Horton et al, 2006). Values are represented as mean ± SEM; *: p-value<0.05; **: p-value<0.01; ****: p-value<0.0001. Sample size: n≥3 wells deriving from three independent preparations; for each well we measured at least 15 cells.


B , C : Five morphological parameters were measured in untreated and treated null neurons at DIV14 . One - way ANOVA indicated a significant treatment effect for the maximal dendritic length ( F = 6 , 306 ; p = 0 , 0039 ) , total dendritic length ( F = 4 , 880 ; p = 0 , 0126 ) , number of intersections ( F = 10 , 70 ; p = 0 , 0003 ) and soma area ( F = 7 , 721 ; p = 0 , 0015 ) . Tukey ' s multiple comparison test was used to assess differences between null neurons and early and late treated neurons . * : p - value < 0 . 05 ; * * : p - value < 0 . 01 ; * * * : p - value < 0 . 001 . The variation of each of the selected parameters is summarized in the radar plot ( C ) . Sample size ( panels B , C ) : n ≥ 18 wells for ctrl group and n ≥ 10 wells for the treatment groups deriving from three independent preparations ; for each well we measured at least 15 cells . Each point is representative of a single well . Values are represented as mean ± SEM .

O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SUBCELLULAR 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 B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B,C: Five morphological parameters were measured in untreated and treated null neurons at DIV14. One-way ANOVA indicated a significant treatment effect for the maximal dendritic length (F=6,306;p=0,0039), total dendritic length (F=4,880;p=0,0126), number of intersections (F=10,70; p=0,0003) and soma area (F=7,721;p=0,0015). Tukey's multiple comparison test was used to assess differences between null neurons and early and late treated neurons. *: p-value<0.05; **: p-value<0.01; ***: p-value<0.001. The variation of each of the selected parameters is summarized in the radar plot (C). Sample size (panels B,C): n≥18 wells for ctrl group and n≥10 wells for the treatment groups deriving from three independent preparations; for each well we measured at least 15 cells. Each point is representative of a single well. Values are represented as mean ± SEM.


Bar graphs represent the expression values ( mean ± SEM ) of 27 genes , selected for their downregulation in null samples at DIV 14 ( p - value < 0 , 07 ; see Appendix Figure S2 ) , after CX546 administration in the two different time windows ( early in G and late in H ) . One - way ANOVA followed by Dunnett ' s multiple comparison test was used to compare the expression of each gene between Mecp2 null early and late treated samples and their corresponding untreated controls ( set to 1 , red dotted line ) . * : p - value < 0 . 05 ; * * : p - value < 0 . 01 , * * * : p - value < 0 . 001 , * * * * : p - value < 0 . 0001 . Sample size : n ≥ 8 wells deriving from two independent preparations . Each point is representative of a single well .

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-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 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

Bar graphs represent the expression values (mean ± SEM) of 27 genes, selected for their downregulation in null samples at DIV 14 (p-value <0,07; see Appendix Figure S2), after CX546 administration in the two different time windows (early in G and late in H). One-way ANOVA followed by Dunnett's multiple comparison test was used to compare the expression of each gene between Mecp2 null early and late treated samples and their corresponding untreated controls (set to 1, red dotted line). *: p-value<0.05; **: p-value<0.01, ***: p-value<0.001, ****: p-value<0.0001. Sample size: n≥8 wells deriving from two independent preparations. Each point is representative of a single well.


F , G : Violin plots represent the average values of MQAE intensity for early ( F ) or late ( G ) treated null neurons and their relative controls . One - way ANOVA followed by Tukey ' s multiple comparison test was used to compare null control neurons and null treated neurons to wt neurons . * * * : p < 0 . 001 ; * * * * : p < 0 . 0001 . The central line of the violin plot is the median while the two dotted lines represent the upper and lower quartiles . Sample size : n ≥ 300 cells from five different embryos per genotype .

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

F,G: Violin plots represent the average values of MQAE intensity for early (F) or late (G) treated null neurons and their relative controls. One-way ANOVA followed by Tukey's multiple comparison test was used to compare null control neurons and null treated neurons to wt neurons. ***: p<0.001; ****: p <0.0001. The central line of the violin plot is the median while the two dotted lines represent the upper and lower quartiles. Sample size: n≥300 cells from five different embryos per genotype.