B Representative immunofluorescence for Myosin Heavy Chain ( MHC in red ) in combination with DAPI ( in blue ) of iPSCs obtained from WT # 1 , GS ∆ 44 ( GS ∆ 44 # 8 ) GS ∆ 44 edited clones ( GS ∆ 44 # 8 . 2 ) differentiated for 9 days into myocytes by MYOD / BAF60c overexpression . Scale bar 100 μm . C Histogram represents fusion index . At least 7 randomly chosen microscope fields of two independent biological samples were analysed ( n = 2 ) . Data are presented as mean ± SD of biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) of iPSCs obtained from WT#1, GS∆44 (GS∆44#8) and GS∆44 edited clones (GS∆44#8.2) differentiated for 9 days into myocytes by MYOD/BAF60c overexpression. Scale bar 100 μm. C Histogram represents fusion index quantification. At least 7 randomly chosen microscope fields of two independent biological samples were analysed (n=2). Data are presented as mean ± SD of the biological replicates.


D extracted from myocytes obtained by differentiation of WT # 1 , GS ∆ 44 ( GS ∆ 44 # 8 ) GS ∆ 44 edited clones ( GS ∆ 44 # 8 . 2 ∆ CELF2a ) was analysed by RT - PCR for CELF2a , CELF2b , CELF2c DMD exon 45 skipping . GAPDH was used as loading control . arrow indicates DMD exon 45 skipped band . Representative results are shown ( n = 3 ) .

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

D RNA extracted from myocytes obtained by differentiation of WT#1, GS∆44 (GS∆44#8) and GS∆44 edited clones (GS∆44#8.2 ∆CELF2a) was analysed by RT-PCR for CELF2a, CELF2b, CELF2c and DMD exon 45 skipping. GAPDH was used as loading control. The arrow indicates the DMD exon 45 skipped band. Representative results are shown (n=3).


E Western blot on ( 40 μg ) extracted from myocytes obtained by differentiation of WT ( WT # 1 ) , GS ∆ 44 ( GS ∆ 44 # 8 ) edited GS ∆ 44 ( GS ∆ 44 # 8 . 2 ∆ CELF2a ) iPSC clones probed with antibodies against dystrophin ( DMD ) . Actinin ( ACTN ) was used as a loading control . For WT 5 % sample , 2 μg of were diluted in 38 μg of belonging from GS ∆ 44 # 8 to reach total amount of 40 μg . Representative results are shown ( n = 3 ) .

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

E Western blot on proteins (40 μg) extracted from myocytes obtained by differentiation of WT (WT#1), GS∆44 (GS∆44#8) and edited GS∆44 (GS∆44#8.2 ∆CELF2a) iPSC clones probed with antibodies against dystrophin (DMD). Actinin (ACTN) was used as a loading control. For the WT 5% sample, 2 μg of proteins were diluted in 38 μg of proteins belonging from GS∆44#8 to reach the total amount of 40 μg. Representative results are shown (n=3).


A accessibility ( ATAC - sequencing ) marks ( ChIP - seq ) signals in Celf2 region ( + / - 50kb ) using UCSC genome browser . ATAC - seq peaks called in two conditions are reported below each track . differential peaks specific for one of two conditions are reported in tracks ATACseq _ specific peaks _ WT ATACseq _ specific peaks _ GS ∆ 44 . ChIP - seq signals for H3K4me3 , H3K27ac H3K27me3 in WT GSΔ44 myoblasts ( WT myoblasts in orange ; GSΔ44 myoblasts in blue ) . ChIP - seq for MyoD in control was obtained from ( MacQuarrie et al , 2013 ) ( GSM1218849 ) . Orange boxes highlight regions containing first exon of each isoform ( a , b c ) .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O 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 DNA accessibility (ATAC-sequencing) and chromatin marks (ChIP-seq) signals in the Celf2 region (+/- 50kb) using the UCSC genome browser. ATAC-seq peaks called in the two conditions are reported below each track. The differential peaks specific for one of the two conditions are reported in the tracks ATACseq_specific peaks_WT and ATACseq_specific peaks_GS∆44. ChIP-seq signals for H3K4me3, H3K27ac and H3K27me3 in WT and GSΔ44 myoblasts (WT myoblasts in light orange; GSΔ44 myoblasts in light blue). ChIP-seq for MyoD in control cells was obtained from (MacQuarrie et al, 2013) (GSM1218849). Orange boxes highlight the regions containing the first exon of each isoform (a, b and c).


D Chromatins extracted from Control ( WT ) GS ∆ 44 myoblasts were immunoprecipitated with anti - MyoD control IgG antibodies . Regions corresponding to MyoD binding sites present in Peak A ( see Figure EV3D ) in MAFA LSTZ2 genes were analysed by qPCR . Fold enrichments of PeakA over MAFA or LSTZ2 immunoprecipitation ( IP ) are plotted for WT GS ∆ 44 samples . mean ± of triplicates from one representative among three experiment is shown . Statistical significance of differences between means was assessed by a two - t - P < 0 . 05 was considered significant . * P = 0 . 0339 , * * P = 0 . 0067 .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Chromatins extracted from Control (WT) and GS∆44 myoblasts were immunoprecipitated with anti-MyoD and control IgG antibodies. Regions corresponding to the MyoD binding sites present in Peak A (see Figure EV3D) and in the MAFA and LSTZ2 genes were analysed by qPCR. Fold enrichments of PeakA over MAFA or LSTZ2 immunoprecipitation (IP) are plotted for WT and GS∆44 samples. The mean ± SEM of triplicates from one representative among three experiment is shown. Statistical significance of differences between means was assessed by a two-tailed t-test and P<0.05 was considered significant. *P=0.0339, **P=0.0067.


B RT - PCR analyses of indicated RNAs in control ( WT ) , ∆ 44 in GS ∆ 44 myocytes collected after 9 days upon induction of differentiation . GAPDH was used as control . Representative results are shown ( n = 3 ) .

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

B RT-PCR analyses of indicated RNAs in control (WT), ∆44 and in GS∆44 myocytes collected after 9 days upon the induction of differentiation. GAPDH was used as control. Representative results are shown (n=3).


C RT - PCR analyses for CELF2a DUXAP8 expression in WT GS ∆ 44 myoblasts ( GM ) myocytes ( DM ) . GAPDH was used as control . Representative results are shown ( n = 3 ) .

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

C RT-PCR analyses for CELF2a and DUXAP8 expression in WT and GS∆44 myoblasts (GM) and myocytes (DM). GAPDH was used as control. Representative results are shown (n=3).


D RT - PCR of DUXAP8 expression in extracted from GS ∆ 44 , GS ∆ 44 ' s mother ( GSM ) GS ∆ 44 ' s father ( F ) fibroblasts . GAPDH was used as control . Lane ( - ) indicates negative control . Representative results are shown ( n = 3 ) .

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

D RT-PCR analysis of DUXAP8 expression in RNA extracted from GS∆44, GS∆44's mother (GSM) and GS∆44's father (F) fibroblasts. GAPDH was used as control. Lane (-) indicates the negative control. Representative results are shown (n=3).


E RT - PCR for DUXAP8 expression in extracted from WT GS ∆ 44 myocytes from WT ( WT # 1 ) , GS ∆ 44 ( GS ∆ 44 # 2 , GS ∆ 44 # 8 ) GSM ( GSM # 1 ) iPSCs induced to differentiation for 9 days . GAPDH was used as control . Lane ( - ) indicates negative control . Representative results are shown ( n = 3 ) .

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

E RT-PCR analysis for DUXAP8 expression in RNA extracted from WT and GS∆44 myocytes and from WT (WT#1), GS∆44 (GS∆44#2, GS∆44#8) and GSM (GSM#1) iPSCs induced to differentiation for 9 days. GAPDH was used as control. Lane (-) indicates the negative control. Representative results are shown (n=3).


F RT - PCR for indicated RNAs on extracts from GS ∆ 44 myoblasts treated with either control siRNAs ( siSCR ) or siRNAs against DUXAP8 ( siDUXAP8 ) collected two days after transfection . GAPDH was used as control . Lane ( - ) indicates negative control . Representative results are shown ( n = 3 ) .

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

F RT-PCR for the indicated RNAs on extracts from GS∆44 myoblasts treated with either control siRNAs (siSCR) or siRNAs against DUXAP8 (siDUXAP8) and collected two days after transfection. GAPDH was used as control. Lane (-) indicates the negative control. Representative results are shown (n=3).


G qPCR of DUXAP8 CELF2a expression in control myoblasts transfected with a plasmid for overexpression of DUXAP8 ( OE DUXAP8 ) or an empty vector ( Empty ) , collected after 96h . Relative mRNA were calculated with delta delta Ct method . qPCR reactions were normalized against an internal control ( GAPDH ) plotted relative to expression level in sample treated with empty vector which was set to a value of 1 . mean ± of triplicates from one representative experiment is shown ( n = 3 ) .

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

G qPCR analysis of DUXAP8 and CELF2a expression in control myoblasts transfected with a plasmid for the overexpression of DUXAP8 (OE DUXAP8) or an empty vector (Empty), and collected after 96h. Relative mRNA levels were calculated with the delta delta Ct method. qPCR reactions were normalized against an internal control (GAPDH) and plotted relative to the expression level in the sample treated with the empty vector which was set to a value of 1. The mean ± SEM of triplicates from one representative experiment is shown (n=3).


Microscope of tissue microinjection pipette before ( top ) after ( bottom ) annotation of tissue surface microinjection micropipette tip by experimenter . Scalebars : top 100µm , bottom 10µm . Ap indicates apical surface BL indicates basal lamina .

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

Microscope images of the tissue and microinjection pipette before (top) and after (bottom) annotation of the tissue surface and microinjection micropipette tip by the experimenter. Scalebars: top 100µm, bottom 10µm. Ap indicates the apical surface and BL indicates the basal lamina.


( Left ) Confocal image of a section of mouse telencephalon fixed stained immediately after microinjection . Dashed lines indicate locations in slice where microinjections were attempted at different pressures . Microinjections were attempted at pressure of 75 mbar ( left ) , 100 mbar ( center ) , 125 mbar ( right ) . ( Right ) of successful injections achieved by Autoinjector at 75 mbar ( 35 % , n = 314 total , 110 successful ) , 100 mbar ( 35 % , n = 185 total , 65 successful ) , 125 mbar ( 8 % , n = 287 total , 23 successful ) .

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

(Left) Confocal image of a section of mouse telencephalon fixed and stained immediately after microinjection. Dashed lines indicate locations in the slice where microinjections were attempted at different pressures. Microinjections were attempted at pressure of 75 mbar (left area), 100 mbar (center area), and 125 mbar (right area). (Right) Percentage of successful injections achieved by the Autoinjector at 75 mbar (35%, n = 314 total, 110 successful), 100 mbar (35%, n = 185 total, 65 successful), and 125 mbar (8%, n = 287 total, 23 successful).


( Left ) Confocal of a section of mouse telencephalon fixed stained immediately after microinjection . Dashed lines indicate locations in slice where microinjections were attempted at different depths keeping pressure constant at 75 mbar . Microinjections were attempted at depths of 10 μm ( left ) , 15 μm ( center ) , 25 μm ( right ) into apical surface . ( Right ) of successful injections achieved by Autoinjector at depths of 10 μm ( 68 % , n = 163 total , 111 successful ) , 15 μm ( 22 % , n = 170 total , 37 successful ) , 25 μm ( 11 % , n = 169 total , 19 successful ) into apical surface .

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

(Left) Confocal images of a section of mouse telencephalon fixed and stained immediately after microinjection. Dashed lines indicate locations in the slice where microinjections were attempted at different depths keeping the pressure constant at 75 mbar. Microinjections were attempted at depths of 10 μm (left area), 15 μm (center area), and 25 μm (right area) into the apical surface. (Right) Percentage of successful injections achieved by the Autoinjector at depths of 10 μm (68%, n = 163 total, 111 successful), 15 μm (22%, n = 170 total, 37 successful), and 25 μm (11%, n = 169 total, 19 successful) into the apical surface.


targeted at a constant spacing of 30 μm , scale bar is 10 μm .

Cells targeted at a constant spacing of 30 μm, scale bar is 10 μm.


Automated microinjection was performed on organotypic slices of mouse E14 . 5 dorsal telencephalon using Dextran - A555 with slice culture for 24 or 48 h After fixation slices were stained for Tbr2 TuJI . of tissue after microinjection with dextran - A555 ( magenta ) . tissue was stained for Tbr2 ( green ) TuJI ( ) . Microinjected at 0 h ; injected show typical bipolar of an AP are mainly negative for BP marker Tbr2 . Two - daughter progeny 24 h after microinjection ; top shows bipolar characteristic of an AP is negative for Tbr2 ; bottom is a newborn BP positive for Tbr2 . Both are negative for neuronal marker TuJI ( ) . Four - daughter progeny 48 h after microinjection ; note that daughter 1 2 reside in VZ , while daughter 3 4 are positive for Tbr2 TuJI reside in SVZ . Distribution of microinjected their progeny in VZ , SVZ CP ( cortical plate ) . n = 147 total for 0 h , 151 total for 24 h , 26 total for 48 h . Distribution of microinjected into 9 bins on from ventricular surface ( 0 µm ) expressed as % of total . n = 147 total for 0 h , 151 total for 24 h , 26 total for 48 h . VZ , ventricular zone ; SVZ , subventricular zone ; CP , cortical plate .

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

Automated microinjection was performed on organotypic slices of mouse E14.5 dorsal telencephalon using Dextran-A555 with slice culture for 24 or 48 h After fixation slices were stained for Tbr2 and TuJI. Fluorescence images of tissue after microinjection with dextran-A555 (magenta). The tissue was stained for Tbr2 (green) and TuJI (white). Microinjected cell at 0 h; injected cells show the typical bipolar morphology of an AP and are mainly negative for the BP marker Tbr2. Two-daughter cell progeny 24 h after microinjection; the top cell shows the bipolar morphology characteristic of an AP and is negative for Tbr2; the bottom cell is a newborn BP positive for Tbr2. Both cells are negative for the neuronal marker TuJI (white). Four-daughter cell progeny 48 h after microinjection; note that daughter 1 and 2 reside in the VZ, while daughter 3 and 4 are positive for Tbr2 and TuJI and reside in the SVZ. Distribution of microinjected cells and their progeny in the VZ, SVZ and CP (cortical plate). n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h. Distribution of microinjected cells into 9 bins based on the distance from the ventricular surface (0 µm) and expressed as % of total. n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.


Automated microinjection was performed on organotypic slices of mouse E14 . 5 dorsal telencephalon using Dextran - A555 with slice culture for 24 or 48 h After fixation slices were stained for Tbr2 TuJI . Expression of Tbr2 in microinjected their progeny at 0 , 24 an 48 h . n = 147 total for 0 h , 151 total for 24 h , 26 total for 48 h . Expression of TuJI in microinjected their progeny at 0 , 24 an 48 h . n = 147 total for 0 h , 151 total for 24 h , 26 total for 48 h .

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

Automated microinjection was performed on organotypic slices of mouse E14.5 dorsal telencephalon using Dextran-A555 with slice culture for 24 or 48 h After fixation slices were stained for Tbr2 and TuJI. Expression of Tbr2 in microinjected cells and their progeny at 0, 24 an 48 h. n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h. Expression of TuJI in microinjected cells and their progeny at 0, 24 an 48 h. n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h.


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 ) RFP poly - A + - mRNA . After 24h in culture progeny expresses RFP ( magenta ; blue : DAPI , n = 17 injected , 8 RFP positive ) . of RFP - positive RFP - negative ( n = 17 injected ) .

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

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 ) Alexa488 ( green ) . Slices were fixed after microinjection ( n = 71 total ) were stained for Tbr2 ( , H - J ) . ( A - C ) Representative examples of a non - coupled ( A , cartoon on right ) , a 2 - cluster ( B , cartoon on right ) a 5 - cluster ( C , cartoon on right ) . asterisks indicate Dx - A555 - positive , microinjected . of microinjected found in a coupled cluster ( coupled , green ) . Cluster , expressed as % of total . Distribution of coupled expressed as from ventricular surface ( 0 µm = ventricle surface . Distribution of coupled divided into 11 bins expressed as % of total . on left are maximum projections ( MIP ) of 18 , 17 , 23 , 12 30 - focal planes ,

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

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

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

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 ) LY ( green , Slices were fixed after microinjection stained for Reelin ( , C ) Left : overview image of a mouse microinjected ( arrow indicates a pyramidal neuron , arrowhead indicates a Cajal - Retzius neuron ; image is MIP of 20 focal planes ) . Microinjected neurons show different positivity for Cajal - Retzius marker Reelin ( ) . Right , top row : pyramidal neuron indicated with an arrow in overview image is negative for Reelin . Right , bottom row : Cajal - Retzius neuron indicated with an arrowhead in overview image is positive for Reelin . on top bottom row left ( LY MIP ) are a MIP of 2 3 focal planes , respectively . three on top bottom right ( LY , Reelin DAPI ) are single optical sections . Scale bar is 20 µm for MIP 10 µm for single focal planes . VZ , ventricular zone ; SVZ , subventricular zone ; CP , cortical plate .

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

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 ) LY ( green Slices were fixed after microinjection stained for SatB2 ( , Overview image of a mouse microinjected . arrow indicates a pyramidal neuron ; image is MIP of 30 focal planes , scale bar : 50 µm . Row on right : microinjected neuron with pyramidal indicated with an arrow in overview is positive for upper - layers marker SatB2 ( ) . are single focal planes . Scale bar is 10 µm . Proportion of neurons positive or negative for Reelin ( n = 12 total ) . Proportion of neurons positive or negative for SatB2 ( n = 39 in total ) . VZ , ventricular zone ; SVZ , subventricular zone ; CP , cortical plate .

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

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 ) LY ( green , Slices were fixed after microinjection Microinjected neurons in 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 .

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

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 n = 37 in others , Mann - Whitney U , * P < 0 . 05 ) . center of boxplots represents median value , bounds of box represent 75th 25th percentile , whiskers represent maximum minimum value .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 locust brain . Rp49 served as a loading control ( n = 4 biological replicates ) . Data information : data are presented as mean ± .

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

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

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

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

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

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

C Representative 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 ) TAGs ( n = 10 biological replicates ) in fat bodies . Data information : data are presented as mean ± ( Student ' s t - , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant ) . assays were performed on four - day - old fourth - instar locusts .

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

E, F 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 ) , data are shown as mean ± ( Student ' s t - , * * P < 0 . 01 ) .

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ILC subsets in humans ( hILCs ) .

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


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

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

(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 cKithigh ILC2s ( C )

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


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

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

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

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of c - Maf occupancy on IL - 4 , IL - 5 IL - 13 promoters in untreated mouse human ILC2s ( IL - 2 stimulation ) in mouse human ILC2s treated with IL - 33 ( n = 3 biological replicates ) .

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

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

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

(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 indicated markers . Each dot represent sample from one donor . Bars represent mean ± .

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

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


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

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

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

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

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

O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O 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).


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

O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O 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 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 ) littermate ChATfl / fl mice housed at RT or 4oC ( CE ) for 6 h . An insert graph in ( E ) highlights mRNA expression of Chrna2 thermogenic genes in IWAT of ChATfl / fl ChATfl / fl ; LysM - Cre mice after 6 h CE .

O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O 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 - oxygen consumption rate ( OCR ) of ChATfl / fl ( n = 10 ) 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 O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O 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 + as a of total IWAT SVF from ChAT - eGFP mice housed at RT ( n = 7 ) or 10 ° C for 4 h ( n = 3 ) . B - Percentages of total T , B 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 + as a of total IWAT SVF from β - less ChAT - eGFP mice housed at RT or 10 ° C for 4 h ( n = 4 ) . D - Percentages of total T , B 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 + as a of total IWAT SVF from ChAT - eGFP mice treated with vehicle ( veh ) or 1 mg / kg NE for 2 h ( n = 6 ) . F - Percentages of total T , B 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-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

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 Adrb3 in IWAT MΦ sorted from WT mice ( n = 4 ) . Expression was measured by qPCR normalized to of Tbp using 2 - ΔΔCt method .

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

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 - of ( D ) total IWAT SVF ( E ) IWAT MΦ expressing ChAT - eGFP in reporter mice with genetic deletion combinations of β - ARs 1 , 2 3 following treatment with veh ( 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 O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of acetylcholine secreted by SVF derived from IWAT of β2WT β2KO mice treated with veh or 1 mg / kg Form for 2 h ( n = 3 ) .

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

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 β2 antagonist ( Buto ) ( n = 6 ) . Chat mRNA expression was measured by qPCR normalized to of Tbp using 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 β2 - AR antagonist ( Buto ) ( n = 4 ) . An equal number of events ( 50 , 000 ) were analyzed by flow cytometry .

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

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

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

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 mice upon exposure to various of oxygen from 21 % to 14 % pO2 . Magnified PLIM of osteoclasts under different peripheral oxygen saturation ( SpO2 , D ) . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ( left panel ) number of TRAP - positive 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-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

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

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

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

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O 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 formation of osteoclast from control Hif1aRank - / - ; Hif2aRank - / - BMMs . TRAP - stained ( left panel ) number of TRAP - positive 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 O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

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

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O 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 ) mice showing osteoclasts ( left , tdTomato ) FLIM image for ATP changes ( middle right , lifetime of EGFP ) . FRET efficiencies of each osteoclast were plotted from 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 O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) mice showing osteoclasts ( left , tdTomato ) NAD ( P ) H autofluorescence lifetime ( middle right ) . Average of autofluorescence lifetime of each osteoclast was plotted from mice upon exposure to low oxygen ( 14 % pO2 ) or ambient air ( right , n = 64 from three mice for each SpO2 ) .

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

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O 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 Tet3 double deficiency on osteoclastogenesis . TRAP - stained ( left panel ) number of TRAP - positive with more than three nuclei ( right ) . ( n = 6 ( control ) , n = 10 ( Tet2Rank - / - ; Tet3aRank + / - ) , n = 3 ( Tet2Rank + / - ; Tet3aRank - / - ) n = 4 ( Tet2Rank - / - ; Tet3aRank - / - ) biological

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

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

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

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

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

(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 ) DMBA / TPA - induced tumors ( n = 3 ) . graph shows of mean IL - 38 expression in tissues . Error bars represent mean ± SD . data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t .

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

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

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

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

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

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

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

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

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

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

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

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

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O 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 ) extracts prepared from 293T 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 O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 ) extracts prepared from 293T 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 O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 ) extracts prepared from 293T transfected with HA - tagged full - 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 O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

(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 ) extracts prepared from 293T transfected with full - 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 O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O

(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 of scratch ( left ) wound closure rate ( right ) of A431 ( n = 5 ) treated with recombinant IL - 38 ( 200 ng / mL ) ( F ) after IL - 1Rrp2 interference . Error bars represent mean ± SD . 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 O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 of scratch ( left ) wound closure rate ( right ) of A431 ( n = 5 ) treated with recombinant IL - 38 ( 200 ng / mL ) ( F ) after JNK interference . Error bars represent mean ± SD . 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 O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 types composing WT Mecp2 null cultures differentiated from NPCs . Antibodies against Tuj1 , Gfap Olig2 were used to recognize neurons , astrocytes oligodendrocytes . In D , G J of positive ( mean ± ) for each marker was counted over total of DAPI positive ( 100 % ) . No difference between genotypes was highlighted ( Two - way ANOVA ) ; variation through time resulted significant for of Tuj1 Gfap ( Two - way ANOVA : F ( 1 , 16 ) = 9 . 542 , p - value < 0 . 05 F ( 1 , 10 ) = 68 . 95 , p - value < 0 . 0001 respectively ) . Sample : 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 O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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

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 I ) their quantitation under basal conditions upon 100µM 4 - AP exposure . magnitude of response after stimulation is expressed either as fold change ( J ) or as averaged maximal values reached after 4 - AP exposure ( K ) . Dotted in J represents untreated controls ( set to 1 ) . Values are represented as mean ± . Student ' s t - , * : p - value < 0 . 05 . Sample 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.